STRUCTURE AND FUNC'MON OF THE HUMAN INSULM

RECEPTOR: QUATERNARY 3D RECONSTRUCTION OF THE

I N S ~ I N S U L I N RECEPTOR - COMPLEX AND THE

STRUCTURAL LOCALIZATLON OF THE INSULIN-BINDING SITE

Submitted to The School of Graduate Studies

of The University of Toronto

ROBERT ZECAO-TIAN LUO

In partial fiilfillment of reqainments

for the degree of

Doctor of Philosophy

O Robert Zhao-Tian Luo, 2000

~ u i s i i s and AcquWons et Bibliographk Services senrices bibriraphques

The author bas grantecl a non- exclusive Iicence ailowing the National Liirary of Caiiada to reproduce, logn, distniute or sell copies of this thesis in microform, paper or electronic fotmats.

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TABLE OF CONTENTS

TABLE OF CONTENTS LIST OF FIGURES LIST OF ABREVIATIONS ACKNOWLEDGEMENT

1. INTERCELLULAR COMMUNICATION

2. TEE INSULIN FAMILY OF PEPTIDES 2.1. liisulin 2.2. -L&e Growth Factors 2.3- Other members ofthe Insulin fàmily

3. MOLECULAR AND CELL BIOLOGY OF INSUUN AND IGF-1 RECEPTOR

3 -1. The Receptor Tyrosine Kinase S u p a f d y 3.2. IasulinReceptor 3.3. IR, IGF-IR and Similarity and Wice

4, INSULIN BINDING AND S I G N a ~ S D U C T I O N 4.1. A f k i t y and. Kinetics of rilsulin Binding 4.2. Autophosphorylation 4.3, Post-Receptor Signal Transduction

5. MAPPING TEE INSULIN-BINDING SlTE 5.1 . Insulin Interaction with the IR 5.2. Insulia Binding Domain on the IR

CHAPTER 2 LOCALEATLON OF INSULIN-BINDING SITE@) ONTHE HUMAN INSULIN RECEPTOR BY BIOCHEMICAL APPROACHES

1. INTRODUCTION

2. MATERIAL AND METHODS 2.1. O v e r ~ i o n of the Extracellular Domain of the HIR 2.2. Purification of the Recombinant IR and Huaian Placenta Membrane

IR 53

3 e RESITLTS 63 3 -1, Recombinant HIR ExtraceiluIar Domain 63 3 2 - A Smaü Labeled Peptide Fragment on the Lnsulin Binding Region 69

4. DISCUSSION 76

-R 3 THE QUATERNARY 3D STRUCTURE OF THE

DE- BY SCANMKG TRANSMISSION ELECTRON MI%ROSCOPYr STRUCTURAL LOCALIZATION OF TEE ms-BINDING S m

1. INTRODUCTION 1.1. El-n M*~~~scupic 3-D Reco-=on Techniqges 1.2. Stnictural Detemunation of IR and Locaüzation of riisului Binding

Site

2. MATERIALS AND METHODS 2.1. BiologicalcalMateriais 2.2. Electron Microscopy and ïinage AnaIysis 2.3 . Three Dimensional Reconstruction

3 e RESULTS 3.1. Specimen Preparation for Electron Microscopy 3 -2- Image processing of NG-BI-HIR Complex 3 3 . Lacation of the uisului-Binding Region 3 -4- Domain Structutes of the HIR

4. DISCUSSION

C-R 4 CONCLUSION AND FUTURE RESEARCH 125

iii

LIST OF ETGITRES

Chapter 1 Page

Fig. 1-1. Predicted tertiary structures of the insalin family of peptides Fig. 1-2. The ptimary structure, the 3D crystai structure and the 3D

space-filiing mode1 of human Fnsulln Fig. 1-3. Schematic representation ofthe insulin receptor gene, the primary

and secondary structure of hIR Fig. 1-4. Human insuiin receptor amino acld sequence Fig. 1-5. A schematic diagram of the Insuiin signahg pathways

Chapter 2

Fig. 2-1, Expression of human insulin receptor extracellular domain with bacuiovirus expression system @ES)

Fig. 2-2. Construction of the BES transfection vectors for the bIR ectodomain Fig. 2-3, Insulin photoprobes Fig. 2-4. Tirne course of expression of =or, and Western blot of hlRa

and hlR Fig. 2-5. Immunoprecipitation of B29-MABI labeled 61Ra Fig. 2-6. Affinity purification of expressxi hIRa with anti-hlR monoclonal

antibody (&W5 1) column Fig. 2-7. Insulin-binding assay of hIRa compared with hIR Fig. 2-8. Tirne course of photolysis Fig. 2-9. Time course and products of trypsin digestion of B29-MABI or

AZAP Iabeled hiRa Fig. 2-10. The sensitivity of ELISA in detecting B-chain fragment of bovine

insulin before and after HPLC analysis Fig. 2-1 1. Trypsin digests of hIRa labeled with '=I-B~~-MABI Fig. 2- 12. Achromobacter protease 1 digests of hIRa Iabeled with

'~1-BBPa-riisuiin Fig. 2- 13. HPLC analysis of partiaiiy purified fractions as identified in Fig. 2-12

Chapter 3

Fig. 3-1. Different electron microscopes used in biomedicd studies Fig. 3-2. An overview of STEM 3D reconstruction Fig. 3-3. Outline of the preparation of NGBI Fig. 3-4. Purification of hIR Fig. 3 -5- Purification and characterization of NGBI

Fig. 3 6 . STEM images of purifid NGBI 101 Fig. 3-7. STEM àarkfield Images ofhWNGB1 cornpla~ 103 Fig. 3-8. Composite raw images ofhIR/NGBI mo1ecdes 104 Fig. 3 -9. Low band-pass nltasd images 105 Fig- 3-10. Finai images used for 3D - d o n 106 Fig. 3-1 1. Histogram ofthe. caiculated mass for 1,625 co1Iected images 107 Fig, 3-12. Caldateci relative anguiar orientation for indIvidual molecular images 108 Fig. 3-1 3.3D reconstruction of the hIWNGBI cornplex (&âce representation) 109 Fig. 3-14. The Seconaary domain stnicture ofhIR and the domain Idon in 3D

stnichae 112 Fig. 3 -1 5, Fitting of biochemkal domairis and their lmown =ray crystal dmctwes

to the 3 D mnstructim 113

OC 2D 3D AZAP BBpa BES BI BSA CD CNBr CR DAG DBI DIPEA DMA EGF ELISA EM FGF Fa1 Fn2 ETLC g HGF hIR hIRa HPLC ID IGF-1 IGF-II IGF-1 R IQAD IR IRS-1 IRR kDa kV L1 LI-CR-L2 L2 LDL M MABI

degree Celsnis twodlmensional tbtee-dimensional do-phenyb-aminopropyl biotinyl-benu,ylphen081anine .

baculovinis eqmssïngsystem bovineinsuIin boviaesenunaibumin connecting domain cyanogen bromide cysteine-rich domah diacylglycerol &-&--insulin di-iso-propylenthyhnhe bimethylacetamide epidermai pwthfactor enzyme-iinked immunosorbant assay eI-n microscope ~ b I a s t ~ w t h f i c t o r fibroneCtinîypeIIIrepeaî1 fibronectintypemrepeat2 rapt @ormance Iiquid chromatography P== hepatocyte growth f'actor humaninsulinreceptor human insuiin teceptor extracellular domain high perfiormance liquid chromatography insert domain insulin-like growth factor 1 insulul-like growthfactor II insulia-like growth factor 1 receptor itaative q--assisted angular daermmation insutinreceptor insulin receptor substtate-1 insulin receptor telated recepttor kiloDalton kilovolts large homologous domain 1 Ll-Cysteine-rich-L2 domain large homologous domain 2 low density fipoptein m o k monoazido-Wyl-insulin

mg Crg MIP ml

mM MOI Mr NG NGBI NGF nm NMR PAGE PBS PDB PDGF PTK RPTK SDS SEM SEI2 SK STEM TEM TK TM

microgram d o s c a n insulin-like pepîide

microlitet mrnimolar mdtipIIcity of Infection mofecular welght nanogold nanogold-bovine insulin nerve growthfactor nanometers nuclear magnetic resomnce polyacryl-de gel eIectmphoresis phosphate bufEer solutio~ protein data bank plateletderiveci g~owth fàcîor pmteimtyn,sine kinase recep~rpro~tyrosine kinase sodiumdoedecyl sulfiite scanniaig elcctron microscope Sn= homology 2 serindthreonine kinase .crnnning transmission electron micmscope transmission electron mic~oscope tyrosine kinase transmembrane domain

My greatest thanks are due to my thesis supevisor, Dr, Cecil C. Yip, It is Cecil's iavaIuabIe, enthusiastic inspiration, and never ending guidance that made my research experience at the University of Toronto most wonderfüi. Through hïs dedication and persistence to scientifïc research, Cecil not only showed me how to conduct great science but also taught me how to be a human bemg with great integrity. 1 am also exceedingiy gratefûi to my thesis co-supervisor Dr. F. Peter Ottensmeyer for his excellent gwgwdance, encouragement and giving me the opportunity to access the state of the art STEM and cornputer image processing technique in his lab.

1 am deeply grateful to other members of m y graduate committee: Dr. Daniel Drucker and Dr. Jennifer Dorrîngton. 1 appreciate their contriibutions and cntical suggestions to the fruition of this research-

1 would particularly thank Dr. Margaret Moule for her helpful discussion, encouragement., enthusiasticdy reviewïng m y thesis and her expertise in research during the project 1 would Iike to thank othet colleagues and members in Dr. Yip's lab: Helga Hsu, Guoqing Zhong, Christine Miasos, Elaine Jack and Dr. Teresa Tallenco-Melnyk. Their consistent encouragement, technihnical assistance and friendship helped me nni-sh my program. 1 wouid aiso iike to thank colleagues and members in Dr. Ottensmeyer's lab: Allen Fernandes, Dr. Yongyi Mao, Michael Burke, Dr. Daniel Beniac, Brenda Rutherford, Yew Meng Heng and David McAiduff. Without their technical assistance, inspiring discussion and noble friendship, my project wouid not get any progress.

1 acknowledge the financial support from the University Toronto, the Ontario Goverment and the Medical Research Council of Can&

FiaMy, 1 would like to give my special thanks to my Iovely wife, Dr. Hongqi Peng and daughter Nancy, for their endless support and companionship. Without their sacrifice, 1 wouldn't be able to stand where 1 am. 1 am extremely grateful to my parents, parents-in-law, brothers and sister for their long t h e encouragement and support.

RECEPTOR: QUA-NEY 3D RECONSTRUCTION OF THE

INSULININSULIN RECEPTUE COMPLEX AND THE

STRUCTURAL LOCALIZATIION OF THE INSULIN-BNDING SITE - PhD. Thesis, Robert Zhao-Tian Luo, hstitute of Medicai Science, University of Toronto,

2000

The elucidation of the mechanism of cellular signal transduction is one of the key

challenges in biology. The first step in this pathway has been particularly refiactory to

study. In the case of insuIin action, it is the binding of insulin to its ce11 s d a c e receptor,

a large integral trammembrane glycoprotein, The binding of insulin d t s in the

activation of the cytoplasmic domain of the receptor as a tyrosine kinase, While much is

known about the interaction of the activated receptor with its substrates Ieading to down-

stream signal transduction, how the binding of insului to the extracellular domain of the

receptor can lead to receptor activation remains unknown. The specific goal of this study

is to localize the insuiin-binding site of the insulin receptor (IR), and, if possible, to

elucidate in molecuiar details the insulin-IR interaction and the structure-fwiction

relationship of the insulin receptor,

To achieve this goal with biochemical approach, 1 used the baculovirus expression

system to produce in large quantity the extraceUuiar domain of the human insulin

receptor, and used various specific photoaffiity probes to iden- the insulin-contacting

sites. Peptide identification methods of high sensitivity were developed to detect the

labeled IR fiagment containing the putative insulin-bhding site by HPLC peptide

mapping analysis. A smali ftagment of 1-3 kDa that might contain the putative insulin-

binding or contacting site on the receptor a sabunit was âetected However, p d y due to

the lack of an insuiin photoprobe with a nigh efficiency of cross-linking, and a high

efficiency peptide mapping method, 1 failed to obtain a sufncient quantity of the fiagrnent

for amino acid sequencing.

In an alternative approach, usuig gold-labeIed insulin as a iigand, and sets of

eIectron micrographs obtained by low-dose low temperature dark field scanning

transmission electron microscopy (STEM) of the in&-IR complex, 1 successfiilly

reconstnicted the 3D quatemary structure of the whole 480-kDa human inSulln receptor

(KiR), complexed with Uisulin, at a resolution of 20 k Contiguous hi& densities within .

the 3D structure indicated a two-fold symmetry for this dimeric receptor and the presence

of subdomain structures which begin with the extracellular subdomain involved in insulin

biading and terminate with the inhracellular tyrosine kinase subdomains. These

subdomains were confinned by fitting known high-resolution crystai domain

substructures. In addition, the 3D insulin-HIR complex provides evidence for the

involvement of only a single insulin molecule in receptor activation. It also explains -

many of the known characteristics of insulia binding to its receptor and many

biochemicd fhdïngs in the studies of the insulin receptor.

INTRODUCTION

Io IiYIERCELLUIAR COMMUNICATLON

In mdticeUdar organisms communications are cruciai ta coordinate their fimction

in responduig to the e n . ~ ~ m e n t and maintahhg homeostasis. The pwth, migraîion

and differentiation of ceiis in the embryo, and theü organhtion nit0 specific tissues,

depend on signais transrmCtted h m one c d to another. In the adult, ce11 signahg

orchestrates normal cellular behavïor and responses to e x t d stimuli- The b i s for the

intercellular communication can be viewed as ligand-receptor interaction. The receptors,

either intraceiiular or ceii siirface, are an by ceiis to enabIe them to detect signal

molecules such as hormones and growth m o r s in their microenvironment, Signal

molecules such as neurotraaîmtters or growth fhctors may k limiteci to interacting with

the receptors on theù own cells or aeighbo~g ce&. Altematively, -y secrrted signal

molecules, including insuiin and many hormones, circulate in the blood and interact with

their target cells elsewhere in the organism. An extracellular ligand b * i to a specific

receptor on a target cell and converts the receptor to an active state. The activated

receptor then subse~uently stimulates intracelldar biochernical pathways leading to an

appropriate cellular response, which may involve changes in ceilular gene expression,

protein trafficking and metabolism. For example, the binding of mwilin to its receptor

activates the receptor on the ce11 surfàce, and subsequently the signal is transmitted to the

celi interior to activate various intraceîîuiar pathways to elicit the pmper biological

responses. Alterations of these signaiing pathways have been found in the pathogenesis of

many diseases such as cancer, diabetes meIlitus and disoders of immune and

cardiovascular systems.

There are genediy two caîegories of mceptors for the circulating signals based on

the lipid solubility of the ligands. Stemids, thyroid hormones as weil as rrtinoids are lipid

soluble signahg m o l d e s . They usualiy induce slowa, long~lasting responses in thek

target cens, *ch are cruciai in the regulation of ceU growth and m o n . Lipid-

soIub1e m o l d e s can cross the plasma membrane and bind to the cytosolic receptors thrt

fiinction as Iiganddependent tmmcriptïon fkctors. On the other hand, water-soIuble

polypeptide signal m01ecules. such as insuiin and growth fktors, are not ablt to cross the

plasma membrane k l y - They must bind to the specinc cell surfkce recepbrs on the

plasma membrane ofthe target tissues and signai via the transmembrane mceptors. These

glycopmfein receptors are stniftmally and biochermermcaIly very diverse, but they al1 have

an extraceiiular iigand-binding domain, mugh wbich the polypeptide si@ molecule is

recognized, and an internai effector domain. The e f f i o r may fimction as an enzyme,

such as a kinase, or a transporter, or it may interact with a cellular coastituent like the

subunit of G-protein, or an ion channel. The first response of the ligand-receptbr

- interaction may involve the production of second messengers intracelluiarly, typidy

small molecules like CAMP, cGMP, diacylglycer01 @AG) and ~ 2 % or, in the case of

the iosuün receptor, the activation of the protein kinase system. The kinase cascade

systems or charme1 Sictivities act as switches and amplifiers of one or more biochemid

pathways controlling ceU rnetabolism, gene expression, celI cycle or cell fate @aulieu,

1990; Fuller, 1991; Ullrich and Schldger, 1990).

Three major types of stmctudy and hctionally distinct receptors have been

intensively studied. They are the G-protein-coupled receptors, receptors with guanylyl

cyclase actIvity, and receptor protein-tyrosine kinases (RPTKs). Members of the 0-

protein-coupled receptor supedmdy famirr seven irausmembrane loops and comprise a

big receptor W y of appmhte ly 8oo/. of known homones and neurottansmitters

(Kobilka, 1992). Ligand-indd activation of these receptors results in the dissociation

of the a- and &- subunits of the heterotrimeric G protein, leading to the activation of

intracelldar e f fwr molecules for signai proproaiion (CIapham and Neer, 1993). The

receptors with guanylyl cyclase activity have been f o u to bind d u r e t i c peptide

hormones. The do- sigoal transduction of tfiese receptors is still not very clear

but an increased cGMP level was fomd foîlowing the activation of the receptor (Gsrbm

and Lowe, 1994). The third gmup of the receptors, whïch bind many polypeptide

homones and gmwth f=tors, such as insulin and epidermal growth f-or (EGF), have

an intrinsic, ligand-sensitive, pmtein-tyrosine kinase @TEE) activity @Jllrich and

Schlessinger, 1990; van der Gea, 1994). These StnrcWy related glycoprotein receptors

belong to a superfamily oi receptor-type PTKs (RPTKs). They feature a large

extracelIda. ligand bindïng domain, a singie transmembrane region and an intemai

tyrosine kinase domain. Ligand binding to the receptor leads to the autophosphorylation

of its kinase domain followed by the phosphorylation of the substrate prote&. Activation

of these receptors induces diverse bioch-cal pathways incIuding the generation of

second messmgers, alteraîion of etlzymatic activity, anaor expression of specinc genes.

As a principal polypeptide honnone controliing ceil metaboLism and growth, insulin

has pleiotropic effects on cells and in the whole organism. Insulin acts on cens to

stimulate glucose, protein, and tipid metabolism. In addition, it stimulates RNA and DNA

synthesis, by mo-g the expression or activity of a variety of enzymes and transport

processes. The giucoregulatory effects of insului at a whole body level are predominantly

exerted by its action on liver, fat, and muscle. Insulin stimulates glucose incorporation

into glycogen and inhibits the production of glucose by glycogenolysis and

gluconeogenesis in liver. By affecthg glycogen metabolism and gluconeogenesis in those

tissues insulin regulates glucose homeostasis. Insulin also stimulates amino acid uptake in

liver and muscle to increase protein synthesis. As weii insuiin acts as a growth factor to

mod@ or augment the action of other regdators of cellular metabolism. Altered insulin

action, such as insulin raistance, plays an important d e in the paîhogenesis of many

disorders, including diabetes meliitus, obesity, hypertension and athemsclerosis.

In the years since the discovery of insulin by Banting and Best in 1921, much work

has been done in an attempt to understand the molecuiar mechanism of insului action-

(Goldnne, 1987; Kahn, 1 W 9 White and Kahn, 1994). The identification of the lnsulin

reuqtor (Yip et ai., 1978, 1980; Yip and Moule, 1983; Pilch and Czech 1979), the

eluciàation of its Pnmary structure (Ebina et al., 1985; Ullrich et al., 1985) and its

characterization as a tyrosine kinase (Kaniga et al., 1982) served as the basis to cl- the

m o l d a r mechanism of insulin aCnacnon. This was followed by the identifidon of

do- transducers (White et al., 1985; Sun et al, 1991). The binding of insuiin to

the extraceliular domain of the receptor d t s in conformational changes leading to the

activation of a tyrosine kinase in the CytOpIamnic domain of the receptor- Activation of

the kinase is mediated by tyrosine phosphorylation of the receptor itseif. The Ievel of

tyrosine kinase activity refiects the senim concentration of insulin. The activated receptor

in tum phosphorylates a substrate calleci insuiin rrccptor substratbl (jRS-1). The

multiple phosphotyrosine residues in IRS-1 serve as docking sites for the signal

transduction proteins in the signaling cascade (White, 1994, Sun et al., 1995). Many

signal transduction pmteins, such as PI3'-kinase, SH-PTP-2, GRB-2, Syp and Nck,

containing a specifïc recognition domain, termed Sn: homology 2 (Sm) domain, can

specXcally recognize and bind to the phosphorylated tyrosines of IRS-1. The interactions

between activated receptors and SH2-containing proteins ampiify the signal produced by

the binding of insulin to its receptor and link insulin signaling to other kinase cascades

involving serindtbteonine kinases and other signal transduction pathwys, such as the ras

pathway. Wltimately, this process leads to stimuiation of glycogen, lipid and protein

synthesis, as well as translOC8fion of insulin-sensitive glucose transporters to the srirf8ce

of muscle and fât celis (Hiring, 1991; White and Kahn, 1994). Mdfûnction of this

signaiing pathwayy fi0111 the nrst step of ligand binding, through receptor

autophosphorylation, to any of the postreceptor signal transducing steps, wiîi lead ta the

manifestation of metabolic disorders in non-insulin dependent diabetes mellitus.

Ih this section, 1 wi i i reviewthe cumnt lmowiedge of the mnilin receptor fiimily in

terms of insulin-activated signai transduction, focusing on the sbirturr o f the xeceptor

and the ligand-receptor interaction.

2. THE INSULIN FAMILY OF PEPTIDES

Iasulin ïs a member of a family of hormones, growth fktors and n e u r ~ ~ ~ d e s ,

found in both vertehates and invertebrates. In addition to inmiin, the f d y includes

-like growth tirtor I (IGFQ and ïnsdhIike- growth f8ctor II QGF-n) as weiî as

the more distantly relateci peptides, the r&xï.ns, the bombysins and the molioscan

insulin-like peptides (MIPs). All members of the Eimily sime a high degree of secpence

homology and probabiy, a cornmon "insuiin fold" in the s tn ichnw. Apart fiom the IGFs,

dl comprise A and B chahs, joined by disuEde bonds, foiiowing the cleavage of the

conmcting (C) peptide in the processing of their precurso~~. Since the C -des in the

precwsors of IGFs are retained, the IGFs are analogous to proinsuiin, with the C peptide

luiking the C-temhd of the B chah to the A chah N-temiinus @ig. 1-1).

These hormones, though sharing very simiIar primary secpence and stmctwe, act

@te differentiy, wïth some overlaps, in their physiologic effécts. While insulin is

primariiy involved in regulating cerbohyàrate, fa and protein metabolian, the IGFs act

mainly as regdators of growth, development and Merentiation. IGF-1 and IGF-II, but

not insuh, circulate as complexes with specific binding proteins. The action of each

hormone is mediated by its distinct, specifïc hi&-afbiity ceii-surfâce receptor with

intrinsic tyrosine kinase activity (Czech 1982; Humbel, 1990).

Insulin: is produced exclusively by the fl cells of pancreas and has a remadcable -

array of biological effccts. Although liver, muscle and fàt tissue are considerrd as its

pNnary physiologic targets, insuiin exerts some reguiatory effectcl on virhially d ce11

types. These effects include the mobiiizatïon of transport systems for hexoses, amino

acids, ions, the acute modulation of enzyme systems sach as the homone-sensitive lipase

in adipocytes, and the modulation of the transcription of specinc genes, such as those

encoduig phosphoenolpyruvate carboxykinase. The time ofonset of these e f f i can Vary

fiom rapid (seconds to minutes) for alterations of enzyme activities to slow @ours) for

the synthesis of protein, lipid, and nucleic acid, and cell pm1Iferation. Iasulin acts through

its specinc insulin receptors expressed by almost all cell types, nomally at a level of 102

to 105 receptor rnoIeniles p a cell The amùio acid sequence of the ùwlùl receptor has

been deduced h m the nucleotide sequence of its cDNA (Ebina et al., 1985; Ullrich et ai,

1985).

One of the structural characteristics of insulin is that it is composed of two

polypeptide chains linked by two disulfide bridges. The shorter chain, caiied the A chain

because of its relatively acidic nature, has 21 amino acids residues and an internal

disulnde bridge. The less acidic and longer B chah has 29-31 amho acids, depending on

the species. There are 30 amino acids in the B chah of human insulin. This dual-chain

structure and the positions of the three disuEde bridges are invariant in al1 the dIfferent

species of insulin that have been isolated and characterized (Blundell and Wood, 1975).

Within the "insulin foid" hnework, the amino acid sequences of the A and B chains are

highly conserved as well. For example, human insulin differs h m porcine insuh by

only one, and fcom bovine by three amino acids.

The genes encoding uisulin have been sequenced and analyzed fiom several species,

including human. The immediate translation product of the gene is a single polypeptide,

preprouisullli. It contains a signal peptide at the N-terminus, whkh facilitates transit of

the precursor into the endoplasmic reticulum. nie signal peptide is cleaved away during

the process. The resuitant molecule, pminsnlin, consisting of Iùiked B, C, and A chains,

lnsulin Proinsulin

Fig. 1-1. Pmlictcd terthry striictiircr of the inrulin f d y af pcptidcr. AU peptides are presented to contain folding simihm to that of insaün as formai by its A and B citain The beavy line and the light line q m s a ~ mpactivcïy the A chah and the B cbain in insrilin, Double lines repriesent the C chain joining the A and B ch- in proinsulin, or the D extaision ofthe Achain found in the IGFs.

is mer plocessod into the mature insulin, a disulnde-Iinked polypeptide with a

moIecuIar weight of about 6 kDa

Since the amino acid quence of insulul was detennined by Sanger and CO-

workers more than 40 years ago @mwn et aL, 1955), om knowledge of insuün bas

greatly increased, In 1969, determination of the X-ray crystai structure of 2&c porcine

insuiin provideci the first direct expdnental evidence for the insulin fold (Adams et al.,

1969). In this stnicture, bulin protomers fonn a hexamer wmposed of t k e identicai

dimers assembled around a the-foId axis, In the dimer the two monomers have their A

and B chains foldeci to fonn compact globular structures with a number of cornmon

features Fig. 1-2a). 'Ihese include @ig. 1-2B): 1. an A chah with 2 helices (A2-7 and

A13-19), joined by an extended loop (A8-12); 2. a B chah with an extended N-terminus,

foliowed by a helix (B9-19), a sharp tum @2û-23) and an extended C-tennural section; 3.

three disulfide bridges (A6-Al l, A747, A2û-B 19); and 4. a hydrophobie CO=.

