IDENTIFICATION OF ROM10 AND PHRI-INTERACTING PROTEINS IN THE MAMMALIAN PHOTORECEPTOR

Rahim Akbarali Ladak

A thesis submitted in confornity with the requirements for the Degree of Master of Science, Gnduate Department of Molecular and Meâicai Genetics, in the University

of Toronto

@ Copyright by Rahim Akbadi Ladak 1999

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IDENTIFlCATION OF ROMl- AND PEKR1-INTERACTING PROTEINS IN TEE MAMMALIAN PHOTORECF2TOR Degree of Master OC Science, 1999, Rahim Akbarali Ladak, Graduate Department of Molecular and Medical Genetics, University of Toronto.

ABSTRACT OF THESIS

The photoreceptor is a unique neuron in the retina, and its highly specialized organelle, the

outer segment, is the site of phototransduction. Elucidation of the molecular basis of

outer segment structure and fùnction are two of the main goals of retinal research. Our

group has described one major outer segment protein, ROMl, and more recently

discovered a second outer segment polypeptide, PHRI, which contains a pleckstnn

homology (PH) domain. ROMl is an essential structural protein of the membranous disks

which fil1 the outer segment. The function of PHRl is unknown, but the presence of a PH

domain suggests that it may be a component of the cytoskeleton or participate in a

signalling pathway such as phototransduction. Using the two-hybrid system, 1 have used

the ROMl C-terminai tail and full-length PHRl in two different screens to identiQ

ROMl - and PHR1 -interacting proteins. The ROM1 C-terminal tail screen has identified

five novel putative ROMl-interacting proteins. Four novel cDNA clones encoding

putative PHR1-interacting proteins were identified, one of which contains a RING finger

domain, a well-known protein-protein binding motif. In situ hybridization revealed that

PHRI is expressed in photoreceptor inner segments, the outer plexiform layer, and the

ganglion ce11 layer of the retina. Immunohistochemical analysis indicates that PHRl is

specifically localized to the photoreceptor outer segments and to the plasma membrane of

ganglion cells. Retinal PHRl also migrates more slowly (-37kDa) than predicted

(-ZSkDa), suggesting that PHRl is post-translationally modified. The proteins which

associate with ROMl and PHRl in vivo are likely to encode important components of

photoreceptor outer segment structure and fûnctioa.

ACKNOWLEDGMENTS

1 would like to thank my supe~so r Dr. Roderick R Mclnnes for his support and

patience. 1 would also I l e to thank the members of my cornmittee, Dr. Sean Egan and

Dr. Barbara Fumeil, for their guidance and help. Special thanks to Danka Vidgen and

David Ng for their help during the writing of my thesis. And 1 would like to thank the

mernben of the McInnes lab for their assistance and scientific support.

To my family, thank-you so much for your patience, support and love. 1 could not

get through these last two years without your unconditional help.

TABLE OF C O m N T S

Abstract of' thesis

Acknowledgments

Table of Contents

List of Tables

List of' Figures

CHAPTER 1: INTRODUCTION

- Introduction

- An introduction to the vertebrate retina

- The vertebrate photoreceptor

- Visual transduction in the photoreceptor cell

- Photoreceptor outer kgment structure

- Outer segment disk rim proteins

- ROMl ami RDS/penpherin

- Associations between ROMl and RDS

- ROM1 and RDS are members of the transmembrane 4

superfamily (TM4SF)

- ~onrl" mouse, implication on photoreceptor structure

and fbnction

- Initial charactenzation of PHR1

- Plec kstnn homology domain

- PH domain structure

- PH domain ftnction

- Identifjing ROMl- and PHR1-interacting proteins using

the yeast two-hybrid system

CHAPTER 2: IDENTIFICATION OF ROM1-INTERACTING PROT'EINS

USING THE YEAST TWO-HYBRID SYSTEM

1 . INTRODUCTION

2. EXPERiMENTAL PROCEDURES

3. RESULTS

- Construction of the ROMl C-terminal tail bait

- Analysis of ROMl C-terminal tail bait expression in the

two-hybrid system

- Two-hybrid screen

- Characterization of the ROM 1 -interacting partners

4. DISCUSSION

CHAPTER 3: MOLECULAR CHARACTERIZATION OF PHRl AND

IDENTIFICATION OF PEIRI-INTERACTING PROTEINS

USING THE TWO-HYBRID SYSTEM

1. iNTRODUCTION 58

2. EXPERMENTAL PROCEDURES 63

3. RESULTS 68

- PHRI alternative splicing 68

- Expression of endogenous PHRI in retinal and brain lysates 72

- PHRl expression and localization in the neuroretina 74

- Presence of a putative C-terminal transrnembrane domain in PHRl 77

- Analysis of PHRl bait expression in the two-hybrid system 77

- The PHRl two-hybnd screen 80

4. DISCUSSION 94

CEIAPTER 4: CONCLUDING REMARKS

1. FUTURE DIRECTIONS

- Localizing expression of PHRl and ROMl putative

interactors in the retina

- Using afEnity chrornatography to confirm PHRl and

ROMl interactors

- Idenemg PHR 1 - and ROM 1 -interacting proteins

using afnnity chrornatography

- Future perspectives for PHRl

- Future perspectives for ROM1

REFERENCES

LIST OF TABLES

Table 1 - 1 Transmernbrane four superfamüy members (TM4SF) 15-16

Tables 2- 1 a e ROM1 C-terminai tail two-hybrid t&ets 47-5 1

Table 3 -A PH domain containhg proteins used or identified using

the yeast two-hybrid system 6 1-62

Table 3- l a-i PHRl two-hybrid targets 82-90

Table 3-2 The weak targets specific in their interaction with

the PHRl bait 93

Figure 1-1

Figure 1-2

Figure 1-3

Figure 1-4

Figure 1-5

Figure 1-6

Figure 1-7

Figure 2- 1

Figure 2-2

Figure 2-3

Figure 3- 1

Figure 3-2

Figure 3-3

Figure 3-4

Figure 3-5

Figure 3-6

Figure 3-7

Figure 3-8

Structure of the vertebrate retina

The vertebrate r d photoreceptor cell

Structural topology of ROM1

Light micrographs of retinal sections fiom 1 month old

~oml"' and ~ o m l " mice

Genomic structure of PHRI

The predicted PH21 protein sequence

Cornparison of PH domains

A. The mouse ROM 1 C-tenninal tail two-hybrid

construct, referred to as MRC

B. Western blot of yeast lysates expressing the MRC bait

Quantitative liquid P-galactosidase assay

Hypothetical mode1 of the rod disk rim

Alternative splicing of PHRI in the retina, brain, kidney,

and liver

Alternative splicing of exon 7 of PHRl

PHRl expression in HEK293 cells, mouse retinal and

brain lysates

In situ hybridization of mouse PHRl in adult mouse retina

PHRl is localized to the photoreceptor outer segment and

to the ganglion cells of mouse adult retina

PHRl protein sequence anaiysis

PHRl two-hybrid bait

Strong PHRl putative interactors identified in the

two-hybrid screen

CHAPTER 1

Introduction

INTRODUCTION

The retina is a highly organized, well-defined, and conserved neurosensory

structure. As the visual transducer of vertebrates, it captures photons and converts the

energy into an electrophysiological signal which is carrieci to the higher visual centers of

the brain (Jindrova, 1998). A thin layer of tissue, the retina, is situated at the posterior of

the vertebrate eye (Wechsler-Reya and Barres, 1997). The retina is composed of two

components, the neuroretina and the pigment epithelium. The neuroretina consists of

three parallel cellular layen: the ganglion ce11 layer (GCL), the i ~ e r nuclear layer (INL),

and the outer nuclear layer (ONL) (Wechsler-Reya and Barres, 1997). The outer nuclear

layer is comprised entirely of photoreceptor cells. The most notable structural feature of

the photoreceptor is a specialized ciliary denvative called the outer segment. The outer

segment is a finger-like structure, composed of a plasma membrane surrounding a stack of

-1000 membranous disks (Morrow et al., 1998). Three of the most interesting features of

the outer segment are that i) the disks are the site of photon absorption and light

transduction (Jindrova, 1998), ii) the disks are constantly renewed at a rate of -10% per

day, with new disks being formed at the base of the outer segment by disk morphogenesis,

and older disks being phagocytosed at the tip of the outer segment by the adjacent retinal

pigment epithelial (RPE) cells (Young, 1976; Anderson et al., 1978), and iii) in addition to

the symbiosis between the RPE and the photorecepton that is manifest by outer segment

phagocytosis, the RPE also provides important metabolites and nutrients to the

photoreceptor (Robinson, 1 99 1). Identification of the proteins involved in

phototransduction, outer segment structure, disk morphogenesis and turnover are three of

the main goals of retinal research.