The crystal, and the later NMR structures, as well as the studies of insuün

sequences, mutagenesis and chernical- modifications have contributed ta our

understanding of the molecular mechauisms of receptor binding. Insulin binding to its

receptor exhiiits a curvÏiinear Scatcharci plot which indicates the presence ofat least two

classes ofbindiag sites or negative fooperativity between sites. It suggests that insului, as

a non-symmeec molecule, may contact its receptor at diffkrent surfàce sites. By

analyzing the interaction of insulin analogues with the insulin receptot, Schaffier pposed

a mode1 for insulin binding to the iiisulin receptor @ig. 1-2 C) (ScMer, 1994). He

hypothesized that insulin has two binding domains to contact its receptor. Binding site

one is the classical bimding ssit, shdied by most investigators and comprishg a number

of residues (kcludirig B24, B25, Al and Ml) in the dimer-fomiing surfàce of the insulin

molea.de. The second binding site wnsists of B 17 and AU, located in the hexamer-

Fig. .l-2. The primaq structure (A), the 3D crystai structure (B) and the 3D space-fig mode1 (C) of human insuih The 3D modd (C) shows the putative receptor contacthg sites PINDING S m 1 and 2). These two biuding sites located on m o separate SmIaces ofthe insuiin molecuie may be hvolved in the the high afîinïty binàing ofinsrilui to the IR.

forming d a c e on the opposite srde o f the molecule %rn the cl-& bindhg site.

Substitutions of these residues wouid dramaîicaily decrease insolln IifFinity to its receptor.

Thus, a hi&-afnnity bindhg of bulin to its receptor may reqaire the h d i n receptor to

contact a different bmding region on the insulin moIecdee

2.2. Insuiin-Like Growth Factors

U . e 0 t h members of insalin family* IGF-I and IGF-II are single-chain

polypeptides of about 7.5 kDa but share similar tertiary süucture and amho acid

homoIogies with inSnlin (LeRoith et al., 1992). Genes encodïng the IGFs have been

cloned and analyzed h m several species (Brisseaden et al., 1984; Humbel, 1990). The

mature fomi of IGF is a singIe-chain polypeptide of about 70 amino acid midues dMded

into contiguous B. C, A and D domains. Its structure is analogous to that of proinsuiin as

shown by computer-assisteci molecular modeling and NMR analysïs (Fig. 1-1). The A

and B domain exhibit about 42% amino acid identity to the correspondhg A and B chahs

of insulin. The C domain is analogous to the comecting (C) peptide of proinsulininsulin A

short D domain, which is not found in proinsulin, extends at the C-teminus.

IGF-1 and IGF-II, though sharing many stmcturai propertïes with insulin, differ

significantiy with respect to their sites of synthesis and biological effîts. The origin of

the circulating IGF-1 is the liver, where its expression is unda the control of growth

hormone. In the blood stream, the IGFs circulate at 1000 times higher concentration than

insulin but, unWre innilni, most if not aii of the cïÏculating IGFs are complexed with

specific binding proteins which are believed to modulate the action of the IGFs and to

prevent hypoglycemia by lowering their effective fke concentration in the circulation. Zn

addition, IGF-1 is also expressed at lower concentration in most tissues, both prenatally

and postnataüy, and thus fiinctions as an important paracrine or autocrine growth factor.

It is clear that IGF-1, by interacting with its own high-dlimity receptor, mediates most of

the effect of growth hormone on longitudinal growth. The IGFs also show insulin-like

metabolic effits hz vitro and bz vivo9 such as glucose transport. m adipose and muscle

tissue, but o d y at relatively high concentrations (Froesch et al, 1985). Like insiilin, IGF-

1 also plays a role in embryogenesis. It is expressed even before the development ofthe

embryonic Lnrer. As in the insului signahg system, the effits ofIGF-1 are mediated by a

specinc hïgh-affbity IGF-1 receptor which is a tyrosine kinase highly homologous to the

insulin receptor.

Though IGF-Iï rnimics most ofIGF-1 effects in vitro, its biological roles are stilI not

clear. It has been suggestd that IGF-II may be involveci in the fetal development since its

expression dramaticaliy decreases just before birth in both amount and the range of tissue

distniution. In contrast to IGF-& IGF-II receptor has no structural similarity to either the

insului or IGF-1 receptor. This receptor is in fact the same molecule as the cation-

dependent mannose-6-phosphate (Man-6-P) receptor, a protein that targets the Mm-6-P-

containhg proteins to lysosome (Massague et al., 1982), and its role in the action of IGF-

II remaius unlcnowm It has been proposed that most of IGF-II effects are mediated

through IGF-1 or insulin receptors (Czech, 1989).

2.3. Other members of the Insuiin famiïy

Several other polypeptides with higiiry homologous sequences to insulin have been

discovered. Some have been found in the vertebrates and 0th- have been identifiai in

proto-chordates, insects .md molluscs. They are bombyxins, relaxins and MIPs. The

sequence data suggest that they share similar three-dimensionai structures and

conformation. UnWEe insulin and the IGFs, these distantly related members show more

divergence. Interestingly, most residues in the extended receptor-binding region of insutin

are weU consmed in these family members, which suggests possible heterologous

ligand-receptor interactions. Relaxin has been identif?.ed in several species, including

hianan. In human, relaxùi has been identifieci rnaialy as a paracrine/autocrine hormone

regulating activities in the uro-reproductive systern (Schwabe et al., 1994). The

interaction of relaxin with its receptor at the molecdar level is still poorly mdersfood,

and the receptor itselfhas not been welf charackrkd.

3. MOLECULAR AND CELL BIOLOGY OF THE INSULIN AND IGF-1

RECEPTORS

As the proteins primarily responsi'ble for transducing the signds for msulin and

IGF-I, the insulin and IGF-1 receptors, Ure their Iigauds, are the products of homologous

genes (Abbott et al., 1992). The characterization ofcDNAs encodlag the uisalin and IGF-

1 receptors revealed a high degree of similarity in the primary nucleotide and amino acid

sequences, their overall structures, and enzymatic activities. Both receptors are

glycosylated hetemtetmmers, composed of two a subunits of -130 kDa and two f!

subunits of -90 kDa Ilnked by disuEde bonds, The a subunit, which is entirely

extracellular, contains the ligand-binding domain, while the fl subunit consists of an

extracellular domain, a trammembrane domain, and a tyrosine kinase (TK) domai..

Among the similarities between the insulin receptor (IR) and IGF-1 receptor (IGF-1 R),

the highest amko acid homology (84%) is fond in theù TIC domains, whiie the overall

amino acid homology between their P subunits is only 4460%. Both IR and IGF-1 R

belong to a membrane receptor superfamily calied the receptor tyrosine kinase @TI().

Members of this supdarnily possess inkirsic tyrosine kinase activity and are stnicturaIly

3.1. The Receptor Tyrosine Kinase Superfnmily

Tvrosine Kinase and Rece~tor Tvrosine Kinase

Insulin and IGF-1 receptors were among the nrSt RTKs identifkd in the early 1980s

due to their Ligand-stimulated autophosphorylation (Kasuga et al, 1982). More than LOO

proteins with the consensus sequences characteristic of TK have been cloned and

characterized h m a variety of eukaryotic species (Hunter and Cooper, 1985). Acting as

an onhff switch In signal transduction, the protein ph~sphory l~od âephosphorylation

cycle plays a key mie in wntrolling various ceUularprocesses- Protein kinases d y z e

the transfer of a phosphate group fhm a donor, usually the y phosphate of ATP, to an

acceptor amho acid in a substrate pmtek Serine/tsreOnine kinases (SKs) phosphoryIate

serine or threonùie residues of substrate proteins whereas tyrosine kinases (TE=)

specincally phosphorylate protein substrates on th& tyrosine residues. Hmter et ai have

demonstrated the existence also of protein-histidine kinases that phosphorylate histidine

residues of substrate protek (Hmter, 1991). Some protein kinases are able to

phosphorylate al l three hydroxy amino acid residues.

Members ofthe protein kinase f2ïmi.I~ share highly consewed amino acid sequences

in their catalytic domain. Based on their cellular location they can be divided into two

classes: cytoplasmic and membrane bound. Cytoplasrnic enzymes seme as molecules of

signal transduction inside the celis. Transmembrane kinases, such as the msulùl receptor

family, transfer signals across the ceïl membrane aud activate cytosolic kinases. It is

interesting that TKs are found only in multi-celluiar organisms, whereas SRs are found in

both single cell and multi-cellular organisms, This suggests that the tyrosine specincity

may have been an evolutionary development related to the acquisition of multi-cellularity

and cell-cell communication.

The overail architecture of iill RTKs is very similac a large glycosylated

extraceiiular domain that binds the polypeptide ligand; a single hydrophobie

transmembrane domain; and an intracellular domai. containing a highly conserved TK

catalytic region. Interestingiy, some cytoplasmic TKs are not transmembrane but anchor

at the Innet d a c e of the plasma membrane and are coupleci to transmembrane

glycopmtein receptors which do not have cytoplasmic kinase domain. This coupling

allows them function like a transmembrane R% T celï antigen receptor-fln, p w t h

hormone receptor-Jak2, and interleukùijaks are examples.

B a d on the wmprehdve SMaIysis of the prlmary -ces, RTKs are

subdivided into many subfhnilies acwrding to and shared stnictural f w

@ a d et al., 1993; van der Geer et al.,. 1994). More than ten subfkdies have been

classinecl over the last few years. The best c- s u b f d y mernbers iuc1~ :

- epidemial growth htor (-F) receptor, phlet-derived growth factor (PDGF) receptor,

insului and IGF-1 receptors, nenre growh f-r WGF) receptor, fibroblast growth nrtOr

(FGF') receptor, and hepaîocyte growth nwnOr (IIGF) rrceptor (c-met). Most of the RTK

f d y membexs are sïngle-chain polypeptide e r s , except for the msuIHi reœptor

subfamily. Members of the insuiin -or subfamily incide de -r, IGF-1

receptor and insulin receptor related receptor @SR) with th& unique a& tetnuneric

structure. The stnictural similarities within a subfàmiIiy suggest that receptors in a

s u b f d y s b similar mechanisms of molecular regulation and signal transduction

pathways, as in the case of insului and IGF-1 receptors.

The cataiytic domains ofprotein kinases are 250-300 amino acid resïdues in length

mostiy located neer the carboxyl terminus or as a subunit of multi-subunit enzymes

(Hanks et al., 1988; 1991). The tbreedimensional stnictine of the TIC domain of the

insulin receptor exhibits a highly conserved bi-lobular stnicture (Hubbd et d., 1999;

Hubbard, 1997). In the large lobe of the TK domain, an extendeci sequence containhg

Tyr1 158, Tyr1 162 and Tyr1 163 fomis a flexible bop, d e d the activation loop (A-

loop). During activation, the A-loop undergoes a major conformationai change upon

autophosphorylation of the three tyrosine residues within the Iwp, resulting in

uarestricted access of ATP and protein substrates to the kinase active sites. Although both

SKs and TKs share a basic struchm, TKs bave the extended A-loop sequeace in the

larger lobe. It may explain the specificity for tyrosine since serine and threonine

hydroxyIs may be too short to reach the phospho-tramfer site within the d y t i c cmter

(Hubbard et al., 1994).

I I a Subunit p Subunit

Fig. 1-3. Schemrtic nprcscatatioa Mthe inrmlin reccptor gene (A) and the primary (8) and secondary (C) structure of bIR Receptor dornains encoded by specinG exons are indicated (A and B), Labels: S=signal peptide, Ll,L2=Ll and L2 dbmahs, CR--teine-nch domain, CD/FnO=Çomiecting domain 0rfibromsi.n ïü repeaq Fnl,Fn2= fibroneah Eï repeats, ID=ïnsertdom8ïn, TM=transmembnme domain, JM+wûamedrane domain, TIC tyrosine b e domain, TL=C-temnîd tail, Disuifide bonds mmecting a-a and a+ subunits are iuriiated by- and possiile phosphorylation sites are mdicated

- b-(C)-

Gene strucîme and EsDression

The inailin receptor @I) is theproduct of a single gene IOcafed on the short ami of

chromosome 19 (I9p7) nearthe gene ofthe LDL receptor (Yang-Feng et al., 1985). The

structure of this gene has been eIucïdated by both restriction mapping @&der-Wieland et

al., 1989) and genomic cloning (Seino and Bell, 1989; Seino et al., 1990). The human IR

gene, composed of more than 150 Kb of DNA, has 22 exons and 21 htrons. Exon I

through 11 are spread over 90 Kb and code for the a subunit, Exons 12 through 22 span

30 Kb and code for the p subunit (Eig. 1-3). The promoter ofthe IR gene is ofthe typicd

-ch %ousekeeping" type with a few bmding sites for edancer protem (McKeon et

al., 1991)-

The receptor is synthesized in the mu& endoplasmic reticulum (RER) as a single

polypeptide cbain proreceptor of either 1339 qr 1351 amino acids, depending on the

differential splicing of exon 11. The pre-receptor has a 27 residue N-terminal signal

peptide that assists in the intraceUuiar translocation of the receptor precutsor. The signal

peptide is subsequently cleaved off. The proreceptor is transportecl through the Golgi

apparatus, where it rapidy undergoes extensive N- and O-linked glycosylation. Two

monomeric proreceptors are then linked by the formation of disuifide bonds into a

dimeric structure. Proteolytic removal of a tetrabasic amino acid sequence (Arg732-Lys-

Arg-Arg735, numberhg based on Ebina et al., 1985) h m each proreceptor monomer

gives nse to the a and f3 subunit Firrther glycosylation takes place before the mature

insulin receptor is inserted- into the plasma membrane (Olson et al., 1988). The ftnaI

active mature receptor is a fiilly giycosylated tetrameric transmembrane protein,

composed of two a subunits and two P subunits (Fig. 1-4). Each a subunit is comprised

of 719 or 731 amino acids. There are 620 arnino acid rtsidues in each $ subunit hcluding

an extcacellular portion of 194 residues, a trammembrane region of 23 amho acids, and a

cytoplasmic domain of 403 residues.

- -

Fig. 14. Human insuhm mceptor amho acid sequence<smmEbUu o t i ~ . 1985) Residues sl to s27 arc the signai seqence. Residucs 1 to 731 ccmstitute the a subunit, and maiducs 735 to 1355 wnstïtutc the B subunit, The polybasic residucm 732 to 735 arc tbe crupatio olenrage site to yield the a and the subunita UnderIined rcsiducs arc d d by Exaa 11-

The heîeroteîramer structure of the insufin receptor was initïaiiy elucidated in the

late 70s by cross-Ii'nkmg radioactive insnlin to either whole ceb o r plasma membranes

Orip et ai-, 1978; Pilch et aL, 1979). Later, pudication of the receptor led to cioning of its

cDNA @bina et al., 1985; UIIrich et al., 1985). While the prhnary sequence of the IR has

been known for some the (Fig- 1-4), knowIedge of the secondary and higher order

structures of the receptor are llmlted, Based on cacrent knowIedge of insulin receptor

hction, and the fûnction of RTK proteins, it seems reasonable to propose that sp&c

bctional properties of the IR reside m its separafe domains. A major advance in

understanding the overall receptor structure was the mode1 proposecl by Bajaj and co-

workers @ajaj et al., 1987)- They proposed that the N-texminal haIf of the IR wntained

two large homologous domains (21 and L2) sepacafed by the cysteine-rkh domain (CR) b

which itself was compnsed of three repeating d t s each containing eight cysteine

residues. A second major development was the observation that the C-terminal portion of

the extracellular region of IR contanis three fibmnecth type III repeats (here designateci

as FnO, Fnl and Fn2) linked to the L2 domain (Marino-Busije et aL, 1998; Malhem and

Booker, 1998; Ward 1999). The fkst repeat, FnO, or identifid as the comecting domain

(CD) by some authors, located in the a subunit wnsists of 123 amino acid residues. The

second repeat (Fnl) is a structure with contriiutions by both the a and the B subunit,

connected with a s o d e d imxî domain (ID) that includes both the a+ cleavage site

and the alternative-spliced exon 11 (O'Bryan et al., 1991). The third repeat (Fn2) is part

of the p subunit. The extraceliular region aiso provides the two disulfide-bonds that

covalently lïnk the a p monomers to form the constitutive IR dimer. One of them is in the

FnO region and the other is located in the a insert domain. The extracellular portion the

IR is foliowed by a short, single transmembrane domain 0 connected to the

intraceiiuiar portion of the B subunit that contains the TR domain. The crystaUographic

structure of the Tg fkgment ofthe fl subu& was detemiùied several years ago (Hiabbard

et al., 1994), but the t h e dimensional of the holoreceptor remaiaf miknown.

The generai structural domab layout of the IR agrees with the suggestion thaî

insulin binds to the extracelluî~ dom- 1-g to the transmembrane activation of the

intracelMar TK of the f3 submk

The a Sabunit

The a subunit of the IR is entireIy exfraceIIuIar wntahhg either 719 or 731 amho

acids depending on whether or not exon 11 is expressed (Seino and Bell, 1989). A

characteristic feature of the a subunit is its cysteine-rkh region which contains twenty

four of the thirty seven cysteine residues in the a subunit The fimction of the cysteine-

nch domain is wt M y understood, but it has been suggested that it is involved in

binding to insulin (Yip et al, 1988, Rafiisloff et d., 1989; GustafSon and Rutter, 1990).

Homologous cysteine-rich regions are also seen in the low density lipoprotein (DL)

receptor, IGF-L receptor and epideanal p w t h factor (EGF) receptor (Goldstein et al.,

1985; Ullrich et al., L984,1986). These regions in IGF-1 and LDL receptors h a . been

shown to bind to their ligands. However, in the case of the insulin receptor, it is d l

controversial as to whether the cysteine-nch domain is directly involved in insulin

binding.

There are 37 cysteine residues in each a subunit monomer- Most of them appear

to fonn intrachain dimifide bonds within the a subunïts, mainly in the Qs-nch region-

On the other hand, some cysteine residues fomi disulnde bonds joining a-a or a-P

subunits, which contribute to the important conformation of the fimctional receptor (Finn

et al., 1990). There are two classes of disulfide bonds in the IR. CIass 1 diSulfide bonds

link a+ subunits, while class 2 diSulfide bonds join the a-$ subunits. The latter is more

resistant to reduction than those of class 1. The mature iasulin receptor is a disalfide

llnked hetemtetramer, including one disulnde on each a-$ wmection and more than one

on the a-a cross-Ilnking8

Based on the structural analysis of the IR sribjected to mild tryptic pmteoLysis, the

C-temiinal25 kDa hgment of the a subunit was found to be involved in a-P didiide

bonding (Shoelson et ai., 1988; Xu et al., 1990)- Cys647 located in this region has been

reportai to be vital to the a-p bond formation (Cheatham and Kahn, 1992). Recently,

Sparmw et ai confirmed that Cys647 in the Fnl of the a subunit forms a diSulfide bond

with Cys872 in the Fn2 of the P subunit (Sparrow et al., 1997)-

Until recently the diSulfide bonds connectiag the two a a monomers together were

unknown. Schaffer and his CO-worker first noticed that the Cys524 upstream of the Fnl

domai. contributes an a-a interchah diSulfide bond (SchafEer and Ljunqvist, 1992).

Other investigators (Macaulay et al., 1994; Bilan and Yip, 1994) suggested that additional

disulfide bonds exist between the a* dimer, which is probably locatted in the cysteine-

rich domains, Surprisingly, the second dîsulnde bond connecting the a-a dimer has been

recently locaiized to one of the cysteine triplet, Cys682,683, and 685 at the end of the C-

terminai region of the a subunit (Spmw et al, 1997).

Bajaj et al @ajaj et al, 1987) proposed, in addition to the cys-rich region, two

similar but independently folded subdomains, LI (encoded by exonl-2, residues 1-1 56)

and L2 (encoded by exon 4-6, residues 3 10-470). each being mainiy B-pleated sheet in

structure. Between LL and L2 is the cysteine-rich (CR) domain Cresidues 157 to 309,

encoded by exon 3-4). Based on sequence alignment and secondary structure prediction,

Bajaj et al. proposed that the LI and L2 domains juxfaposed and extended away h m the

ce11 membrane, with the CR domain ~11der1ying and supporting them (Bajaj et al.. 1987).

The recently detennined crystal structure of a fiagrnent containhg the f b t three domains

(LI-CR-L2) of IGF-IR, a receptor closely related to IR (Garrett et al, 1998), shows that

the layout of the thcee domains was quite diffkent b m that proposeci by Bajaj et al. The

remaining regions of the a subunit (residues 471-731, encoded by exon 8 to 11) consist

of a l3ronectï.n III repeat @no, residues 471-593, also c d e d comectiag domain (CD)),

part of the second fIbronectln ï i I repeat (Fnl, residnes 594461) and a 71 residus insert

domain (ID) (O'Bryan et al., 1991, Schaefer et al., 1992). LI, L2 and CR domains have

been suggested to be involved in insulin bmding @Tapes et al., 1986; Yip,

l988,1992,l993; De Meyts, 1994) and in both a-a and a-p disulnde bond formation

(Cheatham and Kahn, 1992; Scbaffer and Ljunqist; 1992, Bilan and Yip, 1994; Spanow

et al, 1997). Little is known about the function of the insert and connecting domains.

The quatemaq structure of IR has not been determuid- Schaefer et al. (Schaefer et

al., 1992), using electron microscopie image techniqye, reported that the extraceUular

domains of the IR (the entire a subunits plus the extracellular domains of the B subunits)

in solution had a "Y" shape with giobular features at the end of each ann of the T",

Similar studies also showed the s o l u b ~ e d placenta holoreceptor as a "T" shaped

structure (Christiansen et ai., 1991). These studies suggest that the receptor has two

extracellular arms, each of which may represent a separate a subunit- The authors also

suggested that each of the subunits wodd contribute one independent insulin-bindiag

site. There has been no report of the successfiil crystallization of the insulin receptor- Its

large size (b& 450 kDa) and its glycoprotein nature may accokt for the lack of success in

its crystaUization. It is ais0 too large for NMR analysis,

Early studies using the technique of photoaffinity labeling (Yip et al., 1980) and

chemicai cross-linkiag vilch and Czech, 1980) have clearly shown that insalin binds to

the a subunit of IR. Although the entire a subunit could be involved in contacting

insulin, recent studies on -receptor interaction have graduaüy focused on the Ll-

CR-L2 region (White and Kahn, 1994). Since no structural Uiformation on the IR a

subunit or its hgments is available, the structure of the LI-CR42 domain of IR now can

only be inférred h m the x-ray structure of its counterpart of IGF-IR (Garrett et al, 1998).

The overall stmchne of the Ll-CR-L2 domains of IGF-IR was shown as a notch shape

structure. Each L domain is a singie-stranded right-handed P-hek The cysteine-rich

region is composed of eight disuEde-bonded modules, seven of which fonn an elongated

rod shape domain connecfing the LI and L2 dom- The three domains surround a cmter

space of sufncient size to accommodate a ligand moIecdee AIthough this hgment is

incapable ofbhding to KGF-& this structure does provide clear infiormation that the L1-

CR-L2 region is a putative region for ligand bindïng*

Two isoforms of the insulin receptor, différïng in length by 12 amîno &âs near

the C terminus of the a subunit, are daiveci h m the alternative splicmg of exon 11. This

results in a srnalier A isofonn of 1339 amino acids and a larger B isoform of 1351 amho

acids (UUrich et al., 1985; Ebina et al., 1985). Both isoforms (- and + exon 11) are

expraseci in most tissues in various proportions. The fimctionaL difEerences between

the two isofomis are not clear. 1t semm that isofomi A has a relatively higher affinity for

both insulùi and IGF-1 and is more rapidly uitemaiized and d o m regulated (Mosthaf et

al., 199 1; Voge et al., 199 1; Yamaguchi et al, 1991). The mechanian of this change in

mty by a structural variation distant h m the presumed binding site is uncertak

However, mutant insulin receptors with mutations at the cleavage site have been reported

with markedly reduced afftnity to insului (Wifiams et al, 1990; Sasaoka et al., 1993).

This suggests that the C-tenninal of the a subimit may contribute to a confionnation for

high afbity ligand bhding. hterestingiy, Fnisca et al recently reported that insulin

receptor isoform A binds to IGF IL with an &*ty close to that of insulin. It actualiy

serves as a high-aff?nity IGF II receptor affêcting growth of fetal and cancer cells (Frasca

et al., 1999; Sciacca et al., 1999). This finding also suggested that the small variation in

the C-terminal of the a subunit plays an important d e in the conformation for high

e t y ligand binding.

The B Snbnnit

The subunit ofthe Ïnsulin receptor contains an extracellular domain of 193 amho

acids, a single transmembrane domain of 23 amino *ds, and an inkacellular portion of

402 amho acid reslCdues- The htraceflular portion of the subunit can be fbrther

subdivided into three fima*onal domallis: a juxtamembrane domain, a tyrosine kinase

domain and a C-terminal domain. The tyrosine kinase domain bas been recently

c r y s and its stnicture detennined (Hubbard et al., 1994). The main hction of the

$ subunit is to react to the signal generated by extrace1luiar insuih binding.

Relatively littie is known about the fimcti*on of the extracellular domain of the $

subunit It con- a half (Fnl) and a fidi Pn2) fihnectin domain and four cysteine

residues. Cys872 in the Fn2 region fonns the interchain bonding *en the a and

subunits (Cheatham and Kahn, 1992, S p w et ai., 1997). There are also sites for both

N-linked and O-linked glycosylation (.ge et al., 1990). A monoclonal anh'body to this

region allows normal insulin binding but inhibits receptor autophosphorylation and

biologid signaling (Gherzi et al., 1987), suggesting that this domain IikeIy plays a role

in receptor signai transduction.

Besides the putative signai transduction fûnction., the extracellular domain of the

subunit may play a criticai role in the confonnation ofthe high aff?nity binding site of the

a subunits. Schaefer et al. (Schaefer et aI., 1990) demonstilated that truncated insulin

receptors with aU or part of the extracellular domain of the f3 subunit deleted dramaticaily

lost their insulin-binding af%ity. The minimai truncated receptor maintahhg fûli

binding Rffinity is the wmplete extraceiiuizu domain, inciuding both a subunits and the

entire extracellular part of the f3 subunits. They suggested that, even though the subunits

do not dinctly participate in insulin binding, their existence is critical in forming the nght

conformation for the high dûnity binding site in the a submïts.

The transmembrane domain of the subunit is a single a-helicai segment of 23

residues which links the extraceîlular and intracelluiax domains of the receptor. This

physical connection between the extraceiiuiar and intraceiiular region of the subunit is

impLicitly cruciai to signal transductio~ However, mutations or replacement of this

region with the trammembrane region of other receptor fhmiiy members did not afféct the

hc t ion of the receptor oramda et aL, 1992, FrattaIi et aI., 1991)- Thus, it appears that

the structural requirements of this region for sÏgnaL transduction are not restrich

The juxfamembrane domain is a short fbgment of48 -dues at the begùining of

the intraceilUlm portion ofthe B subunit Two essential fimctions have been found for the

juxfamembrane domain: receptor ïntennalization and signa1 transmission Interestingly,

both hct ions relate to the specinc NPXY motif in this region (Feener et ai., 1993). They

are NPEY at position 969-972 and GPLY at residues 962-965.

The NPXY motif bas been indicated to interact with adapttor proteins Ï n coaîed pits

to facilitate receptor endocytosis @ r m and Greene- 1991). Mutagenesis of this domai.

has shown that the ligand-induced IR endocytosis was abolished. More recently, the

NPXY-motif has been identified as the interacting site with ùisulin receptor substrates,

such as IRS-LIIRS-2, and Shc (Keegan et ai., 1994; O'Neill et ai., 1994). Mutations at or

near Tyr 972 inhi'bit insulin stimulated tyrosine phosphorylation of IRS-l/IRS-2 and Shc

(White et al., 1988, White & Khan 1994). The phosphorylation of IRS relies on the

engagement of the phosphorylated NPXY-motif in the insulin receptor (Sun et al., 1995;

Keegan et al., 1994).

The tyrosine kinase domain is the most conserveci region among the family of

RTK (White and Kahn. 1986; Ullrich and ScMessinger, 1990). It contains an ATP-

binding region and a so-called regulatory or catalytic region. The ATP-binding region

composed of an invananant sequence GXGXXGX located at residues 1003-1008 of the

insulin receptor, followed by a lysine residue located distally 10 to 20 residues toward the

C-tennind. The Lys1030 in this region provides an AT.-binding site through a sait

bridge. Point mutation of Lys1030 disrupts both ATP binding and kinase activïv

(Maegawa et ai., 1988), and 1eads to severe insulin resistance (J'ayIor et al. 1992).

There are 13 tyrosine residues in the cytoplasmic portion of each P subunit,

Phosphopeptide mapping demonstrated that 8 of the tyrosine residues are criticai for

exogenous tyrosine kinase activity and insulin receptor biological activities (White et al.,

24

1988; Kohanski et aI, 1993; Feener et al., 1993)- These tyrosine resïdues are IocaCed in

three clusters in the primary stnicture: Tyr96St- 972, and 984 m the juxfamembrane

region, Tyr1 158,1162, and 1163 in the kinase d y t i c domain, and Tyr1328 and 1334 in

the C-terminai taiI. This numbering system includes exon 1 1. The toree tyrosine residries,

Tyr1 158, Tyr1 162, and Tyrl163, lying in the b a s e activation loop (A-loop, residues

1149-1170) are the most important site of autophosphorylatim of the IR

Autophosphorylation of aii three tyrosine resïdues in the YXXXYY motif of the

activation Ioop occurs ~6thin seconds after insvim stimulation and stimidates tyrosine

kinase activity by 10-20-foId (White et al. 1988)- Mutation of one, two or three tyrosine

residues in this region prognssively duces hmlïmstimulated kinase activity and resuits

in a parallel 105s of biological actïvity (Vogt et al 1991; Wdden et al. 1992). The crystal

stninime ofthe tyrosine kinase domain of the inSulln receptor fbsubunit not onIy shows

the fine structure of the kinase domain but aiso provides important u i f o d o n on the

mechanism of kinase activation (Hubbard et al, 1994, Hubbard, 1997). In the inactive

state, Tyr1162 in the A-loop cornpetes with protein substrates for binding in the active

site. Dunng activation, the A-loop uudergoes a major codonnational change upon

autophosphorylation of Tyr1 158, 1 162 and 1 163, resulting in umestricteâ access by ATP

and protein substrates to the kinase active site. The crystai stnicture of the kinase domain

also indicates that autophosphorylated Try1163 plays a role in stabilizïng the

phospborylated A-loop and suggests that Tyr1 158 may provide a dockhg site for

downstream signahg proteins.

The C-terminal region of the IR exhi'bits maximai divergence between IR and

o t k RTKs -ch et ai., 1986). Therefore, it has ben proposed that it wntri'butes to

specificity in transmembrane simialinp. It also contains a second major region of

autophosphotylation uyr1328, and Tyr1334) in the IR B subunit. However, the exact role

of phosphorylaiïons of these tyrosines in insulin action is d l not clear since kinase

activity in the C-teuninai tnincaîed IR is normai Myers et al, 1991; Takata et d., 1992).

It also contains a few serhdthreonine phosphory1ation sites stimdated by several

stimulators, incIudIng insulin, The fimctïon of serine/hnine phosphoryiation in this

region remah controver~l*aL wonezewa et al., 1991; Hotami-sligil et al., 1993).

3.3. IR, LGF-IR and IRR: Simüarity and Divergence

Three receptors, insulin receptor w), IGF-1 receptor (IGF-IR) and insului receptor-

related receqtor W., in the RTK -y share a high level of overail structural

similarity. Although th& gens have been mapped to different chromosomes

(chromosome 19, 15 and 1, respectively)), IR, IGF-IR and IRR have similar amino acid

sequences and identical exodintron o q p h t i o n @bina et aL 1985, Ullnch et al. 1986,

Shier & Watt. 1989, Abbott et al. 1992). AU three receptors are synthesized as a single

polypeptide that is processeci to yield distinct a and P chahs joined by diSuldide bonds

into a 4 3 , heterotetramtxic cornplex. Whereas the IR and IGF-IR have a tetrabasic

cleavage site, RKRR, the IRR has an RHRR at thïs site. AU three have a completely

extracellular a subunit that contains a region of approximately 150 residues which is

particdarly nch in cysteines. However, these cysteine-rich regions show a lower

sequence homology than munding regions (-45% compared to 45%). difference

has been implicated in some shidies as playhg a role in detemiining ligand specificity

(Zhang and Roth, 1991; Kjeldsen et al., 1991; LeRoith et aL, 1994). The p subunit

consists of both extracelIular and intracelldar domains connecteci by a transmembrane

domain. The htracelldar tyrosine kinase domain is highly homologous in all s u b f d y

members.

Wlde the IR and IGF-IR share a very close sequence homology, IRR is less similar.

Most notably, the P subunit of IRR is only 551 amino acids long versus 620 and 627

amino acids for that of the IR and IGF-I& respectively (Shier and Watt, 1989). This sue

dinerence is in large part accounted for by the carboxyl-tail of I R . which laclcs 48 amino

acids including the two C-teLmlLlaI tyrosines- and the seraidthreonln phosphoryIation sites

present in other two receptors, h con- to the highiy comemed ATP-binding and

tyrosine kinase region in aii tbree receptors (79-84% identicai), the sequence homology in

the carboxyl--tail region of the th- receptors is remarkably low (1944%)-

Despite their structural similaütks, the msuIin and IGF-I receptors mediate dinerent

physiological effects. The insulin receptor regdates metaboiism as weU as some

development and growth (Lee and Piick 1994). The IGF-1 receptor, on the other han4 is

noted for rnediating the pwth-pmmoting action of IGF-I, as weU as some of the effects

of IGF-II (Humbel et al., 1990). The ligand for IRR is dl unknown. 1t is interesting that

the insullli receptor, besides binding insulin with high affrnity, a h bbinds IGF-1 and II

with about 100-fold weaka aflhity (Anderson et al., 1990; LeRoith et ai., 1994).

However, the IGF-1 receptor has a very low affcnity for insuiin. There is also a smaiï

amount of naturally occurring hybrid IGF-Ilinsuiin receptors which contani af3 hdves of

both the insulin and IGF-1 receptor (Mosthaf et ai.. 1994 Siddle et al., 1994) with low

aflkity to either ligand The hction of the hybnd receptor is not clear.

- Taking advantage of the similarities and diffèrences in the domain structures and

their big difference in e t y for their ligands, especially in the case of IR and IGF-IR,

investigators have an opportunity to study the iigand binding sites and specinc signaihg

pathways. Several chimera receptors with switched domains between IR and IGF-1 have

been studzed. These studies have revealed important information in the understanding of

the interaction of ligand and receptor (Anderson et al., 1990; Zhang and Roth, 1991;

Schumacher et al., 1993).

4. INSULIN BINDING AND SIGNAC TRANSDUCTION

4.1. Affhity and Kineties of Insaïin Bincihg

Insulin binds to its receptor in a rapid and reversii1e manner. The affinity constants

vary around 109 M-1, and dissociation occm h m within minutes to several hours with

increasïng insulin concentration De Meyts n o n " twenty years g that insalin binding

to its membrane receptor, as andyzed by Scatchard plots, showed an upwardly

curvilÏnear bindlng isothexm (De Meyts et al, 1973). These were Interpreted as indicative

of negatively c o o p d v e bmding, suggesthg that the a£E~Üty of the reaction is not

~ O R R over the saturation range. They pmposed that there were two "buiding sites" on

the receptor and consequently th& the IR c m bind insulin with high anàlor low afnnitty

based on the insulin concentration. The insulin receptor binds one insulin molecule with

hi& afiïnïty @Cd-19 M-1 ) in its high affinity site at physioIogicaI concentration of the

insulin. At saturation conditions, the receptor can bind auother insulin molecule with

lower affinity (~d-108 M-1). This intqretation has triggered numerous additional

binding studies h m many laboratories and resultted in considerable controversy over the

years.

De Meyts's mode1 also provideci an explanation for the kinetics of insuiin bhding.

When the binding site is empty, the receptor can bind to insullli with high afkity. This

binding ieads to additional insulin bhding occuning with lower af3bity and d t s in a

higher dissociation rate.

Considering the fact that the insulin receptor is a fûnctional a& hetemtetramer, it

is highly possible that high affinity insulin binduig requires that uisalin be in contact with

both a subunits, although the contact region in each a subunit may be different (Yip,

1992; Lee et al., 1993; Schaffer, 1993). Binding of one i . molecule to the receptor

may make it more dï.fi5cu.k to bind the second insulin molecule. The exact ligand contact

region on the a subunit is still inwmpletely defineci. The mechanism of negative

cooperative binding will be better explained when more details of the insuiin receptor

structure are hown.

4.2. Autophosphorylation

h s u ï h bhding to the receptor a subuüit rapidly stimulates aatophosphoryIation and

enhances tyrosine kinase actiw ofthe $ subunit of the receptot (White et al, 1988). The

signai of ligand binduig in the extraceiIdar domain is trammittecl through the

hydrophobie. transmernbrane domah, possiay hy a conformationai change. The

uwccupied insuiin receptor acaially inhibits the tyrosine kinase activity of the subunit

(Hubbard et ai., 1994)-

As mentioned preMousIy, aautphosphorylation of the receptor occurs in t h e

distinct regïons: the juxbmembrane region T-72, and possibly 965 and 984; the kinase

catalytic region, Tyr1 158, Tjn 1162, and Tyr1 163; and the C-temiinal region, Tyr1328

and Tyr1334 (White et ai., 1988, Kohanski, 1993; White and Kahn, 1994)- The

mechanism of iasulul receptor autophosphorylation likely occm by mutuai

transphosphorylation of the $ subunits (Lammers et ai., 1990; Lee n ai., 1993; Taouis et

al., 1994) although some cils-phosphorylation has been reported. The recent gystai

structure of the insuiin ~eceptor PTK domain suggests that a cls-autoinhibition is found in

the inactive kinase* Insulin binding moves two kinase domains closer to enable hm-

phosphorylation in the presence of ATP by disengaging Tyr1 162 nom the catalytic loop

(Hubbd et al, 1994; Hubbard, 1997).

The intracellular juxfamembrane domain of the insulin receptor P subunit contains

at least one phosphorylation site -72) in a NPxy972 motif (Feener et al., 1993).

Point mutation at this residue has no effict on the insuiin reccptor's tyrosine kinase

activity but impairs receptor signal transmission (White a al., 1988; Keburagi et al.,

1993). This NP2@72 sequence is also îhe binding site for the insuiin subsîrate IRS4

and Shc, which transduces the signal intraceiiularly (Gusiafson et al., 1995; -Pawson,

1995).

4.3. Post-Receptor Sigul Transduction

The dowastream elements respmsi'ble for the propagati*on of extract11uiar signais

through tyrosine Ianase rnessehger systems have amacted much attention and ban

extensively revïewed (Pawson and Gis& 1992; White and K h , 1994; White 1994;

1996). In this section, 1 briefly Summaaae the cumnt lmowfedge about the insulin

signahg pathways.

The bhding of insuiin stimulates the i n W c tyrosine kinase of the IR, *ch

d t s in autophosphoryIation of the subunits on tyrosine residues and the subsequent

phosphoryIation ofiasullli receptor sribstaite pmteias. including iasulio receptor substratc

proteins @RS-1 and IRS-2). IRS-1. and IRS-2 are proteins with molecular weight larger

than 180 kDa based on theh electrophoretic mobility SDS-PAGE. They err 43% identical

in their primary amino acid SeQuences (Sun et al. 1995). Both of them contain so-called

IRS-homology domains: Mly 2 and 3 (White, 1996). The Il32 domain, sharhg 75%

homology between IRS-1 and IRS-2, has been found to be similer to the phosphotyrosine

binding (PD) domain in Shc and can bind to the phosphoryIlatcd ~ ~ - 7 2 sapence in

the insulin nxqtor (GustafSon et al. 1995; Sun et al., 1995). While M1 and IEI2 are

important for recognkhg and binding to the insuiin refeptor, ïH3 1~3y m o d ï

downstream sigaals (Gustafin et al. 1995). IRS-proteins contain more than 20 potential

tyrosine and 30 serine/threonine phosphorylation sites, including many tyrosine

containing motifs such as YMXM and YXXM recognized by various kinases. Unlike

other RTK family members, such as EGF and PDGF receptors, the insuiin receptor does

not appear to have direct association with SH2-proteinS. Instead, in the case of the inmlin

receptor the phosphorylation sites and motifs in IRS-proteins provide docking sites for

SH.2-proteins, such as PI-3K, SH-PTP2(Syp), GRB-2 and Nck (White, 1994; 1996), and

many other kinases, such as ~8~15i.n kinase II, and MAP kinase (Sun et al., 1993;

Tanasijevic a al.. 1993). These multi-protein interactions provide a meam for signai.

ampiification by eliminating tht s to i~hiorn~c constraints enwuntered by TeCCPfOrs that

directly recnllt SH2-proteins to thQr autophosphorylation sites.

hteresthgîy, in addiition to intaadiag with the IR, IRS-pro* are also engageci

and phosphoryîated by the receptors of IGF-1 and various cfasses of cytokines and growth

f e r s (White a aL, 1985; Myers et al.. 1993, White. 1994; 1996). The shrned use of

IRS-proteins by diffaent recepfOrs likely suggests important connections previousiy

unknown, or observed but unexplained, between various homones and cytokines.

Severai intracellular pathways aie involved in the insulin signaihg system

through the phosphorylated IRS-proteins @ig. 1-5). For exampIe, phosphoryIated IRS-

protein am associate with and activafe PC3K and S&PTP2- The activateci PI-3K may

1ilÙE to p70 ribosomal S6 kinase and specinc isotypes of pte in kinase C Wyers et ai.,

1994)- Activated Pb3K was also found to be mvolved in GLUTQ trans1OC8fIon

(Cheatham et al, 1994). In addition, IRS-pmtehs, mostiy IRS-2, can actiV8fe Grb-2

which regulates a Ras guanine nucleotide exchange fktor called SOS. The Grb-2-

activated SOS initiates a mitogm-activated Lmasc cascade consisting of c-Raf; MAPKK,

MAP kinase. This pathway leads to alterations in gene transcription and 0 t h cellular

activities (Nishida and Gotoh. 1993). It is not yet clear how the metabolic effm of

insulin, such as glucose metabolism, are regdated in the insuiin-signaüng pathway.

Aithough th= is evidence that suggests that PI-3 kinase (Cheatham et al.. 1994) or a

kinase cascade simüar to the MAP kinase pathway may be involved in giucose

homeostasis (Suthedand et ai. 1993), many of the detaïis in this poithwa. are stül to be

elucidated.

Rotein tyrosine phosphatases (PTPs) also play a critical d e in regulating insulin

action in part through dephosphorylation of the active (autophosphorylated) fom of the

insuiin receptor and attenuation of its tyrosine kinase activity (Drake and Posner, 1998).

Recent studies have show that PTPlB knockout mice have increased iiisuün sensitivity

and are resistant to obesity. suggcsting this PTP is a negative regulator in insulin action

and related to the pathogenesis of diabetes and other diseases (EIchebiy et al., 1999).

Sianal Signal Signal

Fig . 1 -5. A scbcmrtic &.gram ofthe insulin signaling pathways. Insalinrecepm contains intrinsic kinase activity that is activated by autophosphoay~ation a h mo~lin b & b g The activated IR then tyrosine-phosphoryiates IRS pmteïns. The rr tMted IRS signahg complex is show11 to compose ofPI3'-Klaase, GRB-2/SOSmaS, ami SH-PTP2 pathways. The activated IR may also stimuiaîe other signalïng pa6iwqrs- @ Phosphqlated Tyrosine- (B StimPlatory eftècts

B e s i k the priecipai IRS subsbeates, 0th- such as Shc, might aIso

exkt in the -signahg pathway. Altemative pdtways ofthe IR may be ptsponsible

for the multiple actions of uwlin. Shc is a substrate protein rapidiy tytghosphorylatcd

by insutin StEmulati'on. Shc contains an wkmimi PTB domain which b'î ta the

phosphoryiated NPxUs72 sequence ip the insuücl xeceptor and can also bd Grb-2 to

link these signais to Ras activation (Skolnik et al., 1993; h n k et al 199e Gustafbn et

ai-, 1995). It has been suggestedthaî Shc, rather than IRS-1, may be the main meàiator of

the mitogenic actions of insuiin and IGF- II Cyhaguchi and Pessia, 1994).

5. MAPPING THE INSULIN BINDING SITE

As discussed insulin initiates signai transduction in target ceUs by binding to a

specific ceil sunire receptor. Photoaffinity labeling Mes have impiicated the a subunit

as the location of inmlin-insulin receptor hbmctions (Yip et al., 1978). Subsequently,

several regions in the a subunits of the IR have been shown by different techniques to k

involveci in insulin binding (Yïp et al., 1988,1991; Anderson et al., 1990; Schumacher et

ai., 1993; Kjeldsen et al., 1991,1994; Schaefei. et al., 1992). The molecuiar details of

these events are still obscure and will require a detailed understanding of the structure

fitnction relaiionships of the IR, in particular those of the extracellular domain. The

purpose ofmy study is to explore the structure and hction of the IR., and particulat1y to

focus on the localization of the insuiin-binding site on the IR. Multiple appmaches were

used to achieve these goals. The information obtained would greatiy help us to

understand the mechanism of the insuhm interaction and signal transduction, the

pathogenesis of diabetes mefitus, and eventuaiiy, to assist in designing new medicine to

treat diabetic patients.

5-1- Insulin Interaction with the IR

As mentioned previously, human bulin cocoasists of two chains, the A-chaÏn (21

residues) and B-chah (30 residues), and it is assumed to bind to its receptor as a

monomer (Brange et aL, 1988). The X-ray crystal structure of porcine insulin provided

the fïrst direct experimental &dence for the insalin folding (Adams et aL, 1969).

Subseqpent studies have shown that hsdh A and B ch- fold to foxm a compact

globuiar structure, stabilîzed by the two interchain disulnde bridges.

A vast number of insulins b m diverse animal specïes, and chemically or

geneticaily moWed insulin analogues have been studied for their bioIogicaI properties

and binding to IR The results indicate that many residues of the insulin molecule may

contact orbe involved in the insulin-receptor bmding (Baker et al., 1988; Murray-Rust, et

ai., 1992; Mynarcik et al., 1997). On the putative weptor-bïnding surfie of uisulin,

GlyAl, IluA2, VaW,A19, AspA21, ValB12, PheB24 and B25, TyrB26 are the major

determinants of the receptor binding site, A few other residues make minor contri'butions

Meanwhile, the N-temiinus of the B chah being the most flexible region of insulùi as

suggested by X-ray and NMR studies, was comïdered to be a characteristic of insulin-

insulin interaction rather than being directly relevant to receptor binding W g e r et al.,

1990; Mul~y-Rust et al., 1992). Since mutations in the C-terminal region of the B chab

insulia have been reporteci to be associated with diabetes (Steiner et ai., 1990), residues in

this region has been suggested to play a critical role in the expression of insulin's

biological activity. Many studies have been done to elucidate the role of this region in the

interaction with the insulin receptor Wakagawa and Tager, 1986, 1987, 1993; Mirmira

and Tager, 1989, 1991; Mynarcrk, 1997). PheB24 and PheB25 have betn suggested to

provide much of the binding fke energy involved in inducing the site-site interaction @e

Meyte, et al., ! 978). Systemic studies of msulin analogs m&ed at B24, B25 (Mimina

and Tager, 1989; Nakagawa and Tager, 1986; 1987) have also supporteci the suggestion

that these residues are responsible for the initial interaction with the receptor.

CombinSng information ikrn the crystaï structure of insuiin and its biological

properties, Schaffer pmposed a model for insulin bindlng to the insulin receptor (Fig 1-2

b). He hypothesized that high afhïty insulÎn binding ropuirrd two receptor-binding sites

on insulin. The nrst bindihg site is the classical bhdïng site, mcluding B24, B25, AL and

A21 in the dimer-fomiing d a c e of the insulin molecule- The second binding site

consists of B I7 and A13, located in the hexamer-forming sudace on the opposite side of

the molecule k m the classical binding site (Schaffér, 1994)- Substitutions of these

residues woald be expected to dramaticaiiy decrease insuiin afE~Üty to its receptor. 0th-

snidies also suggest that insulin binding to its receptor may lead to a coaformational

change in the C-terminus of the B chai. a region which is essentid for high mty

receptor binding- The ernerging concept, therefore, is that receptor bindùig is not a simple

collision process, but rather a multiple step process that may involve successively

different residues in the transition towarâs to the active state-

5.2. The liisuïin-Binding Domain of the IR

Unlike insnlin, the exact regions of the receptor that directly contact its Ligand

remain incompletely defined and are stili the object of much study.

Evidence suggests that the binding of insulin at high aflhity involves multiple

regions in the a subunif and even the P subunit of the receptor- The fke single a subunit,

or the a+ monomer, as well as the a-a subunit dimer, has minimal, or no binding

iiffinity for insuiin (Boni-Schnetzler et ai., 1986; Schaefer et al., 1990; Roach et al, 1994).

The minimal tnuicated holoreceptor which still maintains the same high affinity for

insulin is the solubilized whole extracellular domain of the receptor, Uicluding the entire

a and all extracellular p subunit linked in tetrameric form (Schaefer et ai., 1990).

Although there is stili a considerable uncertainty about where the receptor binds to

hsulïn, these observations suggest that the hi&-affnity insului-binding pocket requires a

conformation consisting of the entire extracellular portion of IR It suggests that the

insulin-IR interaction mpks not only multiple specific contact sites but also some

supporting regions to keep the proper biedmg pocket in the right conformation.

A~nroaches to Studv the hsulin-Bhdhn Site

Attmpts to localize the specinc region for high affinity iiuului bmding on the a

subunit have involved severai different approaches, hluding affinity labeling,

recombinant DNA technology, such as sitedirected mutagenesis, deletion studies and the

construction of chimeric receptors comprised of different domairis of the msulùi and IGF-

1 receptors (Tavare and Siddle, 1993)- Although each approach has its advantages and

dîsadvantages, and the results may not agree, these studies do provide usefril information

about where on the IR insuIin binds.