The cloning and characterization of retinal gpecinc, gbundant, and onserved

(SAC) cDNAs has been an effective strategy for the identification of cDNAs encoding

proteins important for retina structure, finction, and development (Bascorn et al., 1992).

My research is concemed with two photoreceptor proteins, ROM1 and PHRI, identified

by the SAC cDNA cloning strategy. These two proteins are both abundantly expressed in

the outer segment of marnmalian photoreceptor, but their precise fwictions are unknown.

The overd goal of my research has been to begin to determine how these protehs

contribute to photoreceptor structure or hction by identdjing photoreceptor proteins

with which they interact.

ROMl is an essential integral membrane protein of the outer segment;

photoreceptors lacking the ROMl protein die (Bascom et al., 1992; G. Clarke

unpublished). PHRl is a novel retinal, abundantly expressed gene which is also present at

reduced levels in other tissues including the brain, kidney, and liver. The presence of a

plekstnn hornology (PH) domain in PHRl (gleckstrin hornology in the -na) suggests

that PHRl may be a component of the outer segment cytoskeieton, or of photoreceptor

signal transduction, including possibly phototransduction. In addition to identifjmg retinal

proteins with which PHRl may associate, 1 also evaluated the quality of PHRl antibodies

that were used to determine the cellular and subcellular localization of the protein.

The identification of ROMI- or PHR1-interacting proteins will provide critical

information about the fiinction of ROMl and PHRI, in the photoreceptor. The

identification of ROMl- and PHR1-interacting proteins using the yeast two-hybnd system

is the major subject of this thesis.

Chapter 1 presents an o v e ~ e w of retinal anatomy and fùnction, with particular

attention to the photoreceptor cell. In addition, information on ROMl and PHRl is

reviewed in this chapter.

Chapters 2 describes the results obtained from the yeast two-hybrid screen using

the ROM1 C-terminal tail as the bait. Chapter 3 contains further information on the

expression, localization and characterization of PHRI, as well as the results of a PHRl

two-hybrid screen. Concluding remarks are given in Chapter 4, with emphasis on the

further charactehtion of the putative ROM1 - and PHR1 -interacting proteins.

AN INTRODUCTION TO TEE VERTEBRATE RETINA

The retina consists of two major components, the pigment epithelium (PE) and the

neuroretina (NR) (Robinson, 1991). The vertebrate neuroretina is several hundred

microns in thickness. As mention4 in the introduction, the neuroretina is composed of

three parallel nuclear layers (outer nuclear layer (ONL), inner nuclear layer (INL) and

ganglion ce11 layer (GCL)). In addition, the three parallel nuclear layers are separated by

two piexifon layers (imer and outer plexifonn layen) (Fig. 1 - 1). The ONL is adjacent to

the retinal pigment epithilium (RPE), and is closest to the back of the eye with respect to

the other retinal ce11 layen (Fig. 1-1).

Light transverses al1 the layen of the retina until it is captured by the outer nuclear

layer. The ONL contains the ce11 body and nuclei of the photoreptor (PR) cells (Fig. 1-1).

The outer plexifonn layer (OPL) is the region in which synaptic co~ections are made

between cells of the ONL and imer nuclear layer (N) (Fig. 1-1). The ZNL consists of

interneuronal nuclei for al1 horizontal, bipolar, and interplexifom cells, the majority of

amacrine cell nuclei and a few ganglion cell nuclei (Famiglietti, 1990). Adjacent to the

IM. is the imer plexiform layer (PL) where synaptic processes between intemeurons and

ganglion cells connect. The ganglion ce11 layer (GCL) is composeci primarily of ganglion

ceil nuclei and a few amacrine cell nuclei (Robinson, 1991). The axons fiom the ganglion

cells bundle to collectively form the optic nerve which carries the light transduction signal

to the visual centers of the brain. The non-neuronal glial Müiler cells span al1 three ce11

layers of the retina, although their nuclei are located in the iNL (Robinson, 1991).

The neuroretina consists of six major neuronal ce11 types (photoreceptor cells,

bipolar cells, ganglion ceils, horizontal cells, amacnne cells, and interplexifom cells) and

two types of glial cells (Muller cells and in some species astrocyte cells). There are also

different neuronal ce11 classes. For example, ganglion cells can be subdivided into several

subtypes, including a, p. y, small-soma, and large-soma, based on morphology and time of

birth (Robinson, 199 1).

The second component of the retina is the RPE. The RPE consists of a

monolayer of pigrnented cells. These cells contribute to the enhancement and resolution

of the visual image by absorbiig scattered light via melanin granules (Bok, 1990). In

addition, the RPE plays an important role Ui photorezeptor fwictior The presence of

mannose receptors on RPE celis facilitates phagocytosis of rod outer segments (ROS)

(Lutz et al., 1995) whereas the activation of protein kinase C (PKC) in ROS inhibits

phagocytosis of ROS by the RPE (Hali et al., 1991). Rezently, it has been shown that

RPE (Retinal Pigment Epitheiium)

OS (Outer Segments)

IS (Imer Segments)

ONL (Outer Nuclear Layer)

OPL (Outer Plexiform Layer)

INL (Inner nuclear Layer)

1 GCL (Ganglion

8orm Layer)

Cell Layer)

A I 1 I

Light

Figure 1-1. Structure of the vertebrate retina. The vertebrate retina is laminar in structure and consists of five layers as shown. Light tranverses al1 the layers to reach the back of the retina, where the rhodopsin bound chromophore 1 1 4 s retinal, in the outer segments of the photoreceptors, captures photons and initiates the iight transduction cascade. The signai then proceeds to the ganglion cells, whose axons comprise the optic nerve, which relays the signai to the visual centen of the brain. Photograph fkom G. Clarke.

proteins secreted by RPE cells (RPE-CM) are important for photoreceptor cell s u ~ v a l

and development as suggested by retinal explant cultures and by rats injecteci intravitreaily

with RPE-CM (Sheedlo et al., 1998).

The Vertebrate Photoreceptor

The vertebrate photoreceptor (PR) is a specialized post-rnitotic neuron which

primarily functions to capture and convert light into an electrical signal. There are two

types of PR cells in the retina of vertebrates, rods and cones. The average human retina

contains approximately 92 million rods and 4.6 million cones (Curcio, 1990). Each PR

ce11 is specialized for vision under low or high light intensity, with rods fùnctioning

opitmally at low intensities of light and cones under high light intensity. Cones also

fascilitate colour vision.

The distribution of rod and cone photoreceptors is not random. Generally, at the

periphery of the eye there are few cones but many rods groups of which make a synaptic

connection with a single bipolar neuron, making this region of the eye highly sensitive to

light with low acuity (Bumside and Dearry, 1986). In contrast, the fovea of the eye

consists oniy of cones each making a single synaptic connection with its own bipolar

neuron.

Rods and cones cm be divided into an OS, IS, ceIl body and a synaptic terminal

(Fig. 1-2). The OS is the site of light absorption and transduction. The IS, which

connects to the OS by the ciliary process, contains the major metabolic machinery of the

ceIl and cm be subdivided into the myoid and ellipsoid regions (Fig. 1-2). The ellipsoid is

proximal to the OS and is highiy packed with mitochondria which generates ATP

providing energy to metabolic functions of the PR such as ion pumping and disk

morphogenesis (Farber and Shuster, 1986). The myoid region of the IS contains the

major metabolic machinery including organelles such as the endoplasmic reticulum and

Golgi apparafus (Farber and Shuster, 1986). The nuclei of the PR cells are located within

the cell body. Signal transfer to higher order neurons occurs at the synaptic terminal of

the PR (Fig. 1-2).

pigment epithelium

R d outer segment

Connecting cilium .