Affinitv Labeling Chernical or photodlïnïty labeling was one of the earliest

methods used to study the insulin-receptor interaction. Photoaffinity labeling (Yip et al.,

1978; 1980) and chemical cross-linlrùig techniques (Pilch and Czech, 1979; 1980) have

been used to show that insulùi prbarily binds to the a subunit of the IR. These

pioneering experiments also revealed the overall a#, subunit structure of the insulin

holoreceptor CYip et al., 1978, 1980, 1982; Pilch and Czech, 1979, 1980; Yeung et al,

1980; Massague et al., 1980). Although they have advantages in the characterization of

the structure and hction of the IR, the chemical bihctionaf reagents or the early

photoaffinity labeling reagents for cross-linking of insulin to the receptor have low

intrinsic efficiency and/or specificiîy. The disadvautaga include the fact that the cross-

linked insulin-receptor complexes are either typicaliy a low percentage of the total LR and

the non-specific cross-linking products may Increase during attempts to increase cross-

hkhg efficiencies (Waugh et al., 1989). Later attempts to develop reagents with greater

efficiency and selectivity have focused primarily on derivatking the E- or a-amino groups

of LysB29 or PheB1, respectively, with additional modifiai aryl azides (Brandenburg et

al, 1980; Ng and Yip, 1985; Yip et ai., 1988; Fabry, et ai., 1992). These new reagents

have much hi* specificity in cross-Iinking and can provide more picciSc iofomation

on -receptor interaction, but their efficiencies are usuaiïy stül below 100& which

limit thek u s t ~ in fbtbnai studies.. Recentiy, a novel b e n z b y I p h e n y ~ B 2 5 -

iIisulur photoprobe was reportad to have an efnciency of cross-linlnnp as high as 60-

100% (Shoelson a ai., 1993). This new class o f photoafflnity reagents would kilitate

the studies ~ f ~ r e c e p t o r stnichnc and fimction.

m e technique ofphotdbity labehg bas been usedto iden* the insubbinding

site. The approach includes a photo-cn,ss-iinkîng process foiiowed by isolation of

receptor-i.nsuh fhgments to aüow the identification of the regions where the puîative

insulin-receptor contact occurs. Using radioactive photoreactive insuiin derivatives that

maintain mon than 75% ofthe bioectivty ofiilsulin, the Iaboratory of Yip found that the

cysteine-rich domain of the IR a subunit contains the insulin-binding site (Yïp et al.,

1988, 1991). This fhding has ban codkned by another sirniiar study (Waugh et al,

1988) and mutagenesis studies, using either site-directive mutagenesis (Rafiieloff et al.,

1989) or HIR/IGF-IR chimeras (Gustafson and Rutter, 1990). However, using different

photopmbes, other investigators later f o d that other regions rnight aiso be in contact

with î n s u h They are the L1 domain, residues 60-90, (Wedekind et ai, 1989), the L2

domain, residues 300-390 Fabry et al, 1992), and the carboxyL tenninal region of the a

subunit (residues 704-718)@Curose et al, 1994). This variation in the d t s indicates the

complicated nature o f the insulin- binding "pocket" and each of these regions may only

represent part of the whole receptor-binding site. The photoatbity labeling appmach

theoreticaily wuld provide the most accurate information for -receptor wn&ting

sites, but the generdy low cross-ünking efficiency and the -ment for a large

quantities of insulin receptor protein make the pst-Iabeling adysis ofthe products very

diff?cult

Recombinant DNA techniaues Recombinant DNA techniques have

fkequently been applied to mapping the iasulin-binding site. Takhg advantage of the

37

highly conserveci domain stnicturt but a large difference m the &nïty to their cognate

ligands, domain exchanged chim&c lnsullnna-1 receptors have been extensively

studied (Anderson et al., 1990; Schumacher et al., 1993; Kjelâsen et al., 1991,1994;

Schaefer et al., 1992). This technique is a usefiil approach for the study of domain

involvement in ligand binding. R d t s obtained have suggested that various regions of

the a subunit are involved in the üisulin binding- These include the N-tenuid region,

residues 1-68 or 1-137 (Schaffer et al., 1993; Kjeldsen et ai., 1991, Schumacher et al.,

1993), the cysteine-nch region (GustafSon et al, 1990, Anderson et ai., 1990), and

regions in the L2 domain (Schumacher et ai., 1993). 'Resalts fiam several cbimeric

studies also indicate that the Cysteine-rich region is specXc only for high afbity bmding

of IGF-1 but not of insulùl (Anderson et ai., 1992; Schumacher et aL, 1993; Zhang and

Roth, 1991).

Many mutations, both natudy occiming and r d t i n g h m siteairected

mutagenesis in the u subunit, also provide important information on insului binding of

the IR Site-directed mutations in the mino acid residues 240-250 of the cysteine-rich

domain ~-Cy~-Pro-Pro-Prr,~Tyr-Tyr-~s-Phe~ to Thr-Cys-Pro-Arg-Arg-Tyr-

Tyr-Asp-Phe-GIn-Asp) has increased the receptor's aflhity to insulin Waeloff et al.,

1989), while a mutation in Phe 89 of the LI domain almost abolishes insulin binding @e

Meyets et al., 1990). A naturaL mutation in Ser 323 (the beginning of the L2 domain) in a

diabetic patient markedly impairs insulin binding to its receptor, suggestiog its cntical

role in insulin binding (Roach et al., 1994). A series of deletions within the extracellular

domain (Schaefer et al., 1990) and alanine-scanning mutagenisis on the whole L l domain

and C-terminal region of the a subunit (Wiiïiams et al., 1995, Mynarik et al., 1996) have

also provided very usefûi information about the localization of the insulin-bïndïng site.

Mutation analysis also show that glycosylation of the a subunit does not &ect insulin

binding (Caro et ai., 1994).

The observations mentioned above sriggest tbt the mnilin-bmding site is a

complex multiple domain structureture It possiiIy involves the LI, C-R, L2, and C-tenninaZ

regions ofthe a subunit Both a subunits sean to be invovdin f o d g the high a£fjnïty

ligand-binding site, and even the $ subm-t may play a rofe m stabilizing the structure.

C~oe1ectrOnMicrosco~v Recently, with the progress of the eiectron

microscope technology, especiaUy the use of scamiing transmission electron microscope

(STEM) in biological studies, cryoelectron rnimscopy has become a new trend in the

study of structure- The concept and f-'biüty ofreconst~cting tbreedimensional images

fiom electron mimgraphs were introduced 30 years ago (DeRosier and Klug, 1968). A

convergence of technical advances in qoelectron miaoscopy and cornputhg image

processing has now allowed this technique to be used to analyze biomolecular structure

without crystaUization. It is clear that the electron cryornicruscopic and image processing

technique are an ideal and powernil tool to elucidate the structure of large proteins that

are quite dÏfEcult for ccconventi~nai" techniques, like x-ray crystallography or NMR. This

technique also c m be used to map out the fbctional domains in a large complex and to

study their conformational variations in mit hctional states. There is considerable

evidence that high resolution cryoelectron mïcroscopy is able to p&de the structure

resolution at below 10 A level (Bottcher et aï.. 1997; Conway et al., 1997; Chiu and

Schmid, 1997; Cheng et al., 1997; Walz et al., 1997). It is especialiy usefid in studying

large proteins, such as d e insulin receptor, which are very difIÏcult to analyze by x-ray

crystaliüation or NMR spectroscopy. This technique alsu has been successnilly applied

to stmcturally localize the ligand or substrate binding site in protein-protein / protein-

Wus interaction (Zhou et al., 1998; Paredes et al., 1998; Wagenknecht et al., 1994). This

new technique gives us a powerful approach in the study of the insuün receptor structure

and fimction-

During the course of my PaD program 1 have taecï biochemitcat and biophysicd

the insulin-insulin mxptor in-OIL In Chapter 2.1 describe my attempts to use the

conventionai biochemicai appmaches to Iocal iP the insulin-bindiag site on the human

insuün rswptor. A bacdovurus expressing system has also been estabfished to

overproduce the fimctionai extracelluiar domain of the HIR Mirent photoafnnty

labeiing and Iimited proteolysis, as weU as various peptide-mapping methods were used

in an attempt to identify the Pnmary site(s) ofthe însdhIR interaction- h Chapter 3,

using cryoelectron mlcroscopy and cornputer image pmcesshg technique, 1 determineci

the quate~1181y structure of the IR and whue the insulin receptor bhds to its ligand A

Nanogold-bovine iIwlui derivative was used as a mark- to locaüze the insuIin-b'îding

site on the 3D reconstruction of IR We have successnilly obtauied the 3D structure of

human insuün receptor and localized b u h in the 3D immbinsuiin receptor cornplex.

Finally, in the iast Chapter 1 discuss the significance and future perspectives on my thesis

work

LOCAUZKIXON OF INSULIN-BINDmG SITE@) ON THE EUMAN INSULIN RECEPTOR BY BIOCEEMICAL APPROACHES

1. LNTRODUCfION

As desc r i i in Chapter 1, the mature humaii insulin receptor @IR) consists of

two a and two subunits linked by diSulfide bonds, with a m a s ofappmxhateIy 450

kDa (Yip, 1988; Kahn and White, 1988). Revious aiXhïty cross-linlring studies using

photoreactive probes or bifimctionai reagents have identifid relativeIy large receptor

fhgments as putative insulin-binding (Wedekind et ai., 1988; Wsuigh a al., 1989; Faby

et al., 1992).

This Chapter descrii my attempt to identifif the insulin-binding site(s) on its

receptor by using various conventional biochemicd appmaches. The laboratory of Dr.

Yip originaiiy Iocalued the insulin-bindhg site to the a subunit of IR (Yeung, 1980). and

then more specifIcally to the Cys-rich domain of the a subunit by combining afnnity

labeling and immunological techniques (Yïp et al., 1988,1991)- My project was a

continuation of these studies The main objective was to use different insulin photoprobes

with high efficiency of cross-linking to label the insuiin-binding domain on the IR,

followed by proteolysis to obtain smailer cross-Iinked receptor fkgments for peptide

mapping and amino acid sequencing, thus to identify the putative iiwlin-binding domain.

This Chapter describes this appmach and the results obtaind

2. MATERLAC AND METHODS s

Intact rraptor or a tnnicated receptor with niII hsubbindiug activity of tée

intact IR was used in the study. Intact receptor protein has previously ban extraded and

pirn'f?ed h m himian tissues or mammalimammalian di iines (Cauh.cFasa9 a al, 1972; Fujita-

Y-hi et aL, 1983; Whittaker, 1987). However, a dEcient q-ntity of the intact

receptor is difncuit to obtain h m these sources for the identification of the insuiin-

binding site@). Since it is weU estabiished that the entire extraceiilar domain of IR is

requited for the high-aff?nity binding of insulki (Sweet et al., 1987; Schaefer et ai., 1990).

1 used the bacui0vVu.E expression system @ES) to express the entire ectodomain of HIR

to obtain substantial quantities of the pmteh for this study.

Construction of a cDNA ExDression Plasmid Encodine the ExtraceUuïar Domain of

HIR - The clonhg strategy of the BES expression v e r is shown in Fig. 2-1. To take

advantage of the bscuîovinis expression system, the gene of the HIR most be cloned into

a baculovinis transfer vector which is designed to position the EIIR gene immediately

dowmtream of the powerfbl bacuiovirus polyhedrin gene promotor. Ail the restriction

and rno&@hg enzymes in these experiments were purchased h m Pharmacia,

Boehringer-Mannheim, or New England Biolab. AU adapters and linkers were custom

synthesized by the ASC Biotechnology SeMce Center, Toronto

Pre~aration of eonstiucts Preparation of the transfer vector consisted of

several steps. cDNA encodîng the entire ectodomain of HIR (hcluding aii of the a

Signal Cys4ch a subunit p subunit

Encoded Protein

Recombinant Baculovirus

Recombinant HIR Extracellular Domain

Fig. 2-1. Expression of humm insuün mxptor crtrrcclluhr d d n with baculovinu erprtssion systcm. A 2.9 kb cDNA d g the extracclIular domam of hIR was cut than pRSV-IR plasmid and mbcioncd mto a bacdovirus expres~ingvector~ linsect cclls hkted with the recombinant virus were used to express the riecornbinant reœptorprotcin,

subunit and ail. of the extraceiiulairp~~on ofthe Jubmiif except for t h e amino m*dS

N-teaninal to the n'ansmcmôrane domain) war txcised h m the 9-7 Kb p M d pRSVIR

(a genemus gift fiom Dr. Ira Ooldfine, UCSF). Restriction endonuclease Srp I was useû

to digest the pRSVIR to remove tbe CD= sfuthg h m nuckotide 2999 of the receptor

cDNA, whÏch encodes the putative transmembrane domain and the eotin cytophsmic

portion of the subunit of the HIR. Ta fiditate the expression and the purification k,

a stop codon W G ) in axbd linka:

5'-CTArrTrTAGACTAG-3r

3 '-GATCAGATCTGATC-5'

L V s t o D

or, dtematively, an adapter encoduig an extra 6-Histidine amino acids pnor to the stop

codon was inaoduced at the SFpr site. In the latter case, the adapter and the expected

deduced protein sequence at the C-terminus of the protein is therefore:

5'-TCTCATCATCATCATCATCATGT-3'

3'-AGAGTAGTAGTAGTAGTAGTACAGATCS'

S H H H H H H V s e

The resulting intermediase plasmid, designateci as pIRX or piRXH (H represents paly-His

tag), was digested with NcoI, which cleaves at a single site at the beginning of the cDNA

(nucleotide 137) corresponding to the nrst metbiorùne of the signai peptide at position - 27 of the HIR protein. The digested plasmid was then blunted with the Klenow niigment

of DNA polymerase 1 to n11 in the ends. A M linker

5'-ATCGCTAGCGAT-3'

3'-TAGCGATCGCTA-S'

was then ietrodueai at the b I d Ncd site ta gcriercite the second p l d d

ofpIRXN or pïRXNH (4.0 KB)- Digestion of above the phsmid~ ~6th -1 aad X6rrI

generated a 2.9 Kb restriction fiagrnent of the tnmcated HIR, nucleotides 138-2998- It

encodes the entire extradular domain of& HIR, except the three amino acid LeSidues,

1 A Y, prior to the ttansmembrane domain of the $ subunit, wîth or without a 6-histidine

fag on the C-taminal enb The hrhef-Ba1 hgment was then subcloned into a l\rheI c i 9

cal€ intestinal phosphofase-treated bacdovirus transfer vector pBlueBac2 or pBlueBac4

(hvitrogen). The zight orientation of the insert CDNA m the transfér plasmicl, designated

as pHIRa or p H I R a was detemineci by vsrious restriction endonuclease digestions.

These transfer piasmids for M-transfdon were prepared by CsCl gradient cenpafugation

in a Beckman rotor VT801 at 80 K x g, at 22 OC for 4 hours. The purifid piasmid

. * coll~t~cts were steniized with 0 . 2 2 ~ filter and saved at 4 O C .

DNA seaaencin~ of the coastructs

In order to confinn that the nucleotide sequence was correct for the recombinant

protein, the DNA of the 2.9 Rb insert in the transfèr construct was sutscloned into

pBluescript (Stratagene) vector at the XoaI site (PBSHIRa, bp 138-2998), or crb with

restriction enzymes and subcloned into tbree sub-cunstructs in p B l d p t vector

(pBSHIR1, bp 559-1273; pBSHIR.2, bp 1101-1695; pBSHIR3, bp 1695-2606), thus

allowing the whoie 2.9 K . cDNA hgment to be sequenced Fige 2-2). AU constructs

were m a n d y (Pharmocia DNA sequencing Kit) or automatically (Perkin-Elma-AB1

Genetic Analyser) sequenced. AU sequenced DNA showed no e m r in the nucleotide

sequences.

Fig. 2-2. Comstnrcioii of the BES transfiin vcctors br tht bïRcctodomain with (A) or w i ü ~ ~ u t @) 6 Eis tag. A29 kb ~ D N A t h g m a ~ t ~ a ~ cuî&mpSViR mdmodined üyaddingprop~adaptor~, with or without 6 Air rcriducs, and stop axbn. l%c modifiai cDNAhgmcas wat suôcloned into pBlueBac4 @BB4) or pBlueBac2 @BB2) Wovinis aansfection vcctor-

Log phase ùiseet Sp (S. fbgiperdzt) cek @.vitmgen) were p w n as anamiaent

culture at 27OC in modifled Grace's medium suppIemented with 0.33% lactalbumin

hydrolysate, 0.33% yeastolate, Iû% fetsl bovine sarmn (FBS), 10 ps/mL gentamycin a d

250 ps/ml amphotaich B &Se-Technofogy). In some experiments 0.1% of Pluronic F-

68 (L'Se-Technology) was addd to suspension culture of Sfl cells to d u c e the

sensitivity of the culture to mechanid shearing- Awthn insect ceii line, High-five (Hi3

ceiis @ichoplusia nl) (Tnvtrogen), was cuitured in Exce11 401 saum fia medium (JRH

Scientific) containhg 10 mdmi gentamycin and 0.25 m g f d amphotericin B to yield a

higher expression level- AU procedures involving celi culturîng, includhg routine

subculturing, and protein expression, were performed as d e s c n i in the M&~uals of

Methods for Bacuiovinis Expression h m Invitmgen.

Recombinant plasmi& were CO-transfected into SB cells dong with the linearized

wiid-type AcMiWV vual DNA (for p-) or Bac-N-Blue lineanzed Wal DNA (for

pHIRaH) by the technique of cationic liposome mediateci transfection (bvitrogen). In

vivo homologous recombination between the polyhedrin sequences in the Md-type Wal

DNA and the recombinant plasmids d t e d in the generation of recombinant viruses

coding for the expression of HlR ectodomaia, Briefiy, lpg virai DDNA, lpg of

recombinant tramfiet plasmid, and 20 PI of Insecth liposome @mitmgen) were mwd in

1 ml of Grace's media without FBS and suppiements, The mixture added to hshly

d e d Sfl celis in plates and incubated for 4 hours on a sIowly rocking platfom at 27OC.

Then, 1 ml of wmplete Grace's medium was aàded and incubated at 27" C for 48-72

hours. The medium b r n sueoessfiilIy co-traasfected, culnaed ails ans scrrmd by a

plaque assay for tht cccombinant vhs contrllning the msulin ncepor cDNA

Piaaue Assa. and Pre~antian of FliPb Titer Vira1 Stock

For the piaque assay, SP ceils in 60 mm plates were hfkcted with 1 ml of 1 x

102, 103, 104 diluteci media containhg CO-transfected virus for 1 hoia at 27°C. The

media were then removed and repiaced with a 4 ml overlay of 1.5% Sea-Phque agarose

(FMG Biopmducts) containing 0.01% X-Gal. Af€er 4-6 days' incubation at 2 7 O C, weil

separaîed, distinct blue piaqyes were isolatecl and used to hocuiaîe SiJ9 celis in multi-weil

plates for M e r screening and p r e h b r y virus ampMcatioa Usuaiiy, 612 plaques for

each recombinant protein were s e l m and 3 4 clones of the finai purifiecf virus were

saved at 4 O C as P-1 WaI stock for mer characterjzation and amplification. The P-1

virai stock was then used to infect log-phase Sfl celis in a T-25 flask for 5-10 days at 27O

C to generate P-2 virai stock The ampfified P-2 viral stock then was used to i n f i Sp

ceus at a ratio of 1: 10 (vfv) or MOI 0.5 - 1 (see detaüs later), either in adherent culture in

T-225 flash (Costar) or in suspension culture in 500 ml spinner flash (BeUco Glass) et

27O C for 5-8 days. This P-3 high titer vUal stock was analyzed in a plaque assay

following the same schebile as previody desctibed except the viral inoculate was

diluted up to 1 x 10-9, to measure the virus titer. The plaque forming units pet ml

@fu/ml) were calculated using the following formala to set the optimal conditions for

protein expression:

pfii/ml= l/dilution x mean number ofpleqyedplate x platelml

To obtain a synchronous Mixtion for e q m s h g the recombinant receptor* a

particular multiplicity of infection WOI) h c k of 5-10 was iiscd to caicuiated the

volume of the Mirr stock with thefoiIowhg fomuk

[MOI ( v i r o d d ) x number ofcelIs]/titer ofvirus (Vli.oIIS/ml)=ml ofinocuium stock

Hi@-titer virai stock was kept at 4 O C in the dadc mtil use. A smd aliquot was

hzen at -70° C for long-term stomge.

ExDression and Characterimation of the Recombinant IR

MOI o~timization for vinw iafiition High titer viral stock was useâ to

infect the Hi5 ceils in 2 ml of Exceii-401 medium in T-25 flasks at -90% confluence at

vaqing MOIS (2, 5, 8, 10 and 15) for 1 hour. Three ml of Eresh media were then added