Rod inner segment

Nucleus

plasma membrane - Disks

Open disk (evagination)

EUipsojd (mitochondria)

Myoid (golgi complex and endoplasmic recticulum)

Celi body

Axon

Synaptic terminal

Figure 1-2. The vertebrate rod photoreceptor cell. The photoreceptor ceii is divided structurally into four regions, the outer segment (OS), the imer segment (ES), the ce11 body which houses the nucleus, and the synaptic terminal. Functionaiiy, the outer segment is involved in light absorption and transduction whereas the inner segment contains the major metabolic machinery of the ceil. The retinal pigment epithelium ce11 is important for photoreceptor survival and disk turnover.

Visual Transduction in the Pbotoreceptor CeIl

The visual transduction pathway is compriseci of several proteins, many of which

have been characterized extensively. Biochemical and structurai studies have led to a very

detailed description of the events in phototransduction.

The highly sensitive üght transduction pathway is the process by which energy

from photons is converted to an electrophysiological signal. The initial step in the

phototransduction pathway is the absorption of photons by the rhodopsin bound

chromophore 1 1-cis retinai causing it to isomerize to the 1 l-ull trm-retinal form

(Hargrave, 1986). Photoisomerization induces a conformational change in rhodopsin, a

member of the G-protein coupled receptor family (Farber and Shuster, 1986). Activated

rhodopsin then binds inactive transducin-GDP (TaPy-GDP) causing the activation of Ta

by catalyzing the exchange of bound GDP for GTP. Consequently, Ta-GTP dissociates

fiom TBy.

Released Ta-GTP proceeds to activate the heterotrimenc rod outer segment

(ROS) cGMP phosphodiesterase (PDE). PDE is inhibited from functioning by its intemal

inhibitor PD& (Granovsky et al., 1997; Artemyev, 1997; Artemyev et al., 1998). The

displacement of PD& by Ta-GTP bindhg activates PDE.

Enzymatic hydrolysis of cGMP by activated PDE decreases the cytoplasmic

concentration of cGMP which lads to the closure of plamsa membrane cGMP-gated

cation channels (Farber and Shuster, 1986). In the dark, cGMP binds to cation channels

of the plasma membrane thereby maintai~ng them open (Hargrave, 1986). Closure of the

channels results in the transient hyperpolarization of the photoreceptor plasma membrane.

The cation channels and ~ a ' - ~ a ~ ' exchanger are the principal ion transporters in the

photoreceptor (Hargrave, 1986). Hyperpolarization at the synaptic terminal of the

photoreceptor leads to a reduction in ca2' influx and subsequently decreases the rate of

neurotransxnitter release. As a result, the neurotransmitter is no longer capable of

inhibiting post-synaptic neurons dowing them to become excited and continuing the

signal through the retina.

In order to terminate the visual cascade activated rhodopsin is deactivated by

phosphory lation. Phosphorylation of rhodopsin is performed by rhodo psin kinase and

occurs at multiple serine/threonine residues at the C-temiinal end of rhodopsin (Hargrave,

1 986).

Phosphorylated rhodopsin, however, still retains some activity. To elirninate

residuai activation of the phototransduction pathway by rhodopsin, the phosphorylated

form is bound by arrestin. The binding of arrestin leads to the effective blockage of

transducin access to its binding sites on phosphorylated rhodopsin (Palczewski et al.,

1992; GureMch and Benovic, 1993). Arrestin dissociates fiom rhodopsin once the

potential binding capacity for transducin has been removed and the I 1-aff ~r<us-retinal has

been released. Binding of new 1 1-cis retinal restores rhodopsin and the process of arrestin

dissociation is dependent upon the regeneration of rhodopsin.

In addition, restoration of the dark state requires inactivation of transducin, an

increase in calcium concentration, and a retum to dark levels of cGMP concentration

(Farber and Shuster, 1986). Inactivation of Ta is accomplished by the hydrolysis of GTP

to GDP. Reessociation of Ta with TPy pennits the release of PDEy which retums to

PDEaP to restore its inhibitory state (Artemyev et al., 1998). Reopening of calcium

charnels through the binding of cGMP, which is synthesized fiom GTP by guanylate

cyclase, increases ca2' concentration (Hargrave, 1 986).

The concentration of calcium in the outer segment is thought to rnediate the

capacity of the visual transduction system to adapt to varying light intensities. ca2' ions

enter the outer segment by cGhdP-gated channels and are removed by ~ a ' l ~ a " , K'

exchanging pumps. The closure of cGMP-gated cation channels causes a cytoplasmic

decrease in ca2' which is detected by recoverin, a calcium binding protein. Recoverh is

thought to participate in visual transduction by preventbg the phosphorylation and thus

deactivation of rhodopsin (Enkson et al., 1998).

An interesting feature of the light transduction cascade is its abiiity to ampli@ the

signalling process. One activated, rhodopsin protein is capable of activating 500 G-

protein molecules. Furthemore, activation of PDE lads to the hydrolysis of 2000 cGMP

molecules per second. In effect, the absorption of a simgle photon causes the amplification

of its detection 106 fold (Bumside and Dearry, 1986).

Photoreceptor Outer Segment Structure

The photoreceptor outer segment is highly specialized for photon absorption and

phototransduction. It comprises of flattened membranous disks which are stacked on top

of one another. The most abundant protein in the disk membrane is rhodopsin which

represents 95% of the protein in bovine disk membranes (McDowell, 1993).

Morphological differences between rods and cones exist in their OS organüration. In

cones, disks are continuous with the plasma membrane (PM) while in rods the disks are

pinched off from the PM to fonn closed intracellular vesicles, except for those at the base

of the OS (Fig. 1-2) (Cohen, 1970). The continuity of disks and the PM in cones is

thought to reduce the siie of cone disks as they are displaced apically, resulting in the

typical tapered shape of the cone OS (Corless and Fetter, 1987).

Although the mature photoreceptor is not mitotic, it still undergoes the dynamic

process of disk renewal in its OS (Usukura and Obata, 1995). Primate rods contain

approximately 1000 disks making up 95% of the total md outer segment membranes with

the average rate of disk renewal calculated to be 10% per day (Young, 1976; Anderson et

ai., 1978).

The process of disk renewal is considered essential for PR cells in order to reduce

the accumulation of light darnaged components during time. As old disks are removed by

phagocytosis fkom the apical tip of PR cells by RPE cells, new disks are fonned at the base

of the OS. Thus, a balance is maintained so that the net length of the OS remains constant

(Besharse, 1986). The mechanism of disk morphogenesis is mediated by an actin-myosin

motor (Williams et al., 1992). Actin filaments are localized to within the ciliary axoneme

at the base of the rod OS by immunoelectron microscopy (Chaitin et al., 1984; Chaitin et

al., 1989; WiUiarns, 1991). The involvement of actin filaments in disk morphogenesis is

thought to be important because addition of cytochalasin D causes their depolymenzation

and perturbs disk formation (Vau& and Fisher, 1989). Recently, the product of the

Usher 1B syndrome gene was identified to be myosin VIIa and was determined to be

concentrated at the connecting cilia of PR cells (Kubota et al., 1997; Liu et al., 1997).

The role of myosin W a in photoreceptor biology is currently unknown, but it is thought

to maintain a barrier against difision of proteins between the imer and outer segments of

the PR (Liu et al., 1997).

Outer Segment Disk Rim Proteins

The vertebrate outer segment disk can be divided into two domains: 1) the central

lamellae, and 2) the highly curved domain refend to as the disk rim (Corless and Fetter,

1987). One of the key differences between the disk rim and lameliar regions is the

distribution of proteins in the two domains. For example, the integral membrane protein

rhodopsin is only present in the lamellar region, and is excluded fiom the disk rims

(Corless and Fetter, 1 987). Despite advances in understanding the molecular events

involved in the light transduction pathway, the components which provide the structural

integrity of the photoreceptor outer segment are less understood. The cloning and

characterization of RDS, ROMl and recently ABCR has highlighted the importance of

proteins located only at the disk rims in the photoreceptor outer segment. The fùnction of

ABCR is unknown, however, it is a member of the ATP transport superfarnily based on

protein sequence analysis (Sun and Nathans, 1996). In the cases of ROMl and RDS, the

roles of these two disk rim proteins has been greatly advanced by biochernical and genetic

analyses.

ROMl and RDS/Peripherin

ROMl and RDS are both integral membrane proteins which localize to the nms of

the rhodopsin containhg disks which comprise the outer segment (Bascom et al., 1992).

The two proteins are 35% identical at the amho acid Ievel. Despite the low degree of

identity between ROMl and RDS, the two proteins share similar structural characteristics.

Hydropathy plots for both ROMl and RDS reveal that both proteins contain four

transmembrane (TM) domains and a large hydrophilic stretch of amho acids (-140 aa)

between the third and fourth TM regions (Bascom et al., 1992). Furthemore, membrane

topology analysis on ROMl in microsomal membranes in vitro verined that ROMl is a

transmembrane protein with cytoplasmic N and C-te& and that the large central

hydrophilic loop lies within the lumen of microsornes (Bascorn et al., 1992). Interestingly,

the membrane topology of ROMl is consistent and comparable to that of RDS (Co~el l

and Molday, 1990). In addition, both proteins are comparable in site (37 kDa and 39.1

kDa, respectively ).

Polypeptide sequence analysis on ROMl and RDS hiflght fiirther similarities

between the two proteins. The most conserved region between ROMl and RDS occurs in

their hydrophilic loops (49% identity) with seven of seven cysteines present in this region

being conserved (Bascom et al., 1992). In addition, there is a stretch of arnino acids

(15/16) in the hydrophilic loop which is highly conserved and cornposed mainly of

cysteines and prolines (Fig. 1-3). There are also two other regions present in ROMl and

RDS which show consenration. The predicted junctions between the N-terminus and

TM1 as well as between TM4 and the C-terminus are identical between ROMl and RDS

(Fig. 1-3). These two regions of identity, each seven arnino acids long, are of unknown

fùnction, but could be required for important protein-protein interactions.

In contrast to the similarities between ROM1 and RDS, there are signifiant

differences to note which may have functional implications for the two proteins. For

instance, there are differences between the charges, and consequently the isoelectric points

of the two proteins, at the N and C-termini. The N-terminus of ROMl is absent of

negatively charged residues unlike RDS which does not appear to show an

underrepresentation of such amino acids (Bascom et al., 1992). With respect to the C-

terminus, RDS has been noted to be highly charged. ROMl, however, is only modestly

negatively charged in this region. Overall the theoretid pI of RDS, not accounting for

glycosylation, is 5.3 which is considerably lower than that of ROMl (5.98) (Boesze-

Battaglia et al., 1997). Finally, there are no observed Klinked glycosylation sites in

ROMl whereas one such site is present in RDS (Comell and Molday, 1990).

Associations behveen ROMl and RDS

Various independent experiments have been perforrned to show that ROMl and

R D S are capable of fonning disulphide-lied homodimers and non-covalent multimers

(Bascom et al., 1992; Goldberg and Molday, 1996; Goldberg et al., 1998). Bascom et al.

(1992), for instance, provided evidence of ROMl diaulphide linked homodimers with non-

Cytoplasm

Disk Membrane

Disk Lumen

PXXC

Figure 1-3. Structurai topology of ROM1. Amino acids conserved between RDS and ROM 1 are shown as open circles while substitutions are indicated by closed circles. The four domains that are identical or near identical between ROM1 and RDS are shaded in grey. Also shown are the motifs, referred to as the CCG, PXSC, PXXC, and ECG, in the hydrophiiic Ioop which are present in members of the TM4SF (Tomlinson and Wright, 1994). Each letter of the motifs stands for an arnino acid (C = cysteine, G = glycine, E = glutamate, P = proline, and X = any amino acid). Modified from R. Bascom.

reducing and reducing SDS-PAGE. ROMl and RDS non-covalently associated

heterotetrarners were identified through an anti-RDS monoclonal Ab 2B6-Sepharose

affinity column passed with detergent solubilized bovine ROS membranes (Bascorn et al.,

1992).

Recently, Goldberg and Molday (1998) have evaluated the ability of the 13

cysteine residues present in bovine RDS to form disulphide-linked homodimers, associate

with ROMl, and assemble into tetramers by replacing each cysteine with a senne residue.

Mutations involving the six non-conserved cysteines had no effect on dimer formation,

folding or subunit assembly . However, re placing any of the seven conserved cy steine

residues, al1 of which are present in the hydrophilic loop, dismpted these properties. Six

of the seven cysteine residues (including a C214S mutant linked to autosoma1 retinitis

pigmentosa) when altered a k t e d the normal folding of RDS, interactions with ROMl

and self-assembly into homotetramers. The seventh cysteine residue when rnutated

(C 1 SOS) did not affect the association of RDS with ROM 1 . However, it did incapacitate

the ability of RDS to fonn intennolecular disulfide bonds (Goldberg and Molday, 1998).

ROM1 and RDS are memben of the transmembrane 4 superflmily (TM4SF)

The structural similatities observed between ROMl and RDS have been observed

recently in a number of proteins, many of which are leukocyte cell-surface molecules.

Interestingly, each protein has the same membrane topology as that observed for ROMl

and RDS (Table 1-1). Currently, there are 19 TM4SF members which are found in

organisms fiom schistosomes to humans (Table 1-1).

The basic architecture of a TM4SF member is the presence of four hydrophobic

TM domains, cytoplasmic N and C-tennini and a large extracellular domain between TM3

and TM4. The membership of ROM1 and RDS into the TM4SF has been somewhat

controvenial due to the fact that they both contain long cytoplasmic C-terminal tails (65-

70 amino acids) which is uncharacteristic of other TM4SF members (5 -14 amino acids)

(Wright and Tomlinson, 1994). In addition, the hydrophilic loops of ROMl and RDS are

not extracellular but in fact protrude into the lumen of OS disks. It should be noted,

however, that the lumen of OS disks is equivalent to the extracellular environment

Table 1-1. Transmembrane four superfamily membus (TMMF).

Bascom et al., 1992

Protein

ROM1

Expression Pattern Rod photoreceptors, Ou ter Segment disk rims

Peri p heri n i RDS

XRDS3S distant relative of RDS

XRDS36 distant relative of RDS

Size (kb/ae and/or B a ) 37 kDa

Rod and Cone photoreccptor outer segment disk rims

Rods only

Rods only

Function

rnaintaining outcr segment stiucnirc

39 kl)a

maintainhg outer segment structure

345 aa

crds2 (chick)

Uropfakinla

Othtr Points

interacts with RDS non- covalently

345 aa

low expression in photoreccptors

bladder

364 aa

rnaintainiag outcr segment stnicturt and function

forms higher- order complexes with XRDS3S and

27 kDa

developing B cells, platelets, neuroblastoma cell lines, activated T cells, neurons, ocular ciliary epithelial cells

rnaintaining outer segment structure

Kedzierski et al., 1996

not known

Àsyrnmetrical Unit Membrane (AUM) prevents bladder

samc as Uroplaskin Ia

interacts with ROM1 non- covalently

avart of

signal transduction in platelets, cell adhesion, ce11 motility

Travis et al., 1991

forms higher- order complexes with XRDS38 and

expression scen in the retina at

Uroplaskin 11 (15 D a ) oligomerizes

1 interacts with Uroplaskin III (47 kDa)