and the ceUs were cuItured for 4 days at 27O C. Anti-HIR immunoblotting and B29

~~~-[125IJ-iodoinsulin photoafkity labeling ( see details later in this chapter ) were

used to check the levels of protein expression. An MOI of 5-10 was chosen in our

expression infection. On the other hanci, for generating hi*titer virai stock, an MOI of

0.5-1 was used to avoid any mwatlted interference in virus production. Western blots

with anti-IR a subunit antibody and r n o n o - [ 1 2 ~ 1 binding assays were carrieci

out to confimi the expression of protein and protein fiinction (see detaiis inter).

Time course and lime sale exDression of the recombinant rece~tor Hï5

cells at -90% confluence were Ilifected with viral stock at an MOI of 10 in 6 weii plates

at 27O C for up to 7 &YS. Media was rexnoved at O hout h m one weii and at eveiy 24

hours h m other wells to m o d r the infidon and expression process. The media h m

the infected ce& as weil as the corresponding ah, washed with pH 7.4 PBS with

cuitme, ~i5ceiIs weniaféctedatacel1daisityof2r 10~ce~swithviralstock atan

MOI of 10 in a 500 ml spinner flasL at 110 rpm at 27' C. One ml of suspension was

collected at zero time and each day foilowing for 7 days, h m the cuituze. These samples

were treated the same as the adherent culture-

In preparetve culture fôr protein expression, HiS cells were cuitured in Exceli-

401 medium to -90% confluence in T a Basks at 27O (=. A f k removing 25 mi of

media, the ceiis were infectecl with viral stock aî an MOI of 10 for 1 hour. Fresh medium

was repiaced to a -38 ml and the ceiis were cultured at 27O C for 4 or 5 days. The

cultured media were coliected for purification of the recombinant protein.

Pre~arinvceU lvsates and lasm ma membranes of the infecteà insect cells

Cell lysate samples for the tirne course sbudy were prepad by M y adding

SDS-PAGE sample bufEa (Laemmli et al., 1970) containkg 100 m M D'IT to the pre-

washed cells for subsequent SDS-PAGE analysis.

The plasma membrane and miczosome fiactions of the celis were prepared as

foilows: Hi5 cells in 25 ml of adherent culture were coliected at Day 4 after infection and

resuspended in 5 ml of pH 7.4 solubilization b s e r (Bu81er A) containing 50 m M Hepes,

150 m M NaCl, 0.1% Triton X-LOO, 1.5 m g / d Bacitricin, I O m M b e d d i n e HCI,

2mM pepstatin, 2mM leupeptin, 5 mg/mI aprotinin and 1 m M PMSF. The cells were

homogenized with an ice-cooled glass-Teflon tissue grinder (Kontes Glass) for 50 stmkes

then centrifiiged in an SS34 rotor -nt) at 1,000 x g, at 4 O C for 10 minutes. The

pellet containing the nuclei was rrsuspended in Bunet B (8uner A without the 0.1%

Triton X-100). The supernatant then was centrifitged in an SS34 rotor at 35,000 x g, at 4 O

C for 30 min, The pellets were resuspended in B e é r B to yield the sample of plasma

membrane- The supernatant was subjected to M e r ultracentüfügation in a Ti 50 rotor

(8eck;mm) at 250,000 x g at 4' C for 60 min. The pellet was resuspended with buffér B >

as the microsorne sample-

Anti-ER]Immunoblo~g Samples of cultumi media, cell Iysata, nuclei,

plasma membrane and lm-msome wne solubilized by boiiing for 5 min. in LaemmIils

sampie b s e r (La& et al., 1970) containing 100 mM DTT, and then separated by

electrophoresis on 7.5% SDS-PAGE, followed by t r a n s f d to 1mmobilon-P PVDF

membranes @dïUpore)- After transfet the membrane was blocked with 5% skim milk

powder in pH 8.0 TBST buffer (10 mM Tns-HCI, 150 m M NaCl, 0.1% Tween-20) for 60

min at room temperature and then incubated with rabbit anti-IR a subunit anti'body

(Santa Cruz) in blocking bufk over night at 4 O C- After washing with TBST buffer, the

aembrane was incubated with goat anti-rabbit alkaline phosphatase conjugated annibody

(Sigma) in blocking b s e r for 60 min at room temperature- The recombinant proteins

were visualized using the AP substrate color kit pharmacia).

Insulin Binding Insulia binding was measured by incubating - 50 ng of

human placenta membrane insulin receptor or recombinant insului receptor protein, 20-

30 h o 1 of m o n o - [ 1 2 5 I J ~ 1 4 - ( final concentration of -50 pM ), and varying

concentrations ( h m O - 1 x 10-5 M ) of unlabeled insulin in 200 ml of buffer, pH 7.6,

containing 50 m . Tris-HC1, 150 mM NaCI, 011% bovine serum albumin (BSA), 1.5

mghi bacitricin (0.1% Triton x-100 in the butEr for placenta membrane rezeptor) at 4 O

C over night. The receptor-insului complex was separated from unbound insuiin by

adding 50 pl of 0.4% bovine y-globulin and 250 pl of 20% polyethylene glycol 6000

f o i l o d by centnfiigation at 1,SW x g for 20 min at 4" C. Radi08Cfivity in the pellets

was determined in a PJmmmcïeLKB y-counter- The lgdioactbity daccted h the

presence of 10-5 M of uniaôeled iasulin was consicid nomspecific binding and was

always less than 5% o f the total activity. ALI binâing experiinents w m e pediomed in

duplicate on thme seprirate o c c a s î ~ ~ . The cornpetition binding curves were compiied

h m several experiinents, and each point was reported as the mean +/- SD- nie

Scatchard plot was a n a l . with EBDA cornputer software (Muoson and Rodmi,

1980)-

For samp1es of time course and subce11uïar fhctions, insuiin binâhg was

measiued as foiiows: 50 pl of harvested media or resuspended M o n samples were

dispersed in a total volume of 200 pi of the above binding bu&r plus 0.1% Triton X-LOO,

and - 20-30 h o 1 of mono-P2S1]~14ins~lin, and assayed as descnbed above.

Photoaffinitv Iabetinn and autoradionnuhv - Purifieci or Cnide protein

samples, such as harvested media or plasma membrane, were incubated with about 1 x

106 cpm of ~2g-MAB1:-[~~51]-iodoinsuün-o (Yip et al., 1980) in 200 pl insuiin binding

buffer with or without au excess (10 mM) UnlabeIed insuiin overnight in the dark at 4 O C.

Photolysis was carieci out for 15 second using a focused iight source h m a 100 W hi@-

pressure mercury lamp (Yip and Moule, 1983). Photolyzed samples were reduced and

s o l u b i i in Laemmli's sample bUner containhg LOO mM DIT- Cross-iinked rrceptors

were either separateci by SDS-PAGE directly or precipitated with anti-insulin receptor

antibodies pnor to SDS-PAGE. Cross-linked, radiolabeled proteins were identifid by

autoradiography of the nx.d and dried gels. The radioactive bands on the dned gels were

cut out and wunted in the y-counter.

MoliniLr m a d o n SampIes - aarilyzcd by 3012% lïnear

gradient SDS-PAGE imda reduced (10 mM DIT) or no~reâuced conditions. 'Ihe gels

were nxed in Io./; acetic acid and 25% isopmpsinol for 30 min Proteins wae visuaüad

using either a dver stain kit @bRad') or by C o o d e BSUiant Blue staining. The

molecular mass of the detected proteins was eshated by cornparhg the relaîîve mobility

of the receptor bads to molecular mass standards (Bio-Rad, orNovex)

2.2. Purification of the Recombinant IR and Human Phcenta Membrane IR

Prenvrtion of iasulin iffinitv coiumn Twenty five mi of m g e l 10 (Bio-

Rad) were washed with one volume of cold isopmpauoi, foliowed by 3 volumes of cold

EQO and were then equilr'brated with the coupling buffer, pH 7.4, containhg OJM

phosphate and 6 M urea Twenty five mg of zinc bovine insulin (a generous gift h m

Connaught Canada) in 25 ml of coupiing buffer was mixed with AfE-gel10 for 24 heurs

at 4" C. A trace amount ofrnono-p2I]~l4iiwlin was added to m o d r the coupling

process. The suspended gel then was transferred to a coiumn and washed with 20

volumes of the coupling buEer and fkther washed extensively with bufEer containing 50

mM Tris-HCl, pH 7.4, 1 M NaCI and 0.02% NaN3, Total radioactivity in the washed

bufEer and the wlumn was counted to calculate the couphg efficiency.

Pre~aration of MA51 monoclonal antibodv rtninitv column Mouse

anti-insutin recqtor monoclonal anti'body, MA51 (IN1 subclass, a generous gift k m

Dr. Ira Goldfine, UCSF) was prepared h m the mouse ascites the mAb MAS1 at - 1-3

mg/ml. The p H ofthe ascites was adjusted to 9.0 (wlth borate buffer) and NaCl was

added to a concentration of 3 M. Twenty ml of ascites containing MA5 1 were mixeci with

15 ml of protein &Sephamse beads @bunaci&) and hcubated for 60 min at m m

temperatureperature The btads were washed twiCce with 10 volumes of 3 M NaCl, 50 m M

sodium borate @H 9.0) by centrifugalion and aspMollc The beads were muspeded in

IO voiumes of 3 M NaCi, 0.2 M Murn bonite (pH 9.0) and mked with

dimethylpimelimidate @MP) to a final concentration of 20 mM- Afkr a 30 min

incubation at m m temperatme, the coupling was stopped by wasbing the beads once

with 0-2 M ethanolamine @H 8.û) and incubated with the same buffer at m m

temperature for 2 h o w with gentie mirring. The beads w m then extensively washed

with a birffe~ containÏng 50 m . Tris-HC1, pH 7.6, 150 mM NaCl and 0.02% NaN3 at 4

Purification of the Recombinant Eetodomain of IR

ARer 4-5 days infecton with viral stock, low-senim-containing medium was

hanrested and adjusted to pH 7.4 with 1 M Tris-HCI bu&r. PMSF and NaNj were addd

to a finai concentration of 1 mM and 0.02% respectively. The conditioned medium was

centrifugecl in a JA14 rotor meclanan) at 4" C, at 10,000 x g for 25 min to remove m y

cells or debris. The supetnatant was concentrateci -50 fold using a Minitan Uttafiltration

System (Mülipcwe) with a 100 LDa cut-off membrane to remove proteins of lower

moIecuiar as the nrst step of purification, The concentrated medium was then adjusted to

50% ammonium sulfate by dropwise addition of saturateci ammonium sulfate at 4 O C.

M e r ovemight stimng, the mixture was centrifiiged at 10,000 x g for 30 min at 4 O C, to

obtain a peiiet which was resuspeaded in 20 ml of 50 mM Tris b&er @H 7.4) and

dialyzed exhaustively at 4 O C against either the His-binding b&er (20 mM TnS-HCI, pH

8.5,O.S M NaCi, 10 mM imidazole, 10% giycerol, 0.02% NaN3 and 1 m . PMSF for 6-

Ris tagged ectod~main)~ or the buffer containhg 50 mM Tnis-HCI, pH 7.6, 150 mM

NaCî, 0.02% NaN3 and 1 mM PMSF for the ectodomain without the o-His tag.

Ammonium d a t e precipitation folIowed by diaïysis M e r rernoved Iow molecuiar

mass molecules and up to 40% of the BSA in the concentrated medium to facilitate

subsequent purification-

Purification of the ectodomain of the IR ais GHk tag Ni-NTA r e s h

(Qiagen) was washed andpre-charged with 100 mM NiCl and then equilr'brated with His-

binding buffer, pH 8.5. Twenty five mi of concentrated, dialyzeâ was mixed by rotation

with 300-500 pl Ni-NTA resin over night at 4 O C. The resin was t r a n s f d to a 2 ml

mini-column (Bio-Rad) and washed with 10 volumes ofHïs-binding buffer, followed by

50 volumes of washing buffer (20 mM Tris-HC1 pH 7.8, 0.5 M NaCl, 20 m M

immidazole, 10% glycerol,0.02% NaN3 and 1 mM PEIISF). AU b & i were kept on ice

when in use except that the Ni-NTA column was used at mom temperature. Recombinant

protein was eluted by washing the resin 5 tirnes with 0.5 ml of elution buffer (20 mM

Tris-HCI, pH 7.8,0.15 M NaCI, 250 mM immidazole, 10% glycerol, 0.02% NaN3 and 1

mM PMSF). The pooled eluted protein was uitrafiltered in Ultramax-50 (Miiiipore) to

concentrate the protein and change the bUner to 50 mM Tris-HC1, pH 7.5,lSO mM NaCl,

1 mM PMSF and 0.02% NaN3. M e d protein samples were aliquoted into severai

siliconized microfuge tubes and stored at -70" C.

Puriecation of the ectodomain of the IR without dHis tag Twenty five to

fîfty ml of concentrated and dialyzed medium was mixeci with 12 ml of MA51-protein A

beads by rotation ovemight at 4 O C. The mixture was transferred to a column and washed

wita 500 mi bUna containirig 50 m M Trk-HCl, pH 7-4,150 mM NaCL and 1 mM PMSF*

The bound protein was eluted with an- elution bu&r conta- 2.5 M MgC12, 0 2 M

concenfrated in an Amïwn ulaafiIttaSrCon device with a PM-30 membrane- The

wncentrated protein then was loaded on a Dextraa G-25 wlumn to ranove the high

concentration of sait The punfied protein h m the MA51 af'hity column was saved in

siliwnized microfuge tubes at -70" C,

Both Ni-NTA and MAS 1-protein A column purifid proteins were purined M e r

by gel filtration chmmstagrqhy on a FPLC Superdex-200 HR 16/60 column

(Pharmacia) in 20 mM Tris-HCl, pH 7-6, wntain. 150 m M NaCl, and 0.02% NaN3 to

remove either giycerol or/and the small amount of coeluted contamimieci proteinS. The

purifiai ectodomains of IR were a d y z e d by SDS-PAGE (Laemmli ct al., 1970) with or

without reducing agent (100 m . D m to check the punty, and by 12~1-insulin binding

for their activim

Puritication of haman insulin rece~tor

The methods of p r e p a ~ g the placenta membrane human insuiin receptot wae

modlfied h m Fujita-Yamaguchi a aL(1982). Briefly, a fresh n o d human placenta

was obtained within 1 hour after delivery, kept on ice, washed with normal saline (150

mM NaCL) 3 times and 0.25 M se once, and then cut into 4 pieces. The pieces

were tramferreci to 1 volume of 50 mM Tns-HCl buffer, pH 7.4, containhg 0.25 M

sucrose and 1 mM PMSF, homogenipd for 90 seconds with a Polytron homogenizer

(??olytron), and centtifiiged at 15,000 x g in a JA14 rotor (Beckman) for 35 min at 4* C.

MgCl and NaCl were added to a final concentration of 0.041 mg/ml and 5.84 mg/m.i,

respectively, The supematant was then centrsfiiged in a SW28 rotor @eckman) at

100,000 x g for 25 min at 4 O C- The peilets obtained were resuspended in homogenization

b&er and the spin/resuspend steps were repeated three times at the same speed but with

increased times (30,35 and 45 min). The final peUets were saved at -70" C until analyses

or fbrther purification

Placenta membranes (0.8-1.2 g of protein) fbm two fksh placentas was used to

obtain the pUnned human insulin receptor. The placenta membrane pellets were

solubiiized in 25 ml of 50 mM Tris-HCl, pH 7.4, buffer containùig 2% Triton X-100,100

mM EDTA, 1.5 m g h l bacitracin, I O mM benzamidlne HCi, 21x1. pepstatin, 2mM

Leupeptin, 5 p s / d apmti'nin and 1 mM PMSF. The suspension was c e n e g e d in a

SW28 rotor at 100,000 x g for 90 min at 4 O C, The supernatant was then loaded on the

insulin column which had been pre-eqyiiibrated with the washing b u f k of 50 m . Tns-

HCI, pH 7.4, containing 1 M NaCl, 0.1% Triton X-100 and 1 mM PMSF. The coIumn

was rotated ovemight at 4" C. It was then washed with 400 ml of washing b a e r and

eluted with 50 rnM acetate buffer, pH 5.0, containing 1 M NaCl, 0.1% Triton X-100 and

1 mM PMSF. The neutralized hctions with insulin-binding activity were pooled and

fùrther concentrated and desalted by ultrafiltration using Diaflo membrane PM-30

(Amicon). The partialiy purified receptor was M e r purifiecl by gel filtration

chromatography on a FPLC Supercryl-200 HR 16/60 column (Pharmacia) as previously

described, and saved at -70 OC.

The amount of protein was m e d by a modified Lo-s method (Bensadoun

and Weinston, 1976) or Btsdford's methad (Bio-Rsd)* using BSA as a protein standad

Amino acid adysis was pebormed by the SHC BiotecbnoIogy Service Center, Toronto.

Insuün Photoprobes and Photoafünitv Inbelhg

N-e(4'-Azid0-3'-~~iodopheaylau,~3-aminopmpyl iiisuün ( p q - A Z A P -

insuiin) (Fig.2-3) is a cleavabIe radioactive insulùi photoprobe *ch aiiows the iiisuün

moiety to be cleaved h m the labe1ed receptor protein a f k photoaf3hity labeling. Ne-

Monoazido-benzoyl-['Y ]-iodoinsulin (829-[IV-MABr) is iilJulin dgivatizcd near its

putative receptor-binding region, aud it retains more than 75Y0 of the bio-activity of

insulin CYip et al., 1980; Ng and Yip, 1985). Both [13+AZAP-insulin and B 2 9 - [ q

MABI were synthesized in Dr. Yip's laboratory* [q-Iodo-biotinm-p

benzoy1phenylaianinP-insulin (m-BBpa-insulin) was provideci by Dr. Steven

Shoeïsoq Harvard Medical School. It was a new photoprobe with two markets

(radioactivity and biotin) for detestion, and was reported to have much higher cmss-

ünking efficiency of 60-100% (Shoeison et al., 1993). In prrparative photolabeling, the

photoprobes used in photolysis were a mixture of trace arnounts of radioactive '9 labeled

photoprobe and non-iodinated photoprobe.

Photoiysis conditions were optirniPd for high photolabelhg efficiency with the

least damage to the protein. In p r e p d v e photoafbity labeling, the mixture of non-

iodinated and 'DI labled photoprobe and receptor protein at a 5: 1 molsr ratio (for 4

A Chain: GNEQC!CASV&LYQLEWCN \

\ B Chain: FVNQHLCGSHLEALYLV 4 GERGFFITP r

A Chain: G W E ~ ASV~SLYQLEWCN F * C * B Chain: FVNQHLCGSHLVEALVLV GERGFF'VTPW

A Chain: G N E Q ~ * B Chah:

Fig. 23. bul in photoprobcs uscd AZAPBi is a cleavable phoîoproble. Anowhead. indicates cleavage site m the probe. BBpiuBI has a biotin madra on the B29 Lysine. * iiadicates Radioia- beiing Ptcs on the B chin -1 and BBpauBI) a the cross-Iinting p u p (AZAPBI).

HiR) were incribiited atpH 7.5 Tris/NapCT binding biiffi with protease inhibitors at 4OC in

the dark for 20 hours. Photdysis Ïn prepaestive was canïed out imdg W

Iight with 1 0 W m powa for 15 seconds or with a 365 mn nIter for 45 minutes. Dtiring

photolysis the pr~te~photoprobe mhmewas keptonice.

Reduction and AUNhtion

The photoIyzed IR mixture was adjusteci to p H 82,O.l M TSs, 10 m M EDTA

and 8 M urai and kept at 50DC under N, and da& for 30 minutes. A 25-fold molar excess

dithiothreitol @Ti') then was d e d and iacubgtsd at 3PC under N,for 2 hours. Fresbly

prepared iodoacetamide in 8 M inca, 0.1 M Tris, pH 8.2 b u f k was added and the

preparation was incubated at m m temperature for 30 miautes. The duction and

alkylation were Camed out in the da&

To remove excess fke photoprobes, the subunit portion o f the receptor and

salts, the reaction mixture was separated by electmphoresis in SDS-PAGE under

reducing condition. After electmphoresis, the gel was irnmediately h z e n in dry ice and

an autoradiograph of the gel was obtained to 1- the photolabeled receptor band on the

gel. This gel band was then excised, cut into smaii pieces anci soaked in a pH 7.6,20 mM

NH4HC03 bu&r at 4 O C overnight. The extract was lyophilîzed and the gel pieces were

DiSemnt proteases or chemicai reagents were tned to obtain complete

hgmentation of the labeled receptor. Dirrct digestion of the extracted labeled protein or

the electmblotted protein on PVDE membrane was triai to avoid loss &ring s a q I t -

transfer. Before ptadysis, SDS wa+ rcmovcd by using the 8CCtOne-triethanolami'ne

precipitaîion m e & d Typicaiiy, the enzyme to protein ratio was kept at M O (wh) to

maiatain sufEcient digestion while the amount of enzyme was at a minimum, The

digestion tirne for each enzyme or c 1 e a . e with CNBrwas tested. Control digestion with

enzyme only was always cam*ed out at the t h e of a proteolysis experiment Roteh

fhgmntation was.monitored by 9-26% gtadi'kt SDS-PAGE, 40% a h h e PAGE and

autoradiography, AU enzymes used were of sequence grade purity.

For digestion with trypsin, 10-30 pi of TPCT-treated trypeùi, depending on the

quantity of protein, was added to the extmc&d protein dissollved in 250 pl of fkshly

prepared 2 M urea 0-4 M -CO3 bufk(pIi 8.0) and incubaîed at 37 "C for 24 hours.

Endopmteinase G1u.E (Va) was used unda sunilsr conditions to digest the receptor.

Digestion with endoproteinase Lys< or Achromobacter protease 1 (\Kako Chernicals

USA Inc) was d e d out by addïng a srnail volume to the protein solution to give a 150

to 1:lOO (wlw) pmtease: receptor ratio and the murtinr was incubateci at 37 O C for 6 8

hours. The &on was stopped by imrnediately W g the solution at -70 O C .

Cyanogen bromlde (CNBr) cleavage of the receptor protein was d e d out by

dissolving the protein in 200 pl of7W f o e c acid followed by the addition of CNBr at

100-fold m o k excess over the methionine residues of the protein- The mixture was kept

in the under nitmgen, at m m temperature for 24 hours, The sample was diluted 10-

fold with water and lyophilized

Hï?LC Pe~tide Isolation

The enzymatic or chernical digest was ac~*difïed with 10 pi of 10%

trifluornacetic acid ('EA) and then injected into a 2.1 mm x 25 cm Vydac Cl8

narrowbore 300A reverse phase HPLC column in a Waters 410 HPLC system (Waters)

with a detector at a Ieadnig wave length of 214 nm, The co1um.u was eluted at a flow rate

of 0-15 d m i n with a linear gradient h m 0-70% acetonitrile/O0065% TFA in 120

minutes, The fiactions were h z e n immedïateLy after coUection.

Identification of Labeled Fra~ments

Identification by rntibody to the B chah of insalin The m e n t of the receptor

that cross-Linked to the insulin photopmbe should contain a fiagrnent of the B chain of

insulin fier reduction - aikylation and enzyme digestion. To iden- this peptide whïch

spdcalIy cross-linked to the B chain of insulin, bovine insnlin B chain was used to

immunize a rabbit to generate a poIyclonal antiiody for an enzyme-linked

immunosorbant assay (ELISA) experiment to detect HPLC fiactions that contain B chah

or B chain-receptor hgments.

ELISA was established for detecihg peptides conaining the B chain of insulin.

To increase the reduced antigen absorption to the soiid phase of the multiwell plates due

to the acetic, organic solvent h m KPLC @a 2.0, up to 70% acetonittde), a covalent

cross-luiking plate, Reacti-Plates (Pierce) was used to nX the peptide on the plates.

Brïefly, 10 pl of the ftaction h m HPLC was added to the welï containhg 100 pl of PBS,

pH 7.6, and incubated at 4 OC over night. Then the plate was blocked with 3% BSA in

PBS for 60 minutes at rnom temperature. 25 pl ofanti-insulin B ch& antiiody at 1:1000

62

dilution was added and 100 fl o f f ((alkalie phosphatase&mnjugated second antibody

at 1:25,000 düution was used for the color reaction Varied concentrations of insulia B

chain were measured at the same time as the standard curve.

Identitication based on biotin-a~din intera&on To take advantage of the second

rnadcer of biotin on the BBpa-insulln, a sensitive biotimavidin assay method was

established to detect the fiaction contalliing the biotin-B chain signal. Similar sample

fixation was done for the ELISA. AAer blocking, A P - c o n t e d streptavidin wiis added

to bind the biotin-contained peptide onto the plates. The color reaction with the enzyme

then was carried out. Altematïvely, to obtain higher sensitivity, extra biotin was added to

bùid the remaimng binding sites of streptavidin, resuiting in more streptaviidin-enzymes

bound, which, in turn, led to an amplifid signal.

3. RESULTS

3.1. Recombinant HIR ExtraceMar Domain

AU inserts in the virus constructs were found to have the correct DNA sequences

by manmi sequencing (data not shown). The titer of purifid recombinant vinises was 5 x

8 9 10 to 1 x 10 as obtained by plaque assay. Within 24 hours of infection of Hi5 ceîls with

the recombinant virus, uisulin-binding activity was detectable in the culture media and

increased with time until Day 4 or 5 aAer infecton. Then the secreted protein in the

culture media degraded gradualiy when the ceils started to lyse (Fig. 2-4). While the

m u -

Hl- HIR H I b HIR

Fig. 24. Time course d ~ ~ i o n ofthe rccombiiunt atrrcdluiu domain of hIR (bIRar) (A), rnd Western blot (antia subunit of UR) of IüRa rad bnmuiplrnnW inniiin ribocpdor @IR) (B). The haerotetramaic hIR bris a molecuiar mus of450 kDa, rmd a mIacolar mass of 130 kDa fot iis asnbimit 'IhedinnntblRcrsbo~amolecolar~of-280 Wa,andamolearlarxuassofllO Wa fiir its a subunit. A s d amount of uncIcavcd recombinant ~icccptar (440 kDa) am be seen an the duced gel, which tcgreseats the uncleaved siugle chain containhg the a mbanit and the erdnctllurar domain of the p sub11111-t-

ot HIR

: of HlRa

-116 a subunit

4 7

49

4 p ' subunit

Fig. 24. Aftinity p- afexpmreâ tüRatnccUular domah with mti-hlR monoclonal antibody (MASI) columa. A) The purification profile manitami by radtradtcmctive tracer of *=I labeled bovine insalin- E x p d insulin receptor extraceilular domain was eluted with 2.5 M MgCI2. B) Purifiecl recombinant hiRexmceiluiar domain was d y z e d by SDS-PAGE underredacimg conditio11 The geIs were stalned either with silver or Coomassie blrie- Tmœ amotint ofimcleaved ap' single chah receptors and bovine albumin can be seen ni silver stained gel.

O 20 40 60 80 100

lime ( 0 min, O sec)

Fig. 2-8. Time caursc of photdyris. Radioactk IabeIed insuiin photoprobe was incubaîed with hIR in bïnding b-er in the da& at 4 4 for 20 hours, foiiowd by nporiirr to UV Iighî (100 Watts) for 5,10,15,20,40,60,80 and 100 se&, or witha 365 nm filter fbr 5, IO, 20,40,60, 80 and 100 minates- Higher cross-iinking eflilciency and lcss protein damage were obstrved with 365 nmfilter,

seaaed fUy procesd ectodomain of HIR accumdated in the culture media, many

mpmxmd precutsors accumulated in the piasma membrane (data not shown). The

secreîed recombinant receptor was mgniPd by human aur~immune anti-HIR antibody

and monoc10nai anti-HIR anti-es Vig. 2-5). More t h 90% of secmted protein was

shown to have a fbiiy processed a2Ss2 heterotetramer structure (two - 110 kDa a

subunits and two -30 kDa truncaîed $' submits), A smali amouut of protein was secreted

as non-cleaved (ap'h dimer (two -140 kDa af3' monomcrs) (Fig. 2-6). The yield of

expression was - 1 mgfiter of culture media as meesursd by immunoassay (data not

shown). The affjnïty p d e d receptor binds iasulin with a high afflnity @Cd = 1 x IO-'

M) which is oomparabie wîth that of wild-type human placenta IR (Fig. 2-1). This

receptor exhr'bits a linear Scatchad plot.

3.2. A S m d Labeled Peptide Fragment on the Insulia Binding Region

To optimirc photolysis 1 tested m i n t photolabeling conditions. With~ut the

filter, photolysis occ& very fast in seconds. Protein demage could be seen with

exposwes longer than 20 seconds. mth a narrow band optical nIter et 365 which

eliminates most short-wave UV light, the cross-lllikiag efficiency was i n c d 2-fold

and the protein darnages resulting mostly h m short wave W light, was reduced (Fig. 2-

8)-

In a preliminary sady, both the cleavable (rq-AZAP-insulin) and the

uncleavable (829-rq-MABI) photopmbes were used to characterize the pmteolyzed

peptide containhg the putative insulh binding site. In the case of the cleavable

photoprobe, k s u b B chah shouid k cleaved a£€er photolysis, and oniy radioactive

iodine left cross-- to the mxptor. A single radioactive bard containhg no attached

insulin B chah appead aRer dpeptidase digestÎ011, In the case of the other

photoprobes, shce since B chah has haso iodinabion sites, Te16 and T-26, bwo

radioactive hgments should be generaîed in the enzyme digests. A small 1-3 kDa

radioactive iabeled fhgment was gaierated by either trypsh or V8 m e n t of HIR

Iabeled with B29-Cq-MABI (Eig. 2-9). h the case of ELIR labeled with m-AZAP-

insulin trypsin digestion generafed a single small labeled peptide. Most of the digestion