Kedzierski et al., 1996

Weng et al., 199 8

embryonic &y 18 interacts with

Wu et al:, 1995; Finch ci al., 1997

associates with alpha 3, 4, 5, 6 and Bcta 1 integrins, CD19

Yu et ai.. 1994;

Yu et al., 1994; Wu et al., 1995; Finch et al.,

ubiquitious: heart, spleen, kidney, lung, brain, liver, testis, skeletal muscle

1997 Loffler et al., 1997; Bcrditchevski et al., 1996; Shaw et al., 1995; Banejee et al.. 1997; Martin- Alonso et al., 1992; Horvath et

26 kDa Imai et al., 1993;

, Mannion et al., 1996; Seldin et

1 al., 1995;

signal transduction in B cells, ceIl adhesion in B, T and non- lyrnphoid cells

component of B ceIl signalling cornplex CD19KD211CDS l ,neu-13 associates with CD63, P14K and

:O-O29

Tomlinson et al., 1996

2D37

rectal & colorec- tal carcinomas mature B cells low expression in T celis lymphoid tissues

32 kDa

281 aa 1.2 kb

not known

downrcguiated on B ce11 activation role in B ceIl poliferation

see CD82

associates with CD53, CD81, CD82, MHC class II, CD19, CD21 in B ccils

Sala et al.. 1990

Table 1-1 cont. Transmernbrane four superfamily members (TM4SF).

Protein

SAS

CD63ME49 1

AIS-

SFA- 1PETA-3

TI- 1

low expression iri

1 heart

spleen, thymus, heart, lung, kidney lower expression B and T cells, thymocytes number of tumour cell lines platelets, mclanosomes, lysosomes high in kidncy B, T and non- lymphoid cells ocular mclanoma tissues Schistosoma mansoni

Antigen

hem, lung, pancreas & prostate tissue epithelial cclls

human lung, breast, colon, ovarian carcinomas & heaithy epithelial tissue

1 Fonction

signal transduction in B cells

signal transduction in B cells, monocytes, granuloctyes, rat macrophages, NK and T mlIs growth remlation? growth regdation cell motility may link P14K ta alpha3, Betal integrin

not known

not known

metastasis signal transduction? involved in growth arrest

not known

Otber Points

associates with CD37, CD53, CD8 1, MHC ciass II, CD19 & CD21 in B celis & CD4 CD8 & CD81 in T cells expresscd on early CD4" CD&' cells immunoprecipitai es with phosphatase 12q13-14 hurnan

associates wi th alpha3 and Betal and alpha6 and Bctal intcgrins

most homologous to

llp15.S human

negati vcly ngulated by TGF- Beta

most distant relative of 4TM superfamily 3q21-25 human

Reterences

Adachi et al., 1996; Nagira et al., 1994; Imai et al., 1993

Tomlinson et al., 1993; Wright et al., 1993; Carmo et al., 1995; Tomlinson et al., 1995 Jankowski et al., 1995 Wang et al., 1992; Gwynn et al., 1996; Radford et al., 1997; Radford et al., 1996

Wright et al., 1990

Emi et al., 1993; Vinaneva et al., 1994 Hasegawa et al., 1997; Fitter et al,, 1995 Kallin et al., 199 1

Maruyama et al., 1996; Kurihara et al., 1997

(Hargrave, 1986). ROMI, RDS and their homologs may, in fact, represent distant

relatives of the characteristic TM4SF members (Wright and Tomlinson, 1994).

Furthermore, in addition to their structural topology, three other reasons strengthen their

incorporation into the TM4SF. First, both molecules contain three cysteine motifs in their

hydrophilic loops, a characteristic shared in aimost al1 TM4SF members (Fig. 1-3).

Second, uroplakins ta and lb, which are not leukocyte proteins, are restricted in location

and function to a terminally differentiated structure callea the asymmetric unit membrane

in the mamrnalian bladder epithelium. In the case of ROMl and RDS their functions and

locaiization are restricted to the photoreceptor OS in the adult retina. Third, many

TM4SF members participate in interactions with one another, as is the case for ROMl and

RDS (Table 1-1, see the column Other Points).

oni il'" mouse, implications on photomeptor structure and function

ROMl and RDS are related proteins that are physically associated at the disk nm.

The colocalization of ROMl with RDS at the disk rims of the outer segment, where they

are thought to be pan of the disk rim protein complex, suggests that both these proteins

may play a key role in outer segment function a d o r structure. Complete loss of RDS

function has dramatic consequences - (a) no outer segments form, and @) there is ce11

death (Travis et al., 1991a). In addition, RDS heterozygotes have - (a) disorganized outer

segments, @) larger-than-normal disks, and (c) ce11 death (Travis et al., 199ia). To

examine the effect of ROM1 on photoreceptor structure and function a mutant mouse

which is homozygous for the Roml allele was constmcted in our lab (G. Clarke,

unpublished).

Interestingly, a complete loss of ROMl function is associated with a less severe

phenotype than the complete l o s of RDS. Homozygous Roml mutants resemble Rds

heterozygotes, having - (a) disorganized outer segments (Fig. 1-4), (b) larger-than-normai

disks, and (c) ce11 death (G. Clarke, unpublished). The formation of large disks in the

R m l homozygous mutant animais demonstrates that ROMl is critical for the termination

of disk biogenesis, a property it shares with RDS (Travis et al., 1991a). Roml mice

heterozygous for a loss of fùnction dele, on the other hand, have Wtually nomal

Light micrographs of retinal sections from 1 month old ~oml+l+ and ~oml- l ' mice. Outer segments arc f m e d in the ~ornl-'- mouse, but are disorganized. All other layen appear normal. RPE - retinai pigment epithelium, OS - outer segment, IS - inner segment and ONL - outer nuclear layer. A lOOx magnification of the neuroretina is shown. Courtesy of G. Clarke.

photoreceptors (G. Clarke, unpublished). Altogether, these findings suggest that the

ROM1 protein plays a less criticai role than RDS in the biogenesis of the outer segment

and in disk morphogenesis, although it is essential to the mamrnaiian photoreceptor. The

different roles ROM1 and RDS have may reflect diaerences in the protein-protein

interactions in whidi these two proteins participate.

Initial characterization of PERl

The identification of retinal specific, abundantly expressed, and conserved (SAC)

cDNAs for genes has been an important step in understanding the structure, function, and

development of the mammalian eye. Two SAC cDNAs which have been cloned in the

McInnes lab are ROM1 (Bascorn et al., 1992) and CHXl O (Li et al., 1994). PHRl was

also identified in the McImes lab as an abundantly expressed and conserved retinal gene.

However, PHRl is also expressed at lower levels in the brain, kidney, liver and lung

(McInnes and Valle, unpublished). The PHRl gene has been mapped to 1 1 q 13.5- 14.1 by

FISH, oncor mapping panel and radiation mapping panel (Taylor and Mches,

unpublished).

The PHRl gene has nine exons, with exons 3-6 coding for a pleckstrin homology

(PH) domain (Fig. 1-5) (hicimes and Valle, unpublished). All PHRl transcripts encode

proteins that contain a PH domain at the N-terminus and a transmembrane (TM) domain

at the C-terminus. In the retina, two PHRl tninscnpts have been identified. The larger

transcript contains al1 nine exons (-729 bp) whereas the smaller transcript originates fiom

the sarne promoter as the full-length transcript, but is altematively spliced resulting in the

removal of exon seven. Two brain specific transcripts which are produced from an

alternative promoter located in intron have also been identified by RT-PCR (personal

communication, S. Xu) (Fig. 1-5).

The full-length PHRl predicted protein consists of 243 arnino acids (Fig. 1-6).

The PH domain is encoded by amino acids 22 to 127. Exon 7 encodes 35 amino acids

(13 1-165) which are immediately C-terminal to the PH domain (Fig. 1-6). A

transmembrane domain (amino acids 124 to 243) is present at the C-terminal end of PHRl

(Fig. 1-6).

Exons coding for the PH domain

Figure 1-5 . Genomic structure of PHRI. The gene has 9 exons, represented as boxes, one of which, exon 7 (shown in purple). is alternatively spliced. The PH domain is encoded by exons 3 to 6, as shown in red gradient boxes. The brain specific transcripts of PHRI are indicated by the pink lines. Brain specific franscripts are transcribed from a brain specitic promoter located in intron 2, as indicated by the B 1 box.

PHRl contains a region of similarity to seven characterized PH domain-containing

proteins: Akt (30% identity), cytohesin-1 (2g0/0 identity), GRPl (29% identity), ARNO

(29% identity), Cdc25 (23% identity), dynamin (24% identity), and oxysterol binding

protein (OSBP) (24% identity). The region of similarity between Akt (Fig. 1-7),

cytohesin- l , GRP 1, ARNO, and Cdc25 and PHRl is present only at the N-terminal part of

the PH domains of these proteins and ranges in length fiom 59 - 74 arnino acids. The

region of similarity between dynamin (Fig. 1-7) and OSBP and PHRI, however, occurs

over the entire PH domain.

Pleckstria homology domain

In 1993 two groups identified a family of sequences, of approximately 100-120

amino acids, which were originally discovered in pleckstrin, a major protein kinase C

(PKC) substrate in activated blood platelets (Tyers et al., 1988; Mayer et al., 1993;

Haslam et al., 1993). To date, more than 100 proteins involved in a variety of cellular

signalhg and cytoskeletal functions have been observed to contain PH domains. Many of

these proteins cm be grouped into seven distinct finctional categones: 1) senne/threonine

kinases, such as Akt/RAC, and PARK, 2) tyrosine kinases, like Btk, 3) regdators of small

G proteins, such as Ras-GAP, SOS1 and 2,4) endocytotic GTPases, like dynamin-1 and - 2, 5) adaptors, including IRS- 1, and 3BP2, 6) cytoskeletal associated molecules,

particularly spectrin, and pleckstrin and 7) lipid associated enzymes, such as PLC isoforms

(Shaw, 1998). In the majonty of cases, proteins containing PH domains have one copy of

this motif, however two copies of the PH domain have been identified in severai proteins

including pleckstnn, GRF and syntrophin.

Arnino acid alignments of the PH domains frMn diierent proteins reveals that low

identity exists in the PH domains of these molecules, fiom 10-20% (Lemmon et al., 1997).

The only invariant residue of the PH domain is a tryptophan residue in the C-terminal a-

helix. This residue is beiieved to be important for the structural stability of the domain

(Rebecchi and Scarlata, 1998) (Fig. 1-7).

The PH domain is an evolutionarily conserved motif that is present in a large

number of molecules ranging fiom yeast to human, suggesting an important biological role

Met Ser P r o A l a Ala Pro Val Pro Pro Asp Ser Ala Leu Glu Ser Pro

Thr P r o A l a Pro A l a Gly Ala T h r Val Pro Pro Arg Ser A r g Arg Val

Cys S e r Lys V a l Arg Cys Val Thr Arg Ser Trp Ser Pro Cys Lys Val

Glu Arg Arg X l e Trp Val Arg Val Tyr Ses Pro Tyr Gln Asp Tyr Tyr

Glu Val Val Pro P r 0 Asn Ala H i s Glu Ala Thr Tyr Val Arg Ses Tyr

Tyr Gly Pro Pro Tyr Ala Gly Pro Gly Val Thr H i s Val Ile Val Arg

Glu Asp Pro Cys Tyr Ser Ala Gly Ala Pro Leu A l a MetFly Met Leu

Aïa Gly Ala Ala Thr Gly Ala Ala Leu Gly Ses Leu Met Trp Ses Pro

Figure 1-6. The predicted PHRl protein sequence. Deduced primary amino acid sequence of PHRI. The PH domain is highlighted in blue while the amino acids encoded by the al tematively spliced exon 7 are shaded in yellow. Possible sites of protein-protein interaction are underlined. Predicted C-terminal transmembrane domain is bracketed.

Figure 1-7. Cornparison of PH domains. A. A cornparison between the PH domains of the dynamin family (DYN 1 and DYN2) from human, rat and mouse for DYNl and hunian for DYN2 aligned with the entire PH domain fro~n PHR 1. B. The PH domains of the AKT orthologs frorn mouse, bovine rat and human are aligned with the N-terminal pan of the PH domain from PHR 1. Amino acids shown in red and listed as the consensus sequence are identical between PHRl and AKT or dynamin. Amino acids shown in blue are conserved whereas amino acids shown in grey are not conserved. The first 63 amino acids of the PH doniain from PHRl are 30% identical to those of AKT whereas the entire PH domain of PHR 1 is 24% identical to the dynamin n~olecules (Altschul et al., 1997). The only invariant residue (W - tryptophan) between PH domains from differeiit inolecules is iiidicated by the arrow.

for this motif. In addition, many enzymes, such as Akt, Dbl, Vav, Bcr and Rac kinase

(Ingley et al., 1995) possess PH domains and have critical regdatory îùnctions.

Mutations in such proteins are implicated in oncogenesis and developmental disorders

(Ingley et al., 1995). In one of the most well-studied examples, mutations have been

found to cluster in the PH domain of Btk, a protein required for B-ce11 development and

poliferation. These mutations are responsible for some types of human X-linked

agarnmaglobulinemia ( K A ) and m u ~ e X-Linked irnmunodeficiency (Xid) (Rebecchi and

Scarlata, 1998). Interestingly, single amino acid substitutions in the PH domain of Btk,

corresponding to mutations found in XLA, have been associated with distinct fùnctional

defects. Two such mutations, R28C and E41K have been implicated to reduce or

enhance, respectively, the binding of Btk to phosphatidylinositol phosphate (PtdIns)

groups (Fukuda et al., 1996; Kojima et al., 1997).

PH domain structure

Currently, the structures of the PH domains of pleckstnn (N-terminal), p-spectrin,

dynarnin-1, PLC 6,, Son of sevenless (SOS1 - both human and murine), Btk and PARK

have been solved to high resolution (Rebecchi and Scarlata, 1998). In two cases, PLC 6 ,

and P-spectrin, the PH domains were determined in the presence of phosphatidylinositol

4,5-biphosphate (NP2) (Lemmon et al., 1997).

Although the primary sequence of PH domains are loosely conserved, the structure

shows a remarkable conservation of three dimensionai organization. The structure

consists of a pair of nearly orthogonal beta sheets comprised of four and three antiparallel

strands, forming a j3-sandwich. The P-sandwich is closed onand stabilkd at one corner

by a C-terminal amphipathic a-heh (Cohen et al., 1995; Lemmon et al., 1997; Rebecchi

and Scarlata, 1998). Moreover, each PH domain is electrostaticdy polarized. Typically

the positively charged face of the domain coincides with the hypervariable loops (Lemmon

et al., 1997). Loops separating the P-strands are variable in both length and sequence.

Furthemore, they can tolerate large insertions. For instance, the PH domain of PLCy is

split at the P3434 loop of its PH domain by three src homology domains (Rebecchi and

Scarlata, 1 998).

Structural studies pedomed on PARK and SOS 1 have reveaied severaî interesting

features of PH domains. In the case of PARK, a particularly unique aspect of its PH

domain is the presence of an extended C-teminal a-helix that behaves as a molten helix

required for protein-protein interactions (Fushman et al., 1998). The SOS 1 protein

contains a Dbl homology P H ) domain involved in GTP binding. Al1 proteins containing a

DH domain also contain a PH domain. In fact in al1 cases the DH domain is foflowed

imrnediately C-terminal by the PH domain. Recently, NMR studies on the DH-PH

domains in SOS1 indicated that critical interactions between the two motifs occur at the

most poorly conserved regions (Soisson et al., 1998). In addition, the PH domain retains

its characteristic three dimensional fold despite the nearby presence of another modular

domain, indicating that the PH domain is structurally stable (Soisson et al., 1998).