o c c d in the fk t few ho- but wmplete digestion took 20 hom. Similar results were

obtained when using endopeptidase Lys-C digestion. CNBr trratment failed to

completely cleave the labeled receptor (Aatsi not shown).

Antibody against the B chia of insulin was shown to reco- with high affinity

the intact insulin B chain or its tryptic digest at pmole levels (Fig. 2-10). However, when

the same method was used to detect hrigments in HPLC Grattions, the sensitivity of

detection decreased several thousand fol& To obtain enough sensitivity to recognize the

peptide(s) ofinterest in HPLC fkactions, 1 cross-Linked the peptides in the HPLC fiactions

to ELISA plates instead of using the conventional coaîing method. The conventional

sample preparation method showed a dramatic drop in the semitivity of ELISA analysis

due to the very low pH (-pH 2) and high concentration of organic solvent (-30-70% of

acetonitile). The modified method wuld detect the peptide containing B chah hgments

in HPLC hctions (Fig. 2-10) or in digests of receptor labeled with B29-MABI (Fig. 2-

1 1) at the pmole level.

0 2 0 2 0 TTmm (hrs)

Fig. 2-9. Time course and products attrypsin digestion dB29-MABL or AZAP iabckd MR -cellular domain- Panels A d B show that trypsm digestion starts wïdiin an hom and is completed in 20 hours. Complete digestion generstes ladeied @pan& smriller tbrin 3 Id)a (A and C) wïîh both B29- MABI a d AZAP photopmbcs, niE 45% alLaline gel analpis shows that BW-MABI labeling genenites two radiolabeled bgments by aypsin digestion, SnSIng k m thepresence oftwo radioactive iodhe label- h g sites on the msPtm B chah One o f t k labelai ftrrgmats contaïus the patative insulln-bhdhg sité, Ih the case of AZAP photoprobe labehg, since radioactive iodirr labelcd on the cross-lnik gnnip instcad of the insulin B &ah, wbich is c i e a . after photolabebg, a single radolakled rccqtor hgpcnt is seen on the gel @)- Bovine insrilin and the iasulia B chab insulin wen Imded on the nght Iancs of the gel as a control (B),

Fig. 2-1 0. The sensithity of ELISA in detccting B chah fhgmcnt of bovine InniUn More (a and d) and 8ftœ HPLC Pnrlysis @, G 0 and f). Adramatically reduœd sensi* @, c) was obsaved undcr the acidic c œ ï ~ o n m tk HPLC ~~ when asmg regiilarpl&es- Using Reacti-Bind plates merce) restarrs the sensitivity &ELISA to pgkcU 1-1 (e, f), Synthetic B22-30 hgmm d the insulin B chain was used as s t d d ma, b, d and e. Trgptic digests of the insulin B chain containhg the f i a m dB1-21 and B22-30 ~~ wne d a s HPLC sa?npIe.

Fio. 2-1 2. Achromobe pcotcmc 1 digests dla5 1-BBp~Inmlia Wcd hIBoL A) HPLC B) An aliqiiot (10 ml) o f d hcticm was assa- by ELISA ushg a h h c pbosphatasmxmjugaod streptavidin, C) Raâïoadvityofeaeh~(0.5 ml) was de6ermmed Oae SbcptaViddin-positnrepeiiL a n d t w o r s d i & e ~ ~ ~ B a s e d a r t h e E L I S A r e s a l m , ~ - 74(mwherdimnA) aud 75 wem paoled and d y z c â again by HPLC.

pmok 0.2

Fig. 2-1 3 HPLC rndysia ofpooled tractions as iQiidilled h Eîgi 2-12 (A). A) HPLC profile obtainaî using a mobiîe phase dincrenthm tbt used to obtam the prome shown in Fis 2-12- B) An riliqaot (10 ml) of cach M o n was assayed by ELISA ushg altcaline phosphaîaseumjugated streptavidk The ~tre~tavi~pos i t i ivc M o n s showed very poor rtcovexy.

In the case of HLR labeled with BB~insuIin, 1 iiscd bot6 th antibody and thc

streptavidinmahod of detection. Radi08CfÉYity was also used inthe dcttction when

fadlfadloactive photoprobes were uscd, A typicai HPLC profile is show in Fig.2-12. Ibc

fiaction containing the peptiCde o f interest was nniner p&ed by HPLC under diffkrent

conditions, resulting in a great l o s of the peptide (Fig. 2-13). I was d l e to coiiect a

sunicient quantity ofthe peptide for amino acid seqyencing.

4. DISCUSSION

The bacuiovinis expression system @ES) bas ban succcssfiilly useâ to express

many fùnctional proteins &uckow and Summers, 1986). It is a very powerfid eukaryotic

protein expression system, and it is -or to bacterial, yeast and mammalian

expression systems for many reasons. Most recomb'i t pmteins fcom BES are soluble

and fully h c t i o d . hsect ceiis used in the protein expression many ofthe protein

modification, processing and transport systems present in higher eukaryotic ceiis that are

essential for fbll biological actîvity ofthe recombinant protein. Like 0 t h investigators, 1

was not able to express soluble intact HIR in M a or yeast (unpublished dm).

Mammalian cell systems, on the other han& usuaiïy express Iimited puantities of

recombinant protek. My prehinary study and the results h m other imrestigators

suggest that the extraceiïular domain of the HIR expssed h m BES main- full

binding aff?nity for its ligand @ad et al., 1990; Sissom and Ellis, 1989).

In this study, I have successfûlly used the baculovuUS system to overproduce the

truncated, soluble, secretory ectodomain of HIR which reEains fuli insulin-binding

afEity cornparrd to native placenta insuiin receptor (Fig. 2-8). Whik a dsf icmry

quantity (a few mgfiter) was s e d m the media with full fiaidon, a sigdîcant

amount of uucIeaved av precursor accumulated mside the ceU @ig. 2-9, Otha

investigators (Paul et ai., 1990; Sissom and Ellis, 1989) have previously reported the

expression of similar versions of the ectodomains of HIR in the bacuiovirus -*on

system, and mlmimalian expresion system (Cosgrove et aï., 1995). The molecular mas

of the a subunit expresd in the baculovinis system is d e r (1 10 B a versus 130 kDa

for the its native cotmterpert), likely due to d i f f i giycosylation dirruig the pst-

translation process @ad a al., 1990; Sissom and Ellis, 1989). Nevertheles, the

carbohydrae content of the receptor has been demoIlSfrZIted to have no effèct on the

ligand-receptor interaction (Caro et al., 1994).

S e v d investigators have reported that the sccraed HIR ectodomrrin pduced by

BES &'bits a hear Scatchard plot (one c h of hi&-afEnity site only) whereas the

native membrane HIR exhibits a curviiinear plot, suggesting one high-affinity and one

low-atFinity site. Interestingiy, the Scatchard plot obtained with the secmted HIR

ectodomain expressed in ' celis was curvilinear (Whittaker and Okamoto,

1988). This difference in the Iigand-binding behavior is not understood. It might d t

fiom the different pmcessing of the heterotetilamer in the d i f f i t expression systems.

AIternaîively, the relative abundance of the uncleaved prorecepfor produced by the insect

cell may e t the binding of a second iasulin m o l d e . Tt is hown that the af! half

receptor exhibits a linear Scatchard plot, but with low affinity for iasulùl (Sweet et al.,

1997). The fact th& only the heterotetrarner stnictute of the HIR can bind insulin with

high afnnty suggests that high afnnity hsuihbinding sites require both halves of the

receptor to participate. Insulin itselfhas no obvious intdnsic symmetry but must interact

with two identicaï a$-receptor halves for hi& affiaitp K i - 1 beiieve thet mat may

exisc identicai but more than one contact site in each a subunit of IR Hi@ afIinity

insului bi~lding Fepairrs contacting diBiirent sites on each half of the tecepfor. At low

physiologid insuijn concentrations, hmib can contact Ïts biading sites on both a

subunits for hi@-affinity binding- At higher concentrations of hsuiin, a second insulin

m o l d e can ody- contact some of the sites requïred for high afnnlty binding, -l&g to

Iow aff?nity biading. The fhct that the IR b i i one insulin with high efnmty and the

second with low affinity suggests that asymmetry may k induceci in the receptor by

insulin binding. It also suggests that one insulin moIecule biadmg to the recep&~ with

high affinity e i t k leads to a conforrnatioI181 change of the receptor, or simply one

insulin ocaipies the space dowing for high atnnity biiding making it dBicult to bind

the second insulin. The d i f f i t pst-transIational processes in the bacdovirus

expression system may lead to some c o d o d o n a l varhtion in the recombinant

receptor to allow either one insuiin molecuie oniy to bind or two insulin molecules to

bind with similar af£ïnity.

M t y labeling of the receptor with photoreactive insulin derivatives followed

by isolation of ~eceptor-inmlin fkagments has ailowed the identification of regions of the

a subunit as potential hormonerecepf~r contact sites. S e v d regions have beai

suggested by vaxhs investigators (Wedekind et ai., 1989; W b et al., 1989; Yip et ai.,

1988; Fabray et al., 1992). Most ofthe studies redted in relatively large hgments ( k m

14-55 kDa), making it diffIcuit to pinpoint accurately the contacting location. Resuits

obtained in the present study indicate tbat a very small -ent (-1-3 LDa) containai

the insuiin biidhg site, or part of the bhding site on the a subu& ResUlts obtained h m

HPLC -de mapphg based onthehighly d e detecthg metho& suggesteâ that it

may be possible to identifjr this hgment by it9 amino acid sequence if dficient

qyantities of the peptide could be obtained, ûn the other hand, d e r peptides are more

dScult to isolate and idenMy. West+m blots used by many investigators to daect their

larger receptor fragments, would be difncuit to use to isolate such a smd peptide for

sequencing. Reversephase HPLC adysis has been succediliy used to isoIate a s d

peptide &action h m otherpeptiCdesdes However. the low photoIabeIing efficlency makes it

difficuit to load enough protein digests on the column to yield a d c i e n t q d î y in the

Graction wntaininp the labeled fiagrnent Iae large percentage of non-productive, non-

labe1ed receptor also contri'butes to the di86cuity of purification. It will require a second

or third re-pudication of the HPLC -011s to obtain a pun hgment for amho acid

sequence anaiysis. The cuxrent amino acid sequencing method aiso requirts up to ten

times more to have reasonabIe d t s for a smaller peptide compared to larger

polypeptides. Since most photoaffkity probes have low cross-linking efficiency (cl-

IO%), either more starting material or a probe with higher efficiency is required to isolate

enough m e n t AIteniatively, a higher efficiency purification method or a more

sensitive amino acïd sequencing method n&ds to be developad The new BBPa-insuiin

photoprobe with bïgher cross-linlo'ng efficiency has shed new light on this project

aithough we failed to achieve the efficiency claimed by the onginai investigators @ee et

al., 1993). Unfortwiately, the new BBPa-insuiin photoprobe we obtained was only

dEcient for developing the method01ogy. It is clear that more wodr would need to be

done to overcome the obstacles in this biochemical approach,

QUATERNARY 3D STRUCTURE OF THE INSULIN- INSULIN RECEPTOR COMPLEX DETERMINED BY

SCANMNG TRANSMTSSTON ELECTRON MICROSCOPE: STRUCTURAL LOCALIZATION OF

INSULIN-BINDING SITE ON THE HIR

1. INTRODUCTION

Rotein-protein intcractons et the molecular Ievel are largely reletcd to th&

three-dimensional (3-D) structures Elucidation of 3-D structures of proteins at high

resolution is cruciai to the understanding of many fimdamental fimctions of

macromolecules and macfomoldar complexes. Besides its significance in

understanding the chemisiry of Mie, the d n e d stmctme o f a protein also provides highly

valuable infîodon for medical appkations, such as new drug design (Gisanda a al.,

The most commonly used methods for solWig macromolecuiar stnicturr are x-ray

crystallography, nuclear magnetic resomnce (NMR) spectroscopy and electron

microscopy (EM). While the 3-D stmcturt of a great number of proteins has been

determineci by x-ray crystallography, if they crystaljize, and by NMR spectroscopy, if

they are sdicientiy small, the vast majority of biologïcai 1118~10molecuies have not been

crystallized or are too large for these appmaches. X-ray crystailography has been

successfÛUy employed to determine 3-D structure at a nsohrtion of 1.5 - 3 k However,

due to the di86culty in growing crystals of proteins suitable for x-ray crysnillography,

most proteins whose 3-D stnwtme have been sohred by this techaique are d e r than

100 kDa Using NMR spectroscopy, investigaiors can solve the struchne of a protcin at

atomic resolution but the molecde must be wen d e r , around 25 - 30 kDa, and hlghly

purined. Howevr, electron microscopy, combined with cornputer pmcessing algorithms,

has successfirlly salved the 3-D structure of macromolecules with molecdar weight Grom

several to several thousand kDa at A to nm level (ûttensmeyer, 1982; Schrag et al., 1989;

Chiu and Schmid, 1997; Rosenbag et al., 1997). This technique offm a powedul hîgh-

resolution tool in structural biology, and Ïs especially useful for large protein molecules

that cannot be studied by either x-ray crystallography or NMR method, The EM-based

3D reconstruction enabIes investigators to solve the molecular architecture of proteins,

and to detennine the overali size and shape of th& constituent protein subunits with

atomic details. In addition, this technique ais0 allows researchers to investigate distinct

fbctional States by monitoring conformational changes or structural reorganizations

(Whittaker et al., 1995; Jontes et al., 1995; Unwin N, 1995, 1998)- Such studies have

provided insight into the structure and fünction of many proteins, including virus

structure and mocphology (Shaw et al., 1993; Zhou et al., 1994). localization of

individual protein components (Zhou et al, 1995), identification of cellular receptors

(Olson et al., 1993) or monoclonal antibody binding sites (Wang et al., 1994; Chiu and

Smith, 1994) and protein-protein interactions (8eckmann et al., 1997).

The electron microscope provides much higher magnincation than the iight

microscope by the combination of magnetic lenses and short wavelength electmns at high

voltage. However, compared to the case in materiai science, biological specimens are

more diffi.cult to image in the electron microscope because of their low contrast and their

sensitivity to radiation. Much effort has been made in the Iast few decades to make EM

more useful to biology. High contrast heavy atoms (osmium, lead, uranium, etc) are

uçuaILy required to stain or coat the bioIogical sampfes in a controlled way to make them

become "visible". However, the object being imaged is acbially an "ertif&ct'' cornposed of

the distn'bution of heavy atoms suuoming the features of hiterest.

Scannin~EIecfrOn M ~ ~ O S C O D ~ One of the collzmody used eiectron

microscopes in biology is the scanning electron microscope (SEM). SEM images arz

obtained by using a very narrow electron beam to scan over the surface of the specimens

and by mapping the signal of emitted secondary efectrons (or of back-scaîtered electrons)

into the corresponding pixel of the recording/display image (Fig. 3-1). Although SEM

provides much higher resolution and a much greater depth of field than the light

microscope, biological specimens usuaiiy need to be coated with a conducting layer of

heavy atoms to avoid charging under the scanning electron beam. This approach limits

the detailed interpretation of smali structures and provides only the surfie feature of the

specimen. In addition, although improvernents are being made, the resolution commody

at about 20 nm is also not high enough to reveal detail in individuai protein structures,

which usudy requires resolution at nrn or sub-nm level. SEM has been mostly used in

the studies of organ or tissue structure in biomedical research.

Transmissio~ Etectron Microsco~e and Scanninn Transmission Electron

Microsco~e Compared with the SEM, the transmission electron microscope (TEM) is a

more powerfiil tool available for the investigation of the microstructure of bio-

macromolecules (Fig. 3-1)- The TEM is somewhat similar to the SEM in that both use a

hi&-energy beam of electrons and a series of magnetic lensa to control the beam.

However, the electron beam voltage is much higher (LOO KeV or pater; shorter

wavelength) in the TEM, and the high energy beam passing thmugh the specimen is used

Fig. 3-1. Dienmt ckctmn miemscqm Prcd ia biomedid rtudier, Tbe samning electron microscope (SEM) is maïdy used to saidy the strrictme oforgan or tissue at relative low tesolution The transmission electron ~ÏQOSCOP~ (TEW is usai to investigate the micro- structure ofbioniolecriles. The scanning aansmissioa elatrm mïczos~ope (STEM) camb' ï the ahmages ofboth SEM and TEM and is more efficient in the use of scat&& elecbons, and CO- can reduce specimcn damage STEM has beai uscd fbr high resolution 3D reconsbniction of profein ~lcculcs.

to form a direct image. A magoi6ication of about 500,000 x is typical and the resolution

can srpproach L mn or bet-

Within the last few years, tremendous pmgress in EM technology and computer

image processihg have been made. The introductfon of low-dose and cryo-presemation

technipes into electron microscopy has opened a new era for 3-D structure

determination of biomacromolecules, Cryo-fixation, ice-embedding or fkze-drying the

protein sample in its physiological b&i7 permit d k t Miaging of the protein itseif m a

near-physiological state. It has been widely accepted that these methods reduce

adsorption artifacts and reproduce structural details more accurately and faithfdly- Most

importantly9 in addition to mapping out the overaiL size and shape ofbiomacrornolecules,

cryo-transmission electron microsmpy, and cryo-scanning transmission electron

microscopy (STEM) enables investigators to look "uiside" biomacromolecules and is

beginning to image theV secondary structural elements, or even thw atomic detaüs. The

3-D EM structural information can, under some conditions, be obtained at a resolution of

as good as severai hgstroms9 sunilar to the resolution in X-ray crystallography, to

directly reveal the a-hek structural details of snme proteins @ü.hlbrandt et al., 1994;

Cheng et al., 1997; Walz et al., 1997). Conseq~ently~ more researchers are beginning to

use electron cryo-microscopy for structural and fiuictional studies of bÏomacromoIecuieses

The 3-D image reconstruction of EM has become a new tool and a very powerfùi

approach in biomedical research.

The uniqueness of the STEM is its ability to visualize individual biological

molecules directly without staining, fixing or shad~wing~ Ih the STEM, image acquisition

is digitized as a small electron probe (3 A beam size in the HB601UX STEM), produced

by a cold-field emiemissÎon gun, a condenser and objective lem, sans in raster fasbion

across the specUnen* The microscope operates in a dark-field mode with high efficiency

amular detectors coilecting virtuaily aU the scattered electrons. The high efficiency of

STEM pennits da&-field images to be obtained d g Iower radiation doses (ten to

hundreds times less than by dark-field imaging in 'ïEl& resuitïng in less damage to

protein structures)- The number of scattered electrons received by the detector is directiy

proportional to the mass thickness ofthe scanned molecules, Thus the molecular mass of

the individual molecule c m be caIcuIated by subtracting the background and using a

scattering cross-section or cali'bration standard- Another unique feature of STEM is that

individual heavy atoms can be visualized and thus specinc sites on biologicd molecules

can be mapped @aidielcl., 1992). Recently, a monofimctional reactive gold cluster (1.4

nm in size), Nanogold, has been successfùliy used to localize specinc fùnctionai sites

with STEM in several proteins (Boisset et al-, 1992; Wilkinson et al., 1994). With low-

dose electron beam scanning, high contrast dark-field imaging and digitally recordhg

features, STEM can collect hi&-resolution images fiom a specirnen as s m d as a single

heavy atom to proteins larger than a thousand kDa. Combined with novel computer

processuig techniques developed for the microscope, STEM has beeo successfüily used

to reconstruct 3-D structures of several proteins, such as SRP54 and Klenow fitapent of

DNA polymerase 1 (Farrow and Ottemmeyer, 1992; 1993; Czarnota et al., 1994) at a

resolution of 12 - 20 A.

1.1. Electron Microscopie 3-D ReeoutrPction Techniques

3-D electron cryomicroscopy of biological macromolecule consists of several

l!lllh 111 STEM El.cboir B..m

A IIR

Specimen Pmparation &i Digital lmage Collection

Cornputer Image Processing

30 structure visualkation and intwpretation

Fig. 3-2. An ovemew of STEM 3D reconstruction Single particles ofthe insulin/insulin receptor cornplex, represented as a han4 were subjected to STEU, Images were collected and analyzed and processed by cornputer involving (A) background subdon, (8) low pass filtering, and (C) alignment F m e r processed images were back-pmjected and displayed as a 3D structure.

major steps: specimen pqmdon;. image coiiectio11, nomielization and pn>assiag; and

3-D reconstnrtion and -*on @ig- 3-2.). Briefly, msny large images of di-

single molecules are coiiected and placed in a cornputer file with himdrods or thousamis

of individual particles. The individuai images then undergo twodbensional(2D) image

dysis , including variauce nonnalization, backgn,und subtraction, low band pas

filtering, as well as statisticai d y s i s . The pmcesed 2.D images thm are d y for 3D

image processingins The backgmumi has been completely removed d&g ?bis pn>cess. A

sinogram, a stack of one-dimensional projections of the 2-D iinage rotated 3609 amund

its Y axis, of each singîe particle is dculated and is used to reiate the orientation of one

image to any other. A f k the relative angular orientation of tach image is calCUI8ted, all

images are back-ected to produce the final 3-D reconstru~*on.

A number of recent developments in wmputational techniqys and experimentai

approaches have greatly improved this pnness and for speciLai cases, increased the

resolution of the electron microscopy reconstruction h m the early 30 A (Dryden et al,

1993) to a few ~ngstroms (Bottcher et al., 1997; TIUS a al., 1997). One of th

approaches, which greatly increases the structure resolution in 3-D feconstruction, is the

use of 2-D electron crystallogmphy. In Tii methd each image contains thousands of

identical units in identical orientations, wbich can be accinately and easily averagd As a

consequence, structurai analysis by electmn cryomicroscopy of 2-D crystais can extend

the resolution to th atomic level. For example, by using this technique, the structurai

resolution of bacteriorhodopsin with a redution approaching 3.5 A peLLlZits visualization

of a-helical bundles in this membrane protein @enderson et al., 1990). Anothet

technique, hown as the random c o n i d tilt method, is able to use samples in which

molecules assmne one, or a few. preferred orienmCons so that-a hi* resoIubLon cau be

attained @&macher et al., 1992; Serysheva et al., 1995). Ho-, tb technicpe is

Iimited by the reqpimnent that the specimen adopt recognizable p r e f d on-ons

withrespectto the supportnIm.

NoveL rnethods *ch do not nquirr crystauization, shuchaal symmetry or

preferred orientations have ban developed and iinplemented as weil, and used in om

work (van Heel, 1987; Ottensmeyer and Farrow, 1992; F-w and Ottaismeyer, 1993;

ûttensmeyer et al, 1994). This approach was designed for random orientations of non-

symmetric molecdes, and maLes use of qmemîon mathematics to sdve the stnicbat of

biomacromoIecules even h m noisy images of single particles. An advantage of tbis

method is that it pemiits stnietural detenninations of macnrmolecuIes under many

different conditions incIuding diffgicnt ionic envbnments, molecule s b or pst-

translational modifications, wbich ovacome many limits in crystallization or pre-

requirement in sample p r e p a d o ~ ~ Recently, a signal stqyence binding protein has been

successfiilly resolved to its 3-D at 12 A res01irtion with this technique (Czarnota

et al., 1994)-

1.2. Strilcturaï Determination of IRand Loaliutbn of riwniin Bindîng Site

Insuiin binding to the ce11 siiraice IR activaies its in%c cytoplasdc tyrosine

kinase through the autophosphorylation of specïfïc tyrosine midues, initiatUig the

intraceUular signal transàuction cascade, wbich is essential to insulin actions such as

glucose homeosmis, protein syntbesis, p w t b a d development (Taylor, 1991)-

However, IR IR not been crystallized, probably due in pazt to its molecular ma98 of 450

88

kDa, M g a membrane protein, eild being highiy glywsylaîed. A f k more than 25 yëars

of intensive stuâies by many Investi*gators, the quatcmey sinidure of IR and the

stmcaaal basis for the mechemsm of IR activadion by b s u h binding remains u n l c l M a It

is known that insulin binds to the extrace11ular a subunit of the whole receptor, and also

with unaltered afflnity to the isolated ectodomain (Schaefer et al., 1990). Several large

regions of the ectodomain have been impLicafPA in binding hdh, but a more detailed

description of the bindhg site is not yet maiiab1e becarise the crysîai structure ofneither

IR nor its ectodomain is biown. On the other hend, for an anaiogous feceptor, IGF-IR,

the crystai structure of a m e n t containhg the first three domains &l-Cy~-ric~L2)

involved in ligand binding has been solved (Garrett et al., 1998). IGF-IR and IR are

closely rdated members of the tyrosine kinase superfamiiy and highly homologous in

their primary stmctwe (ülirïch et al., 1986). The structure of the ment, which Iacks

Ligand-binding activity, shows that the tbree domains form a centrai notch sdïcient for

ligand binding. In addition, the structure of the hgment of the intracellular tyrosine

kinase domain of IR has been detemUned by x-ray crydography, both in the

unphosphoryiated and phosphorylated state (Rubbard et ai., 1994; Hubbard, 1997).

Since the IR is a mode1 protein in signal transduction system, and is involved in

basic metabolism, in development and in many human diseases, solving L structure is

very important, Since detesminiig IR by x-ray crystalîogxaphy or by NMR analysis hes

proven ciifficuit, electron cryomicroscopy seems to be a possible alternative appmach to

solve this problem. As mentioned before, the large molecular s h and the high degree of

gIycosyIaîion of IR should not present a problem in STEM d y s i s . Another advantage

of STEM is the visualization of heavy metal atoms, such as the 70 atom gold cluster,

Nanogold This havy metaI marlrer can be risad to map specinc hctionai sites on

protein moIecules shce it will appear microscopidy as siilgle "spots" of about 1.4 mn

in diameter m e I d and Furuya, 1992). If insulin is labeled with NanogoId, this iiisuliri

derivative can then be used to Iocate the hsu&binâing site on the IR with electron