PH domain runction

Identifying PH domain ligands has largely involved in vitro studies exarnining

possible protein-protein or protein-lipid interactions. Based on structural similarities

between PH domains and retinol binding protein, it was speculated that PH domains may

be capable of binding to lipophilic molecules (Harlan et ai., 1994). To examine the site of

interaction between the PH domain of pleckstnn and vesicles containing PtdIns(4,5)P2, 'Il, 13 C, and 1 5 ~ chernical shifis were followed as a finction of added iipid ("Harlan et al.,

1994). Residues K13, K14, S16, V17, N19, T20, W21, K22 and G46 were al1 detennined

to be involved in PtdIns(4,5)Pz binding. interestingly, Harlan et al. (1 994) noted that al1

residues involved in lipid binding were located in the N-terminal region of the PH domain.

Furthemore, substitution of each lysine residue for asparagine caused mutant pleckstnn

molecules to exhibit a -1 0 fold loss in binding afanity for PtdIns(4,5)Pz indicating that the

positively charged lysines are required for interacting with the negatively charged

phosphates (Harlan et al., 1995). Further evidence has also been collecteci fiom the X-ray

crystai structure of the PH domain fiom PLCG1 complexed with PtdIns(l,4,5)Pi. The

solution of this complex revealed that the positively charged face of the PH domain is

required for interacting with phosphatidylinositol phosphates (Ferguson et al., 1995).

The specificity of interaction between PH domains and phosphoinositides has also

been shown through comparative binding analysis of PH domains from different proteins.

Such studies utilize 3~-labelled phosphoinositides or examine quenching of intrinsic

tryptophan fluorescence afler binding. For instance, Akt has been observed to interact

with PtdIns(3,4)P2 and PtdIns(3,4,5)P3 specificaily, whereas dynamin is capable of

associating only with PtdIns(4,5)P2 (Frech et al., 1997; Rameh et al., 1997; Hirata et al.,

1998; Barylko et al., 1998).

The involvement of the PH domain with phosphatidylinositol phosphates suggests

that a role of this motif is to recruit proteins to the membrme, thereby targetting the PH

domain-containing molecules to the appropriate cellular cornpartment. Evidence for the

plasma membrane association of PLCG1 and PLCy has been obtained by

immunofluorescence studies using recombinant proteins in Cos and MR33 cells,

respectively (Paterson et al., 1995; Falasca et ai., 1998). Positively charged N-terminai

residues in the PH domain of PLCGi have been altered to show that they have a critical

role in phosphatidylinositol phosphate binding (Yagisawa et al., 1998). The plasma

membrane recruitment of pleckstrin and spearin has also been assessed by

immunofluorescence (Wang et al., 1995; Wang et al., 1996; Ma et al., 1997). In the case

of pleckstrin, Ma et al. (1997) have shown that pleckstrin is required for the formation of

membrane projections in transfected Cos-1 cells, indicating that the recruitment of

pleckstrin to the membrane is of structural and functional significance.

The specific binding of phosphatidylinositol phosphates to the PH domain of

proteins such as Akt and GRPl has been associated with the function of these molecules.

Binding of PtdIns(3,4,5)Pi, but not PtdIns(4,5)P2, makedly enhanced the guanine

nucleotide exhange activity of GRPl (Klarlund et ai., 1998). Activation of the kinase

domain of Akt requires the association of its PH domain with PtdIns(3,4)Pz7 and not

PtdIns(3 ,4,5)P3 (Klippel et al., 1997; Franke et al., 1997).

As with protein-lipid binding, protein-protein interactions are another major source

of signal transmission in various intraceliular signalling cascades. Associations between

PH domains and proteins such as PKC and G protein Py-subunits have been observed.

The ability of the PH domains of Btk and RAC-protein kinase to bind to PKC has been

demonstrated both in vivo and in vitro (Yao et al., 1994; Yao et al., 1997; Konishi et al.,

1996). The functional consequence to Btk when PKCP binds to it is that Btk becomes

phophorylated (Yao et al., 1994). As a result of phosphorylation by PKCB, the activity of

Btk is dom-regulated. In addition, Yao et al. (1994) have exarnined the effm of PKCP

binding with altered fonns of Btk. The GST-Btk(xid) protein with the Ala28Cys mutation

showed lower PKCP binding, suggesting an essential need for the arginine residue in Btk-

PKCP association. Reduced Btk-PKCP binding may in fact have a major impact on the

disease manifestations of Xid rnice by reducing B-cell poliferation and, thus the immune

system. In the case of RAC-PK, binding has been observed in vitro between its PH

domain and the PKCG isofonn resulting in the phosphorylation of PKCG (Konishi et al.,

1996). The association of the PH domain of RAC-PK and PKCG was found only aîter

cells were heat-treated, suggesting that RAC-PK may participate in the cellular response

to stress through its PH domain (Konishi et al., 1996). Interestingly, the region to which

PKC isofonns bind to the PH domain has been shown in vitro to map to the amino-

terminal portion of the PH domain of Btk (Yao et al., 1997). In other proteins this region

of the PH domain also binds phosphatidylinositol phosphate groups. Yao et al. (1997),

suggest that molecules such as PtdIns groups, which bind to the N-terminal portion of the

PH domain, may act to regulate the binding of the PH domain to PKC isoforms.

The ligand-induced activation of many receptors leads to the dissociation of the

py-subunits from the a-subunit of heterotrimeric G proteins, both of which regulate many

different effector molecules involved in signal transduction. In many cases, proteins

involved in intracellular signal cascades contain PH domains. In vitro studies involving

GST fusion proteins containing PH domains fiom several signalling molecules such as

Btk, Akt, oxysterol binding protein, PARK, IRS-1, and PLCy have been observed to

interact with GPy subunits (Touhara et al., 1994; Konishi et al., 1995). Studies involving

truncated PH domains have indicated that the critical region for interaction with GPy

subunits includes only the C-terminai portion of the PH domain and sequences just distal

to it (Touhara et al., 1994). Furthemore, GPy subunits are known to contain W 4 0

repeats, a 40 amino acid motif with multiple trp-asp dipeptides. Interactions between the

PH domains and WD40 repeats have been noted in vitro, suggesting that the WD40 motifs

are important for protein-protein associations with PH domains (Wang et al., 1995).

Recently, the IRS-1 and IRS-2 PH domains have been show to bind to acidic

motifs in proteins (Burks et al., 1998). Using the yeast two-hybrid system and the PH

domains of IRS-1 and iRS-2 as baits, Burks et ai. (1998) identified three proteins Lon

protease, myeloblast protein, and nucleolin. These results are the first exarnple of the

identification of PH domain interacting proteins using the yeast two-hybnd system.

Although the roles of these molecules in insulin action are not known, each protein

contained an acidic motif of aspartate and glutamate residues capable of interacting

specifically with the PH domain of IRS-2. Only the acidic motif present in nucleolin

bound to IRS- 1, suggesting that the PH domains of IRS- I and IRS-2 are not identical. By

using peptides containing the acidic motifs identified in the two-hybrid system, Burks et al.

(1998) were able to show that binding of the acidic peptides dismpted IRS-1 and IRS-2

coupling to the activated insulin receptor.

Understanding the function of the PH domain has relied mostly on in vitro studies.

Analysis in vitro has dissected the PH domain into two functional components. The N-

terminal region of the PH domain is required for phosphatidylinositol phosphate and PKC

binding, whereas the C-terminal region is essential for G protein association. In vivo

experiments showing interaction between the PH domain of the Btk and G proteins, or

PKCP or c, have been obtained using co-irnrnunoprecipitation (Yao et ai., 1997; Jiang et

al., 1998). To further elucidate the significance of various protein-protein or protein-lipid

associations observed, more detailed in vivo investigations are required for this

biologically important domain. Subsequent in vivo analysis can help determine the

physiological relevance of observed interactions involving PH domains in terms of

functional effects on signalling pathways, cellular morphological changes or regdatory

events.