cryomicroscopy. In addition, available structural information on the TK domain of IR

and the L1-CM2 domain of IGF 1 receptor (Habbard et ai., 1994; Garrett et al., 1997;

Baron et al., 1992) couid be helpfirl in analyzÏng the results obtained fiom STEM sndy

ofthe whole recepfOr.

In this Chapter, 1 descfl'be the result h m experiments d e d outto daamùie the

3-D qusteniary structure ofthe human insuün receptor by 3-D reconstruction fFom STEM

images of singie molemles. Nanogold-bovine insuiin as an EM market was pqared and

used to localize the insulin-binding site in the structure of the intact insulin-insulin

receptor cornplex. Results describeci in this Chapter have been published in abbreviated

form in Science (Lu0 et al., 1999).

2.1. Biological Materials

Preparation of Nanoeold Bode Insuiin =BE)

Nanogold @Janopmbes, Stony Brook, NY) is a newly developed gold duster

label, prepared by using a discrete gold cornpoumi rather thaii the traditional coiloid

Nanogold is an exîremely U13iform 1.4 nm diameter gold clusta particle wntaining

appmximately 70 gold atoms with a molecular mass of 15 kDa This heavy metaï particle

BOC 81 DBI NO NO-DBI NG-BI

can be easily sccn as a bright baU shape spot unàer the electmn at a

magnification firtor higher than 200.000 r The sulfo-whydrov-do (NEIS)

NmogoId vig.3-3a) used to label bovine insulin has a monocreactive p u p which is able

to cross-iink to an accessible reacthe amine group on a protein,

An iosuün moIecule contams three reactive amine groups, one each at -*due,

Al, B I and B29. Since the amino termiripl of the B chain (BI) is known not to be

involveci in binding to the receptor (Nakagaw8 and Taga, 1986, 1987. 1993; Schaffer,

1994) the amino group of PheI was derivatïzed with a gold cluster. This was done using

bis-(t-Bot)-bovine irisului @BI) in which the d o group of Al and B29 are protected.

DBI was prepared in Dr. Yip's laboratory (Yetmg et al., 1980). One mg of DBI (-150

nmole) and 30 nmole of mono-NHS-Nanogold were dissolved in 200 pi of f-

dimethylacetarnide @MA) containing 2 pl di-iso-propylethylamine PIPEA), pH 7.6-

nie mixture was vigorously mixed at room temperatme for 60 minutes thm dned in a

SpeedVac (Savant Instruments). The pellet obtained was dissolved in 20 pl of

tci£iuoroacetic a d , kept et m m taperahue for 5 minutes, and then dried in the

SpeedVac. The peliet was washed twïce with 100 pi of 1 M acetk acid, resuspended in

120 pl of 1 M acetic acid and cbmatographed on a Bi&l P-10 column (1.7 x 25 cm)

in 1 M acetic ad. Fractions of 0.5 ml were collected, and their absorbency at 280 n m

was measrned. An aliquot of each M o n was subjected to electrophonsis in 10-200h

SDS-tricine gel in tricine/glycine b u f k (pH 8.8). The fiactions containhg NGBL (Fig. 3-

3b) wem pooled and rechromatographed on the Bio-Gel P-10 colimui. The purifiecl

NGBI was storeci at a concentration of 0.5 pmoYml ai -700 C.

Preyrition of Nia0~01d-ib~~lWiiimlh ~ C C C D ~ ~ CO-

Insulin receptor protem @IR) was solubilized h m human placenta membranes

and pmffied by afnmty cinomatography on an iiwlin co11m.n followed by M e r FPLC

purification on a Sephacryl S-200 HR column (1.6 x 60 cm, Phannacïa FPLC set and

column) as d e s c n i in Chapter 2. The p d e d HIR was immedïately quick fiozen in

liquid nitrogen in aliquots of 20 pl at a protein concentration of 20 pg/jd and kept at -

70°C mtil needd For gold labehg, EIIR was incubated at 4OC oveniight in 20 mM

=ES buffa @H 7.5) with NGBI (final concentration of - 0.5 x 1 0 ~ M) at a mdar

ratio of insulin= of - 10:l- Free NGBI was removed h m the mixture by

d ~ t r a t i o n through a microfilter with a cut-off of 300 kDa (Sigma) aAer incubation-

The NGBI-hIR cornplex was then diluted to 7.5 pg of receptor proteintml with 20 mM

HEPES buffer, pH 7.5, prior to loading on the microscope grid support. The presence of

one NGBI per one hlR in the majorïty of the complex particles was confirmeci by

preliminary TEM and STEM imaging before loading the specimen for the final STEM

analysis.

2.2. Electron Microscopy and Image Anaiysis

Pre~aration of s~ecimen for STEM

Copper grids (300 mesh) coated with a holey plastic nIm (pore size 5-10 p)

overlaid with a carbon film 23 A thick were used to support the protek in both TEM and

STEM analysis. The thin carbon film was prepared by high vacuum electron gun

evaporation of carbon onto h h l y cleaved mica The thin carbon nIm was then floated

on a dish of double distilleci, de-ionized water that had been passed through a 0.2 p m

mUïpore £iI= The tfiin carbon film wis picked up with the piastic coated gnd and th

grid was air*-ed.

To determine the optimal concentration of protein for STEM analysis, 20,10,7.5,

5, and 2.5 nfil of HIRdiluted with 20 mM HEPES, pH 7.5 b e , were testad in

902 TEM using electmn energy l o s dadceld condition at the magnifïcation fhctor of

20,000 r The specimen (5 pl) was injected into 5 pl ofdilution M e r preloaded on the

carbon film of the grid. The specimen was washed with 3 drops of 20 mM, pH 7.5

HEPES buffg and 1 drop o f 10 mM ammonia acetaie bu@- eH7.5). For purposes of

concentration optimizaton, the grid was Ieft to air dry. The concentration of75 ng/ jd of

HIR was then chosen for the specimen concentration for STEM study.

For STEM anaiysis, the same sample preparation was perfomed, except that a&r

washing, the grid was draineci by wicking with Whatmam No. I £ilter paper to luive a

very thin layer of solution, as o b d with an opt id microscope. The sample grid was

immediately quick-hzen by plungïng the Md into liquid ethane at -150" C. Quick-

fÏeezing to liqud nitmgen temperature assud immobilization of the molecules in a solid

glas-like environment similar to the aqueous state. The hzen specimen was trmderred

at liqyid nitrogen temperatme into the STEM (Vacuum Generators, Mode1 HB601UX).

The specimen was fieeze-dried within the cold-stage holder of the STEM at -140 OC.

Freezedrybg at this temperatme resulted in a contrast-rich specimen that has not been

distoaed structuraUy by phase bomdary forces during dehydration.

Imane acauisition and anriysig

mes in a 480 x 480 pixel format were acqpbd in the STEM with the

specimen at -lSOS=, using culd-field anission at en acceleraîing voltage of 100 kV, a

dose of 6e/A2, and a magnïfication of 252,ûûû x, conespondnig to a pixel size of 65 k

The beam size was 3 A per pixel. Both melastic and elastk antlular dadc field s i w s

were detected and dïgitally recordeci Smidtaneously- The sharp Nanogold signal was

used to adjustthe stigmatism and focus on the images.

The paired electron images acquired h m the eIastic and inelastic detectors were

combined to increase the signal-to-noise ratio two-fold compared to the elastic signal

alone. To process molecular images: singie particles were ùiteractively selected in 64 x

64 pixel windows using the program WEB (Wadsworth Laboratones, Albany, NY). In

preparation for orientation determination the selected images were low-pass filteteci to 11

A using a Gaussian filter in the program SPIDER (\Kadsworth Laboratories). The

molecular mass was calculated from the seIected particle images in relation ta the mass of

the 23 A thick carbon support with a carbon density of 2.0 g/&. The particles had a

Gaussian mass distribution with a modal mass of 570 kDa, which inchded the mass of

480 kDa for HIR and NGBI plus the weight of an estimated 150 Triton X-100 molecules.

To detennine the molecde bomdary, particle images were CCgrown7' fkm a center high

density point in expanding contiguous contour levels until a global cut-off was reached

that corresponded to contiguous structures with the average mass. 704 Particles with

apparent structural integnty, appropnate relative molecuiar mass and showing the

presence of the high-density Nanogold spots were selected for M e r processing.

2.3. Tbree Dimensional Reconstruction

Iterative Ouaternaw-assisted Aneolar determination

Three-dimensionai teconstructions were. wmputed h m the mes of randomly

oriented RIR/NGBI: cornplex particles ushg Iteratbe Quatemary-assisted Angufar

Deteminafion VQAD) method @amw and Ottensmeyer, 1992; 1993a; 1993b; Czarnota

et al., 1994; 1997) based on skogram correlation and the common axk theorem (van

Heel, 1987; Crowther et al., 1970; Crowthe~~ 1971).

The IQAD approach to determinïng macromolecular structure permits the

determination of the tbree-di.-ol structure of a biological macromolede by

cdculating a poste no^ the anguiar relationship of a biological macn>molecule in a

particular image with respect to many other images of an identical macromolecule at

different random orientations. Briefly, in Fourier space any 2D Fourier transforms

(fiequency space representations) of corresponnding 2D projections of a 3D density

distribution will be centrai sections of the 3D Fourier transfoun of the 3D density

distniution, and thus will share a cornmon line of intersection in 3D Fourier space. By

calculating the relative position of common lines to one another in a 3D coordinate

systern, the relative orientations of the images can be determine& The addition of IQAD

processing, sinograms, sinogram correlation fûnctions and qyaternion mathematics to

o p e e the fitting togeheer makes it possible to use even sets of noisy image to obtain

the optimal orientation arnong them of any individual image @arrow and Onemneyer,

1992, 1993). 3D reconstnicctions then are performed by fI1tered back-projection using an

angular distniution-dependent filter. Calculations were carrieci out on an SGI Indigo

work station (Silicon Graphks hc.)-

Resolution measurements and results dis~tav

Mea~u~ements of resoIutïon for ~ns t ruc t ions were dculated by cornputmg 3D

Po- transforms for two independent n x o ~ * o n s and h m a set of haif of the

images. From these 3D transfonns the average differenca in phase between them is

calculated in radiai shelIs h m the center of each transfonn outwards. This phase

differences related to the correlation of StnicturaL detail at each shelî. Since the radius of

the sheiî corresponds to a resolution level (rager radius, better resolution) the loss of

correlation at a given radius mdicates the resolution E t -

The program IRIS EXPLORER 2-0 (Silicon Graphics hc) was used to display the

3D reconstructions at a chosen intensity-based contour levei, a threshold which

corresponds to the calculated volume of a 450 B a protein, assuming a protein density of -

1.3~/crn~. To show domah relationships and structural b, the collStNctions were

displayed with intermediate densities 5% to 10% higher than the average density of the

fùll volume.

Crystallographic modd or co-oduiates of LI-Cys-rich-L2 domains of the IGF-IR

(Garrett, et al., 1998), the tyrosine kinase domain of IR mubbard, et al., 1994, Hubbard,

1997; PDB: L I E X , 1IRp) and human nbronectin III repeats domain (Baron et al., 1992;

PDB: I W ) were fined into the reconstruction volume of the insulin-HIR complex by

modeling the electron density of the x-ray crystallographic structures into the volume of the

assignecl domains of the 3D remntruction using program INSIGHT II (Molecular

Simultations Inc.). Since the CO-ordinates of the first three domains of the IGF-1R were

not available, a cornputer draw 3D mode1 of approximate cylinders based on the x-ray

structure idormation in the publication of Garrett et al. (1998) was used instead.

3. RESULTS

3.1. Specimen Preparatioa for ElQfffOn Mfcrooropy

The purity of the receptor protein @IR) was found to be better than 95% as detennined

by sodium dodecyL solfate polyacryIamïde gel electrophoresis (Fig. 3-4). The punfied

HIR showed the same binding a£hity as placental membrane IR for insulin. (Fig. 3-4).

In the preparation of nanogold-bovine insullli, we carried out the reaction in organic

solvent instead of aqueous solvent as suggested by the manufacturerturer Our method

significantly increased the efficiency of derïvatization to greater than 70% as compared to

about 15% in aqueous condition (data not shown). The derivatkation and purification

processes wae monitored by SDS-PAGE and acid-urea gel electrophoresis (Fig. 3-5).

The purifÏed NG-BI was found to be more than 95% pure and to have a molecular mass

of 19796 Da by manix-assisted laser desoxption/iozation time-of-fîight (MALDI-TOF)

mass spectrometry Pig. 3-9, a mass consistent with one insulin molecule having been

derivatized with one Nanogold cluster. Compared with native bovine insulin, NG-BI

showed only a slight decrease in binding affinity to human placental membrane insulin

receptor (Fig. 3-5). As a compact particle containhg about 70 gold atoms surromded by

an organic matrk, Nanogold was visible as a bEght ball shape spot in the STEM at a

magnincation higher than 200,000 x. The purifieci NGBI showed no visible damage in

the STEM at 2,000,000 x (Fig. 3-6). The insulin signal was too weak to display when

compared to Nanogold at the same time.

3.2. Image Processing of NGBI-HIR Cornplex

A SDS-PAGE of HIR

F ig. 3 4 Purification of hunirto placeataol insulin nxeptor @IR)* hIR protein was pmi- fied by aflFiniîy chromatography using an insulin-AIE-gel column, foiiowed by gel filtration A) SDS-PAGE of the pmified hIR, stained with Coomassie blue- Lam 1 shows a band corre- spondmg to the unredusedhIR of450 kDa- Lane2 shows two stained bands conespondkg

obtained showthai there was is no detet&abk diffei.ence in me binding &ty for bovine insu- ün between the purifieci hIR and thehIR in placenta membrane (HPPM hIR)-

Fig. 3 4 Purificatfan aad chmtccizatkm of bdll~gdd imuün (NGBr). A) Gel-fiIîratlon sepdm of the reaction mixture- Solid bars represent the OB- OfNGBL and fb NG, and open bars reptesent the OD- of i k inSnlin, B) SDS-PAGE in 10120% tricine gradient gel ofthe reactïon mixtint and hctions obtained in (A). Tbe gei was stained with Coomassie Blue, The staiued band seen in fhction 1 L shows that NGBI can be partdiy purineci fiom the reaction mixîure. C) MALDI-TOFF ana@s established that the NGBI obtaiiëed h m repeat geF nIiraîion was more than 90% pure, rmd that the mass of the NGBI was cansisteat with a stoichionietry of one Nanogold per one Uisulin molecule- D) Receptor- binding assay s h o d that NGBI binds to hIR wiîh a siightly redlKXd af6nity compared to nonderiva- tized nisalin.

Fig. 3 4 STEM images of puriîied NGBI at a msi&nification of2,000,000 X ( i i r t ) . The brïght m d images seen are signals h m Nanogold, Signals h m insplin wodd be very weak compared m*th Nanogold There is no obvious damage of Nanogold particfes as evident i2om their weii-round sphericai .chape.

To increase the si* to noise --O for a given dose, the simuitaneously recordai elastic

and inelastic detector signais of the STEM were combind A representative unprocesseâ

STEM datk-field image containhg a few NGBI-HIR complexes is shown in Fig. 3-7A

As indicated by arrows, obvious intense sigaals can be seen h m the Nanogold marka.

Randody orientated complex parficles have an average size of about 15 nm. Most of the

complexes exhibit one brïght spot (or two in less than 10% of the complexes) on the

particles, that can be seen directly or can be visualized by varying the threshold on the

displayed intensity. Figures 3-7B and 3-7C show that brÏght NGBI locations are even

more strikùig in the high-density threshold representations of the extracteci particles after

low pass filtering. When two NGBI particles are detected, they are in close proximity to

each other (Fîg. 3-7B, lower two iinages),

Figure 3-8 shows a group of selected complexes extracted into a circular rnask

h m the raw images. Figures 3-9 and 3-10 show two m e r processing steps for the

same group of particles: low pass filtering to 11 A using a Gaussian filter, and molecular

boundary determination. The latter images are ready for 3D image processing çuch as

orientation deteunination.

The relative molecuiar weights for approximately 1,600 particles, ushg the 23 A

carbon support film as a m a s standard, are shown in the histogram in Fig. 3-1 1. Only the

particles having a calculated molecular weight no greater than a difference of 18 % fiom

the average were chosen for reconstruction. The calculated average mode1 mass is about

570 kDa, which ïncludes the mass of 480 kDa for AIR and NGBI plus the weight for an

estimated 150 Triton X-100 molecules.

Fig. 3-7. STEM dark6ehï images of human insuün nccpttor bound to Nanogdd insulin @RNGBI)- A) A field of view showing severaï complexes- Anowheads point to the Nanogold markers, Scale bar = 20 nm- B) IRNGBI images extracted fbm Mage fields, low-pss filterd to 1.0 nm (lefi oolumn), and obtaiaed at high daisi*ty thrrshold (nght cd-) showing one or two Nanogold matkers indicating the binding of one or two Naaogold-insulin (Luo et al. Science 285:1077,1999),

Fig. 3-9. Low band-pasr nItercd images. The sime images as in Fig. 3-8 were low-pass fil- tend to 11 A ushg a Gussian filter and background was subs~raded to increase bu signal-to- noise ratio, Scale bar = 20 ma,

FIg. 34 0. Final images used for 3D recoustrucîion. To finthet r&e the images for 3D reconstruction, the ssme images as in Fig, 3-9 wece processeci using "grown" software to detennine the molecuie mass center and boundaqundarv Various shapes of the images were obtaMed which repre- sent the same molecule king vie& in different on-ans, The location of NGBI is seen as a bnghter spot on the image- After tbïs refinement, images were ready for 3D image processlng. Scale bar = 20 nm,

S.

mîaüve Molecular MISS

Fig. 3-1 1. Histogram of crilcuïatcâ mass for 1,625 coilectcâ images. llv nlrtive molecalar weights werc calcuiaaed using the canboa supportmg EiIm as a mas standard. The partides in the shndad area reptesent the ones having a calcalated rnoIccuk weight witbin 18% of the average. 704 IWNGBI particies k m the shaâedarea were chosen fbr reconstruction-

Fig. 3-13. 3D reconstruction of the IR/NGBI complu (surhce repfesentation). A) Density threshold representing the tutal expexted volume for the complex (AL); intermediate density tbreshold, unsymme- trized (A2); high density threshold of (A2) showing only the Nawgold label (A3). Circles indiate loca- tion of the gold marker within the ~ ~ t i o n s . Resolution was 20 A by phase residual analysis. B) Reconstruction with two-fold symmetry shown at -70% of full volume, indicating relationship and con- wctivity of structurai domab. Labels, for only one af3 monomer, d e r fo biochemical domallis. Arrow- head indicates proposed plane of ceii membrane. LI, C-R, L2 = Ll-Cysteine-rich-L2 domains; CD =mm nechg domain; Fnl, Fn2 = nbronectin Hi repeats 1 and 2; TK = tyrosine kinase; TM = trammembrane domain (Luo, et al. Science 285:1077, 1999).

The rcletive orientations of the selected m o l d e mes of NGBYEW aim

O#ensmeya, 1993). They are plotted in Hg. 3-12, iadicating a -y rdom didriibuaon

of angles for these images with some tendency t o m a p r e f d orientation- Such

tendencies away h m riBILdOmness in orientation are compematd prier to 3D

r e c o d o n by orientation dcpendcnt weighting ofthe images.

The 3D recomtruction ofthe NGBI/HIR wmplex is shown in Figure 3-13k The

shucnne at the fWy expeskd volume, shown in Figure 3-13A (top panel), is compact

and giobular in overall shape. The domain-like fcatines of the structiin are shown in

Figure 3-13B at intermediaie densîty thresholds-

33. Location of the hnlCn-Binding Region

The location of NGBI on îhe recoLiStNCtion was detcnnineed by increasing the

density tbreshold without imposiag symmetzy (Fig. 3-13A, pane1 2 and 3) to pinpoint the

site of highest density @me1 3). Based on the d t s of biochemical evidence, insuIin

binds to the ectodomain of the nceptor, picucularly in the region of Ll-Cys-nch-L2, the

srst three contiguous domains of the IR (Fabry et al., 1992; Murray-Rut et al., 1992).

The hi- derisity in the 3-D volume indicates the l d o n of the NG clusta and, with it,

the location of insuün binding @ig. 3-13A, panel 3). It identifies this region of the

structure as the receptot ectodomain and, more specifIcally, part of the LI-Cys-rich-L2

domains.

When we fit a NGBI molecule into the tentative region (see details in Cbapter 3.3.

and 3.4), the best fit ïs o h h e d with a molenile of iasulin king in contact with the L1-

Cys-rich domaias of one a subunit and with the L2 domah of the other a subunit, T&is

isw mistent with the previously proposeû mode1 invohnDg both a subtlIUlfS in the high-

3.4. Donuin Structures of the HIR

The domain-like features of the sbcuctiae becorne evident d e n the stnrcture is

viewed at intermediate density threshoids (Fik 343& panel 2), and, except for the NO-

BI region, indicaie a strong 2-fold verticai r o t ~ * o d symmeky as would be acpeaed

h m the oligotetramerlc primary stnicture of IR. This symmetry was used to d u c e noise

in the recomtmctions and render the stmcbaes shown in panel 1 and in Figure 3-13B.

This Figure shows the top, bottom and side views of the stmdurr. putatively as being

viewed in the piane of the membrane, and in the extraceflular (top) and intracellular

(boîiom) perspectives.

In the side views, the top part of the stmctwe. where NG is Iocaîed, is identifiexi as

the ectodomain of the a subunit The dog-bone-shaped substnicture of the 3D reconscniction

(Fig. 3-13B, top view), and the equivaient topmost, bw-tie-shapecl stnicture (Fig. 3-13B,

OO), are designated as the two L1 domains of the dimeric receptor on the bask of the x+ay

stnichire of the Ll-Qs-rich-W d0main.C. The si& view at 65" shows the putative LL-Cys-

rich-12 domains as contiguous su- ~CLTOSS the upper centrai region ofthe molecule.

with enough additional volume in this region to sccount for most of the remahhg mass of

the two a subunits. primarily the ibnnecth-like comecting d0msin.c (CD) (O'Bryan et ai.,

1991) and the _cmaller insert domain (ZD) where the second diSulfide bond fom between

the two a subunits.

I a subunit

Fig. 3-14. The secondary domah structure of LIR (A) and the domain location in 3D structure. A) Linear locations of domauis in the dimeric hIR B) Schematic representation of the domain structure of one a p monomer based on the comectivity in 3D reconsûuction, the primary domain ~equence, the symmetry reqyirement for two disulndes on two-foid axis, the fit of known domain struchues, and on the principle of keeping unknown domains compact Di-stances between modeled locaticms of CD, Fnl, and symmetrid diadfides cornmensurate with numbers ofintemening amino acids (stn>caires not to scale; alpha subunit: red; beta subunit: blue and green; unknown structures: spheres or Lines): Labels: A = TK activation loop; 1 = Cys524; 2 = -82,683,685; 3 = diSulfiide bond betweea Cys647 and Cys872; arrowhead = cleavage site of pecursor recepta protein (A and B); other labels as d e s c n i in Fig. 3- 13)- C) Approximate locations of the domains on the 3D structure ofhlWNGB1 cornplex

Fig. 3-15. Fitting o f bioehemical domaios and their h o w n r-my structures to the 3D recon- struction (alpha subunit: red; beta subunit: blue and green). A) Fitting of the Ll-Cys-richu &CL) domains as approximate cyllnders to ectodomain of IR (wiremesh representation). One insu- lin rnolecule @urpIe nibon) inserted with its receptor-biuding domains contacting the LlICys-rich domains of one a subunit (fuchsia) and the L2 domah ofthe other (mi). Nanogold market (yeilow) on insulin B chiain wincides with highaensity site. C) hage of right angle view of (A) with LCL domains (iiisului @y hidden), fitted TIC stnicture (green), two dimeric F a structures @lue and red), A-hop @lack) of left TK domain in crystauographic position, and A-Loop (dadc blue) of symmetric TK extended to overlap peptide substrate ofopposite TK B) and D) Image of right angle top view of (A) with (B) or without @) LCL damains showing F m domains (bludred), TK domaius (green), and crystallogmphic (bIack) and exteded (dark blue) A-loops. The two a sut+ unie are show11 in rd, f3 subunits in blue and green. One wke me& square is 6.5 k Accession nubers: insulin PDB: l B W TK stnicaae (PDB: lIRK), andFm (Pm: 1mFn). AL = TK acti- vation loop 6110 et al- Science 285:1077,1999).

The contiguity ofthe domain stmctme @ig. 3-13B, top ami side view 9û"), dong with tbe

residuesftomboththea andB subunit, is assignedtdteupperleftendoftûecres~enr(side

view, 0°) where it is contiguous with the CD portion of the a submit @op view). It bas been

s u g g d xecentiy that the CD has a fibmI1CCfiil-like stnicbae ~~ et al, L998). Fig. 345C and 3-15D (cf Fig. 3-138, 9û0, top vKw, iespcctively) show the fïtting of the

crystal stnichaeoftheTKdomain(gr0en) ofthe $ subunitdofthetwo FnIlIiepeats

@lue/.ed) modeled as the canonid ~~ type ï ï I stnictims (O'Bryan et aï., 1991;

P a s p i e , 1991).

The spatial relationsbip beîween the domains of the a and f3 subunits (e-g. side view,

90°) suggests the location of the celi membxane lipid bilayer as the space below the a

subunits and above the bidge linking the two dgned TK domains- Iosiead of a flat open

@on, this space in the 3D rrcoastnrction foms a thick dome-like slab above the bridge

with a thichess variation of 2.2 to 2.7 nu^

Starhg fbm the putative transmembrane domain, the $ subunits &me slightly

twisted towarü each other- H a t the intracelïularr TK domains of IR would then occupy this

portion of the (ide view, 6650, bottom view). There is a sepration of several

nanometers between the fkes of the masses of the two putative kinase domains. Around the

4. DISCUSSEON

By blocking two ofthe three fkee amine grow on iasulin m o l d e , we d e r i .

the B1 phenylalanine of bovine hsdh wïthNaaogold. The amino temiiaal region ofthe B

chah of insulin is beliwed to be minimaBy invoLved in receptor binding. Birxiing

experimemts showed that the Zeceptot-binding afniiity of NG-BI a f h dablocking was

decreased only slightly compared to native bovine insrilin. The con&ions ofderiVaSiZafion

were gentle enough for Nanogold since the pochrt showed a0 stmctud damage of the gold

clusta aftrrward î l e bigh pirrity ofNG-BI, mm> c o n î m b h g k e insulùi or Nanogold

molecules, ensured the specificity o f NG-BI bindiirg to HIR Aiîhough we iwbated both

NGBI and the bIR at relatively h i g k concentmtïon (10% " to to maximal bbiadirig

of NGBI to the IR, the rnibsequent removal of fine ligand a d the dilution of the bound

wmplex establishexi a new equili'bntun concütï011 The new equilibrium wodd result in the

binding of one NGBI to one hZR STEM h@ng confinnecl the presence of one NGBI

bouud to one hlR in the of the complex @cles. There was aïso very few fine

NGBI particles seen in the background (Fig. 3-7A).

To achieve hi@-redution reconstruction of the NGBI-HIR complex, we applied

the recent advances in elecûon cryo-rnicro~copy to out study, including the instrument,

sample -on and image processing. To pqyre the specimen, we d d out the

Ligand-receptor bmding in physiologicai biinir, followed by qyick freezing, and fnac-

drying inside the STEM at -130 to -140. C. This maintainecl the proteIn in the near

phNoIogicaI hydrateci w-011. dmhg image aqiidion. At a vezy low dose (+d'A!),

imagnig at -1500 C marLsdy rrdueed the radiation damage. Th use of both elastic a d

inelastic detectors to captine morr signais in the S m nir(ha breased sienoise ratio.

A sharp Nanogold signai was used to mainiein di the images m sharp f m - These

improvements are fidamentai for the successfiil3D remuüudon. As a dt, for the frst

tune, the 20 A resolution obSamd in this study d e d clear domain stnrctims of the

HIWNG-BI cornplex. A strong two-fold verticai rom*onal symmetry as anticipated h m the

dimeric configuration of the oiigotetrameric (apb dmctme of IR can be seen in Fgmr 3-

13B. A clear Namgold-insulh sisignal can be . * * on the 3D cornplex stmdme and

localized on the assignai L1-Cys-richL2 domah

The o v e d 3D structure of the ~ - m S a h rece~tor eomnles

In this shdy, we have showun thaL the ovaaIL shape of the 3D reconstniction of the

insulin-IR compIex by STEM is compact and g l o b e (Eg. 3-13A-1,). In studies by othas,

the HIR images obtained by e l w n mic~os~opy have been shown as X-. T- or Y-shaped

particles (Chnsnansen . et al., 1991; Schaefer a ai, 1992). Following the pubIicaîion of our

present study (Luo et al, 1999) Woldin et ai. Folclin a ai., 1999) reported their TEM

imaging of vesicle-reconstituted HIR cross-Iinked to a biotinyl daivatiaed insuiin

photoprobe @Bpa insulin) wtiose localbtion was visuaüpd by b i i to Nanogold

streptavidin. The TEM images were rather fûzzy and not clearly discemable h m the

background. Nonefheless, they showed the image of the nxeptor as Y or T shape with very

thin cW-Wa" Jhuctinw of O@ 1.5 nm in width for the armg the putative extracellular

domains of the receptor. 'Ibis is signifïdy d e r than that reporteâ (about 3 m) for the

appsrrntly d i f f i nOm ours- Nevextbless, Waen Wewiqg the domablike dmctmes m

some orientdons at mtmnediate deaüty ttii.esho1ds (Fik 3-13B), we do fbd images with

shapes- * . of X- or Yshspsd particIes- AIthough Woldin et aL used about 7 times

kger dose Ur o h i n h g their TEM mipses, the difference between our observations made in

this stuciy and observations by others ising TEM may be the d t of sample prepadon

For example, the negative-stain methoà, which was used in TEM studies may intduce

artifacts byhighlighang thebomdaûes or nIliiig i n d spaceq or alteringthe shape of

m o l d e s by iimoduaiig a nomphysiologicai ionic environment, Fiaehennore, stabhg a h

limits the potential resolution by the large sîze ofthe staining ions,

Superimposiilg h w n aysEal stmctms of d e r domains of the q t o r on

substnictures of the ~econstniction has macle it possiale to deduce the spaaal relationship

among the domainw in the overail cornplex. There is no crystal structure available for a

protein with such a big moleculat s iz . However, crystai d m û w û m s of severai domains of

the human insuiin receptor are available. Tbey are either h m the insuiin receptor or its

closely homologous family members. These hclude the TK domain of the insulin zeceptor

@ubbard et ai., 1974; Hubbard 1977), Ll-Cys-rich-L2 domain of IGF-1 receptor (Ganrett et

ai., 1998) and fibrrctin III repeats domaias (O'Bryan et al, 1991; Pasquale et al., 1991). Al1

these domains cau be properiy Mted into the correspondhg domain volume of our 3D

reconstniction Taken together, the o v d stmctue shows the division of the complex into

the extracellular and the cytopWc segments dong a plane, the putative cell membranes on

which the fibn,nectïn type ilI npa ts lie. These repeats appear pontoonlike to support the

d y 1- insulin-binding segment of the ectodomaim Undarvash the putative

tnmsmedllbraae mon, TK domaihs oc- the major voIirme of the htracellular portions

of the f3 subhts @ig- 343B),

Stnictunl correlation wîth the ~rinrrirv stm- of HIR

As a complex Ileteroîeîratner membrane protein, the tW CO- svei.l fîmctional

domallis. 1t bas ban proposecl that the specific fimctionai properties of the IR reside in its

separate domains. B a d on itp Pnmay stnicaae @ig. 3-14A)¶ the stmctud relationship

of the various domains in one or$ monomer is schematïcaliy show in Figure 3-14B.

Beginnins at the N4erminaI, the LL-Cys-n'ch-L2 domain (tadi contains about 150 amino

acid residues) le& into a comectïng domain (a9 a structure of Fibnectin III repeat (see

Ward, 1999), assigned FnO) ofabout 140 amino acid residues, foilowed by part ofone F a

repeat @nl) and the insert domain. The comecting and insert domains contain -24,682,

683 and 685 that form at least two disulfrde bonds between two a subutüts (Spamw et al.,

1997). The FnIU repeat in general is a p süucture of s e v a strands (Baron et aL, 1%

Leahy et al., 1992). The second F a repeat is constitiited by amiao acid sequences nMn

both a and subunits, and is htempkd by the ckavage site between the two subunits. The

N-terminai ofthe $ subunit contains about the of the seven f3 straods ofthe second rrpeat

(Fnl) and the entire third repeat Wn2). Fmm symmetry consideration, the disuüïdes am

located at the center portion of the 3D stnictiae. The two a p dimers are linLod by the

disuifide bonds between FnO and insert domains and the a anci subunits are connected by

a disuifide bond between Fnl and Fd? repeat domains. The third repeat is îinked to the TK

domain by a secpnce of 70 amino acids, incliidinp the 23-residue transmembrane sequence.

acid residues) oOcuplLes the majorvolinne ofthe mtracc1lu.k portion of aie subunit

S-on of known crystal stmchnes of d e r domains of tbe meptot on

substnrcaae~ of* 3D zec~OStNCtion would codkm the domain asSigment as cfisaissed

above and thus hlps to deduce the spahi relationship among th do& in the cornplex.

Availeble x-ray coodhates weze fitted to the respective subshuetine by modehg the

electron density of the xiay crystsuographic s&u%ms mto the volume of the assigned

domains of the 3 0 riecontrutïon. Figures 3-15C and 3-1!5D (CE Fig. 3-13B, 9û0, and top

Vimy mspectively) show the Mtiag ofthe crystai stmctme ofthe TK domain Cgreen) ofthe

subunit and of the F a ~epcais (bldred) to the reconstriIction. When fitted into the top

of the two crescent-shaped ap rnommers, the type III repeats Jie on a horizontal plane

£lankhg on two sides, pontoomlikey the nmaining strucûm of the a subuuits.

Two symmetrical TIC crystal stnictures (Fig. 3-15C a d D) can be fitted into the

putative intraceiiular space of the receptor stmctwe. nie masses of the putative kinase

domains are connatcd via a slender hotizontai bridge (Fig. 3-13B, side view 90") that was

not ob~exrved in the x-ray stnichms ofthe TKs The prrsaice of this bridge can be explained

in temis of the reconstniction being in a transition between fk IR aad its ligand-activateci

form. In this situation, the TK donains of the IR have undergone a confomrsitionai change

such that the A loop of one TK domain wouid k reaching out to the utalytic loop of the

opposing TIC domein However, since thae was no ATP in the biading buffer in the

prepouaton of the NGBI-IR complex for STEM, the activation of the IR stopped in this

stage and no bm-phosphorylation occumd The catalytic loops in the two fitted TIC

domains are separaid by 4 ma hterestingîy, this distance ik just SuffiCient to pemiit the

tyrosine triplet m 1 5 8 , 1 1 6 2 a d 1163) m a fiüly extendeci flexible activation loop ofone

TR, to reach the catalytic loop of the opposite TK as modeIïed Ecm the x-ray coordmates

(PDB lIRp). The extemsion of the activation loops wuid account for the Lmking deaSity

observeci between the lower portions of the subunîts m the structure assignments.

Our 3D stnictine showed a Iimited space for the transmembrane region of the

putative plasma membrane- This spacing is a change in shape, and a decrease in the

thickness h m that expected for a ~anbrane bilayer that would accommodate an aipha-

helicai transmembrane domain CTM) of 23-26 hydrophobie amim acids. However, since

the p d e d IR in the absence of its native membrane was Wly active, the relative positions

of the extraceiidar and intraceiiular domains must still represent a close to native

arrangement-

The mssing LI-Cys-rich-L2 domains of the dimerk a subunits are presented in

the reconstruction only in their general shape (Fig. 3-154 B and C), since the known x-

ray coordinates were not available (Garrett et al., 1998). An approximate mode1

composed of cylinders for the LI-Cys-rkh-L2 domains was generated instead. This

approximate structure was fitted into the extraceiiular portion of the 3D structure. Based

on the location of the Nanogold signal in the cornplex structure, one, or two if the

condition alIowed, molenile(s) of insulin should be fitted into this region discussed

below.

Structural Iocalizrrtion of the insnlin-bindinn site on the insulin rece~tor

By labeiïng the receptor withNGBr, we cleady demonstrated the stoichiometric

relation of inSulln to its receptor and showed the stnrcalral IOC8fI*on of insulin on the

receptor in the pnicess of-on. As me~ltiolied above, the crossing LI-Cys~ric~L;!

domains of the dimeric a subunits are presented in the mconstNCtion only in their

general shape (Fig. 3-15A, B and C). Nonetheles, using this sfnietirrr, the localizaton of

the gold cluster, and the known receptor-biading domain of ùrniün (Murry-Rust et aL,

L992), a NGBI molecule is tentatively fitted i . this region. The ntted insulin molecule

is in contact with the L1-Cys-icch domains of one a subunit and with the L2 domain of

the other a subunit. A mode1 imrolving both a subm*ts in the high9tnnty bindhg of

insulin has previously been proposed based on studies of insuiin analogues binding to the

IR and WiGF-1 R chimeras (Yip 1992; S c W i 1993; De Meyts et al., 1993). The 3D

recoIIStNCfion provides the structural evidence for th ïnvolvemmt Although two

mo1ecuies of ursulin can be fiîted to tbis configuration, two molecules of Nanogold-

labeled insulin were observed only rarely in the STEM images. The h i ~ ~ t y binding

of the fïrst insuün molecule to the IR has likely induced a conformational change in the

binding domain so that the second msuün molecule would bmd only et low affinity.

Likewise the binding of a second moIecule of insulin could effect a conformational

change that enhances the dissociation of the bound insulie Thus the cwyilitlear

Scatchad plot and the negative cooperatvity of insulin binding (De Meyts et al., 1976)

can be explained on the b i s of the 3D ieconstmdon. The reconsûuction also explains

why only low-afEdty binding is obtained with pmified a p monomer (Boni-Scnetzier et

al., 1987; Sweet et al., 1987)-

Evïdence of insiilUi. rcüvatson of the HIR

The 3D dmcture zweais that a siender horizontai bridge Vig. 3-13B, side view 9@')

wmects the masses of the prdative lrinaJe domain?c. It was not observed in the xiay

stmctms ofthe TKs, but, as stated above, can be expiaïned in terms of îhe r e m ~ C o n

being in a transition between the fke IR and ito Iigand-BCfivafed f o m h the two

symmetndy filied TK crystal structrires @ig. 3-L4C and D) the catalytic loops are

separated by 4 nm. This distance is just d c i e n t to pemnit the tyrosine triplet flyr1158,

1 162 and 1163) in a fully extended flexi'ble -on lmp of one TIC to reach the d y t i c

bop of the opposite TK as modeleci k m the x-ray coordlnates (PDB lIRp). The extendon

of the activation loops, eqiiident in mdon to four adadod po1ypeptide chains,

d y accounts for the iïnkïng Iuiloag oeasiryed betuem the lowerportions of the subuds

(Fige 13B, 90"). Ti& is an important dinkence h m the =ray stm%mes of the inactive and

activateci TKs as discussed below.

Monomeric inactive receptor TKs such as EGFR are brought together by ligand

binding and becorne activated as dimers resulting in TIC a u t o p h o h o o n (Canaals,

1992; Schlessinger aad UllrichJ992). Ln the inainsicaüy dune8c I R - M y mxptors, the

distance between the two cytopasmic ksubunït TKs within the dimer must be too great

without ligand b i g for the ectivetion of the kinase. Hubbard and bis coileagues

pubbard et ai., 1974; Hubbard 1997) suggested thet insulin binding to IR d e a e a d this

distance by dknpgkg Tyr1162 hmthe cataiytic loop to enable tram phosphorylation in

the presence of ATP. Zn the reconsûuction a good fit to the ligand-receptor complex is

obtained when the two TK domains are orienteci with th& caîalytic Ioops juxtapod In

this orientation the exîeded fiexiie activation loop of each TK, which moves 30 A

Hubbd lm, canjustreachthe catdytic loop ofthe opps&TKto be a c t i . 'Ibese

two l o o p s c a n d y f o r n t n e l i n l a n g m a p s ~ b c t a n a n t b e ~ s e n i p t h e 3 D

recollstrtlcttConin the absence of ATP.

The 3D stmctme obtsiaed nwi images of the HIR-compIex contains ody a suigîe

NGBI. This suggests that one m o l d e of insulin is sufncient to bring the two a$

monomas to an activating confTgiiration, Ln light of this supposition, it is signifiant to note

that the dimexic q r with a Sei.323- mutation in the L2 domain of both a suburiits

showed a sevete Hiipaimiem in insulin bhding (Roach a al, 1994), whereas a hytxid

reoeptor with ody one ofthe two a subunits miitattd was found to bhd insuiin with high

nffinity and was M y active as a tyrosine kinase Umuis a al., 19W). Based on the 3D

reconstru&on we would suggest that insuiin is bound to the L1 domain of the mutant a

subunit and the wild-type L2 domain ofthe hybrid IR and that the bindïng of only a sïngïe

molecule of insuiin is sufEcient for TK a c t i . o a

In this shdy the 3D guetemary stmctwe of the Ikinsulia cornplex f o d in the

absence of ATP was obtained Thus the smrhnr was expected to be an intemediate

between the insuiin-- IR and the fXiy activated, phosphorylated IR. 'Ibe recoostruction is

d y interpretd as a receptor poised for activation by *uns-phosphoryiaîio~. The fidi

extent of conformational changes induced by insuiin b i i remains conjecbiÿa, without

simiiar reconstnictions ofthe initial state of insulln-iiee IR and of the W activated state for

cornparison. Nonetheless, in the absence of a aystailographic stnicane of the insuiin

recepttor, the 3D recoastnrction presented here provides concrete stnicttnaI information

towards the fidi undersCanding of trammembrane signai trairpmission in insulin action.

CONCLUSION AND FUTURE RESEARCH

The nntstep inthepathwayofmwilinactionis thebindingofirrpulinto its ce11

sinnice receptor; an integrai transmembrane glycoprotein, leadhg to the activation ofthe

cytoplasmic domain of the nxeptor tyrosine kinase- Mueh is lcnown about the iateraction

of the acfivated receptor with its dsûaks leadhg to dom-stmam signal transduction.

On the o t k han& how binding of insuiin to the extraceliuiar domain of the receptor can

lead to receptor activation rernains unknow11, Understanding w h e insulin binds to the

exbraceiluiar domain would provide the foundation to anmer this Question.

Many approaches, biochemicai and biophysîcai, have ken applied to elucidate

the site(s) of insulin-insulin zeceptor interaction Diffbnt d t s were reported, and

various regions in the a subunit of the IR have ken suggested to be involvesi in the

insulin-IR interaction. These regions inchde almost every major domain, such as N-

terminai, cysteine-rich domain and C-tenninal of the a subunit, An accurate location of

the insulin-binding site on the xeceptor has not been fortbcoming.

The specific goal of th& study was to locaüze the insulin-binding site in the IR,

and, if possible, to elucidate the molecuiar detaifs of insulia-IR interaction and the

relatiomhip of the stmctme and fbnction of the iilJulin receptor. To achieve this goal by

the biochemicai appmach, 1 have expred the human insulin receptot extraceîlular

domain in a large s a l e in the baculoinis expression system and used various specinc

photoa5nity probes to identifil the insalui contact sites. The photolabeled receptor

protein was digested with proteases, and high-sensitivity identification methods wae

developed to detect the labeled IR fbgment contahihg the putative insulin-binding site

by HPLC -&mappiag d y & - 1 have fOCUS4d on a fbgment of 1-3 kDa that mi*

contain the putative -binding or 0 0 ~ site on the reccpf~r a subunit;

However, 1 I d to obEain SLlfncient qymtin'es of the fkagment for emino acid

~ e ~ ~ e ~ l c i a g , partiy drr to the ladr ofa high efficiency photoprobe of iosului and a high

efficiency peptide mapping method AvaiMiIity of inoalin photoprobes with higher

efficiency is a kcy in fÙture studies ushg this approach. The bcnzoylphenylalsinine

photoreactive group in a anv msulin photoprobe has shown a much hi* cross-lnilring

efficiency than the traditionai phenyIazido photoreactive group. A cross-luiliag

efficiency of more than 50.A has been reportecl for this photoprobe compared to Iess t h

10 % reportai for other insuiin photoprobes (Shoelson et al., 1993). The use of this high

efficiency photoprobe should lead to a higher yield of the cross-hkhg productsC Tii

addition, methods for the purification of the cross-linked fhpnent and for higher

sensitivity in amino acid sequeaciag need to be developed The use of MALDI-TOF to

determine amino acid sequence of srnall peptides is clearly a p d c a l alternative.

M y next approach to the goal was cryoelectzon microscopy, spedically STEAK

By using insulin labeled with an electrondense gold cluster to bind to pirrified human

placenta membrane insulin receptor, 1 successfiilly detennined the 3D structure of

insulin-iasulin receptor complex at a resolution of 20 A and localized the site of insuiin

binding on the complex. The stnifam was fitted with available known high redution

crystal domain substnicanes to obtain a detailed contiguous mode1 of the

heteitotetrameric (aph transmembrane receptor. The 3D ieconstniction shows that the

two a subunits jointly participate in insuiin Endhg and that the two S s u b d kinases in

this receptor-ligand complex are in a jiatsposition thet permits trans-autophosphorylation

on finding only one molccule of insulin (as visiiaüzed by the gold-labeled insulin), ami

oniy rarely two. the zecoIlStNCtion provides shactmal evidence fot the involvement of

only a single iasulinm01ecuIe in receptor activation.

The <iuiitcmery stnrane ofhuman insaün receptor wmplex detnmined bythu

study provides the shuctiiral expianaiion for many of the known c-CS of insulin

bmding to its receptor* Fitting the crystai stnictirrG of hdh into the fec~nstructioa

mode1 shows that the L1, CR and L2 domains of both a subunits contili'bute multiple

contacthg sites for high9ninty insuiin-IR intaacton. The stnictiirr a b shows that

exbraceIlular Qmains of the B subunits play d e in s t a b i i the highdlh& insulin.

binding site. Moreover the stnicbne provides a mechmistic basis for the mechankm of

The stmctue ais0 e x p W many biochemicai findings in the studies ofthe insulin

receptor. A recent study indicateà that insutin receptor isofonn A in which exon 11 is not

expressed, besides its high dlinity to insulin, binds to IGF II with an affinity close to that

of iosulin. It appears to serve as a high-afEnity IGF II receptor affecting the growth of

fetal and cancer ceb (Frasca et aï., 1999; Sciacca a al., 1999). lkis Gnding suggests that

the 4 variation in the C-terminal o f the a subunit of the insuiin receptor plays an

important role in the wnfonnation for high-ety ligand binding. Interestingly, binnding

prolacth, are composed of the two fibronectin repeats of each monomer that fomio the

dimeric receptor Web, 1996; BoleFeysot et al., 1998). The skucture we bave obtained

for the wmplex shows that the C-terminai ofthe or subunit and îhe adjacent N-terminal of

thep subunitwntsinsasùnilarstniehncoffibro~rrpeatlidlmer~Howthe~

or the absence of these 12 rcsïdaes ccxied by the exon LI, in the C-tenainal region d d

change the IR'S affinity to IGF-II is uuknown. H o m , the stnictural similPr@

between the IR C-terminus and growth fhctor receptots Qes provide a stniduraI b i s for

a suggestion that the C te rmï~L region of the insuiin receptor isoform A could serve as

&other Iigand binding site in acUti011 b the LL-CR-L2 region for insalia This

hypothesis wuid be confimeci by STEM Smüar to the present study, but using

Nanogold-IGF-II-

Insuiin and impaireci insuün d o n play a key d e in the pathogenesis of di-,

in type II diabetes which account for more tfLen 85% of the diabetic

population. While a combination of genetic and environmentai fktors contn'butes to the

development af type II II, defects in the insulin si@ transduction pathway

beginning with the intetaction of iasulia with its receptor play a major d e in the

manifestation of the disease. The 3-D recoIIStNCtion of the insuijn-IR complex describecl

here presents the fïrst mode1 to explain signaLing process fion ligand binding to tyrosine

Ianase activation The sirucairal localization of the insulin binding sites in the IR

provides evidence of how and where iiisulin interacts with its receptor. Combining with

information hrn crystai structure ofthe i s o W receptot domains, such as LI-CsL2 of

the IGFI-R, it wodd be possible to obtain more details ofthe insulin-IR interaction. This

would eventuaiiy heip to develop (1i. new antï-diabetic medicine.

Recently, a non-peptidyl fimgal compound L-783,281, was reporteci to have anti-

diabetic activity in mice (Zhang et al., 1999). M e r than interacting with the insulin-

binding sites on the a subuints, this compound appears to interact with the cataiytic

stül mimic the efEcts of the ligand of the Lcccpbr. Our ramnstniction mode1 may

provide a shucaiiril basis for sbuiying the potential mechaniSm by wbich tbu fimgai

compound actives tyrosine Linase ofthe IR. Otheroral (or non-oral) antidiabetiCc agents

have aiso ban reporteci to have insuiin mimimimic e f f i such as vanadates, PTP inhibitors

and phosphoinositoIgLycaus Frick et ai., 1998; Shdev, 1999; Chen et ai., 1999; Iversen

et ai., 2000). Some of them dirrctly ïnteract with the IR. It is still not very clear how these

cornpo~~~ds mimic insulin action at the m o l e leve1. Neverthelesss more detailed

structure and fiinction studies and the nhecpent stmctwe-based design for new dmgs

wouid help to develop rational therapies for trrating insulin resistance-

Besides the principai IRS substrates, other nhtmtes, such as Shc, might also

exist in the insulin-signaling pathway. Altemative pathwmys of the IR may be responsible

for the multiple actions of insulia Shc is a substrate protein rapidly tyr-phosphorylated

by insuiin stimuiation. Shc contains an Noteminai PTB domain which binds to the

phosphorylated N P X Y ~ ~ ~ sequence in the insului receptor md can also bind Grb-2 to

link these signais to Ras activation (Skolaüc et aL, 1993; RonL et al 1994; Gusta£Eon et

al., 1995). It has ken suggested that Shc, rather than IRS-1, may be the main mediator of

the mitogenic actions of insuiin and IGF- II (Yamaguchi and Pessin, 1994).

Aithough our observation represents a signiîïcant advance in elucidating the

structure of the insulin receptor, and provides a structural basis for the mechanism of

receptor activation, additional saidies remain to be done. First, the present study was

d e d out on detergent-solubiï membrane protein in the absence of a bilayer

membranet Althoughtht s o l u b i i protein has been show11 to k fUy active in ligand-

binding and autophosphorylstion when cornpprcd tu placenta membrane receptor, the

sauctine of the comp1ex obtained did show a cornpresseci tninsmembrane domain in the

3D r e c o ~ o ~ ~ A receptor in a biIayer q q o x t would p v i & a more naturai

recomtruction. S W e s on 2D crystai of the protein supported by a iïpid monolayer or

bilayex would greatly increase the final res01ution of the 3D rewnetnicdon. Severai 3D

reconstructions obtained by electron cryomicroscopy based on 2D crystd mes have

resolved the stmdure of a nunber of proteins et resolutions below 7 A ( m g et al.,

1997; Walz a ai., 1997), very close to the resolution obtalned by x-tay crystdlography.

Since the insulin receptor has so fiu not been crysîaked the 3D - d o n of this

protein at a higher resolution is especially miportant to elucidate the molecular daails of

the fiulction related stnictun, such as the insulhbinding site. Understandiog the

molecular mechanism of the insuh-IR interaction would help in designhg new drug foc

treatment of diabetes meIlitus.

Insuiin bi~lding to the receptor leads to codonnation change of the receptor. This

conformafion change is the key step to receptor activation. A reanstruction of the fke

receptor without bomd iiwlin would provide very usefhi information Cornparison of the

activated and the pre-activateci conformation of the receptor wiii provide the information

required for undexstandhg the mechankm of receptor activation. Cn this study we

obtained the structure of a receptor bound to iasulin at the pre-phosphorylation stage in

the absence of ATP. Addition of ATP to the insulin-binding b s e r could reveai the

conformation of W y phosphorylated receptor kinase. Combining informaîion obtained

h m different activation states should help us to understand the mechanism of insulin

receptot a c t i . 0 1 ~ Furthermore, in addition to monitoring the conformatio~ change

during the receptor dvafi*on, electron ~ ~ ~ O I I I I * C I O S C O ~ ~ wodd also k an exceiient t d

for visuali;nng * . the c o n f o ~ * o n change ofthe receptor resulttîng h m mutagenesis, This

visuaiization wouId provide the direct explanlition ofthe chan@ relationship in stnictun

and firnctïon Visdhtion of clown-stream interaction with signal transduction elenicnts,

such as IRS proteins is also v a p important for mkstanding the mechanism of insuiin

receptor activation and the uisulin s i m g systemstem IRS proteins are another grwp of

proteins that phy important des in signal traasdm*on but are dficult to crystaUize

structure for d y i n g their stnichae and fiincti011, W e c a n imegine that th cornbidon

of molecular g d c s in sampIe preparation, stateof-the-art electron cryomimscopy for

data acquisition, and pownful image proceshg will open a new era of structure and

fiindon studies for the iiwlin receptor at close to the atomic resolution level.

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