Identifying ROM1 and PHRI interacting proteins using the yeast two-hybrid

system

The two-hybrid system relies on the structure of particular transcription factors

that have two physically separable domains: a DNA binding domain and a transcriptional

activating domain. The DNA binding dornain targets transcription to specific promoter

sequences (UAS, upstream activating sequence), whereas the activation domain serves to

initiate transcription by facilitating the assembly of the transcription complex. The fact

that a fùnctional transcription factor can be reconstituted through non-covalent

interactions of two hybrid proteins, with one containing the DNA-binding domain and the

other containing the activation domain, is the basis of the two-hybrid system (Bai and

Elledge, 1996). The hybrid proteins are usually transcnptionally inactive alone or in the

presence of a non-interacting hybrid protein. However, if two interacting hybrid proteins

are co-expressed, a reconstituted transcription complex can be assembled at an upstream

activating sequence, thereby activating expression of a testable reporter gene (Bai and

Elledge, 1 996).

The two-hybrid system has three major advantages over biochemical methods such

as co-immunoprecipitation and chromatography in detecting protein-protein

interactions. First, the yeast two-hybrid system was designed to identify cDNAs encoding

proteins that physically associate with a given protein in vivo. Second, with the

development of cloning vecton such as pACTXI, large representative cDNA libraries from

a vanety of tissues can be constructed and screened efficiently. Third, this method of

identifjmg protein-protein interactions can be used to define or test the domain necessary

for the interaction of two proteins. For these reasons, 1 chose the two-hybrid system as

the method to identify putative ROM1 and PHRt interacting proteins.

CHAPTER 2

Identification of ROM1-interacting proteins using the yeast two-hybrid system

The vertebrate photoreceptor cells are unique neurons, both stmcturaily and

functionally (Besharse, 1986; Bumside and Dearry, 1986). In particular, the

photoreceptor outer segment is a highly specialized component of the photoreceptor. In

primates, the outer segment is composed of flattened disks stacked one on top of the

other. Each disk is independent and separated from the plasma membrane in rod

photoreceptors, whereas in cones the disks are exposed to the extracellular environment

(Besharse, 1986). Each disk is composed of a rim and a centrai region called the lamella,

the latter containing the majonty of the proteins involved in the phototransduction

pathway (Farber and Shuster, 1986). The nms have generaily been thought to have only a

structural role, seMng as points of attachent for proteios to the plasma membrane and to

other neighboring disks.

Presently, only three disk rim proteins have been identified: ROMl, RDS and

ABCR. The ABCR protein is a member of the ATP transport farnily (Sun and Nathans,

1996), which generally mediate ion transport. Although, the ligand transported by ABCR

is not known, mice homozygous for mutant abcr show no outer segment abnormality at

least to two months of age (personal communication G. Trais) suggesting that ABCR

may have no critical structural role in maintainhg the integnty of the outer segments.

Outer segment structural roles for both ROMl and RDS, however, have been identified.

Mice homozygous for mutant R h fail to develop outer segments, even though an aborted

attempt at disk biogenesis is made, indicating that RDS is essential for disk formation

(Chaitin, 1991). Mice homozygous for mutant Roml, in contrast, do form outer

segments, but they are disorganized and contain enlarged disks (personal communication

G. Clarke), demonstrating that ROMl plays a criticai role in disk rnorphogenesis.

ROMl and RDS are related protehs, each capable of forming disulde linked

homodimers (Bascom et al., 1992). Moreover, interaction between ROMl and RDS

homodimers lads to the formation of non-covalent multimers (Bascom et al., 1992).

Both ROMl and RDS share the same structurai topology (Bascom et al., 1992). The

presence of four transmembrane dornains, two cytoplasmic t d s and a large loop between

TM3 and TM4 places ROMl and RDS in the four transmembrane superfamily (TM4SF)

(Wright and Tomlinson, 1 994).

There are currently 19 known TM4SF members, seven of which are expressed by

leukocytes. Generally, more than one TM4SF protein is present in the sarne cell, as is the

case for ROMl and RDS (Wu et al., 1995). In addition, almost al1 TM4SF members are

capable of interacting with another member of the superfamily (Table 1-1). Furthemore,

TM4SF members are also capable of interacting with other proteins. For example, CD9

and CD63 associate with alpha and beta integrins (Rubinstein et al., 1994; Nakamura el

al., 1995; Radford et al., 1996), uroplakin la and uroplakin 1 b interact with uroplakin II

and uroplakin III, respectively (Yu et al., 1994; Wu et al., 1995). Identifjmg protein

associations involving TM4SF members has generally relied on co-immunoprecipitation

expenments, however, characterization of the TM4SF proteins regions mediating the

obsewed interactions studied have yet to be determined.

ROM1 and RDS appear to be part of a multi-protein cornplex at the disk rim. In

order to elucidate the function of ROMl through the identification of proteins present at

the disk rim capable of interacting with ROMl besides RDS, a two-hybrid screen was

initiated. Identifjmg ROMl interacting partners is a first step to begin to understand the

biochemical role of ROMl in the outer segment of the photoreceptor. In addition, the

ROMl interacting proteins may in fact be essential disk rim components. Thus, mutations

in such important disk rim protehs may be the moleculai defect behind uncharacterized

retinal degeneration diseases.

Since ROMl is a transmembrane protein which traverses the disk membranes four

times, it cannot be used in its entirety as a bait in the two-hybrid system. For this reason,

only hydrophilic regions were chosen as bait to test for protein-protein interactions in the

two-hybnd system. Hydrophilic regions of transmembrane proteins have been used to

identiQ protein hteractors successfbily using the two-hybnd system (Bauch et al., 1998;

Shiratsuchi et al., 1998).

1 chose to do the first ROMl two-hybnd screen with the C-terminal tail for two

reasons. First, its placement in the cytoplasm offers a putative site for protein-protein

interactions with cytoplasrnic proteins present near or at the disk rims. Secondly, the C-

terminal tail is the lest conserved region between ROMl and RDS, suggesting that

unique ROM1-interacting proteins might be identifieci by using this part of ROMl as the

two-hybrid bait. In addition, there is a perfkctly conserved seven peptide sequence in

ROMl and RDS, present at the boundary between the Gterminal tail and the fourth

transmembrane domain (Fig. 1-3). This region of conservation between the two proteins

may be a site of protein-protein interactions.

The C-terminal tail of ROMl was fused to the G U binding domain and used as

a bait to screen an adult bovine oligo-dT retinal library. From the screen 1 have isolated

five different cDNAs which encode polypeptides capable of interacting specifically with

the ROMl C-terminal tail. Whether the interaction between ROMl and these proteins is

physiologically relevant will require confinning the interactions with direct in vitro

approaches such as afijnity chromatography and detennining their expression in the retina

by itt siiu hybridization.

EXPERIMENTAL PROCEDURES

PCR

The DNA encoding the mouse ROMl C-terminal tail (amino acids 285 - 35 1)

encoding DNA (Bascom et al., 1993) was amplified Rom plasmid DNA containing full-

length Rom1 cDNA cloned into pBIuescript KS (Stratagene). The primer set used in the

PCR reaction consisted of the 3 1 -mer 5'

GGAATTCCATATGGGTTTGCGGTATTTGCAG 3', which contained a NdeI linker and

the 26-mer 5' CGGGATCCCTAGGCCTCAGCTAGAAC 3', which contained a Pst1

linker. The components used in the amplification were: 10 mM Tris, pH 8.0, 50 mM

KCI, 1.5 mM MgCli, 0.01% gelatin, 10 pM of each (MTP, 50 ng of each primer, and 2 ng

of plasmid DNA. Folowing an initial denaturation at 98OC for 5 min, 2.5 U of PFU

polymerase (Stratagene) were added and the tube was sealed with mineral oil. Each PCR

cycle (total 30) was carried out as follows: a denaturation step at 9S°C for 30 sec, an

a~eal ing step at 59OC for 1 min, and an extension phase at 72°C for 2 min.

PCR products were purified using the Qiagen PCR purification kit according to

the manufacturer's specifications (Qiagen, CA). The ampliecl product was then digested

with NdeI and Pstl (BRL) at 37OC for 2 hrs and purified by a phenol:chlorofotm

extraction method (Sambrook et al., 1989).

Su bcloning

The two-hybrid vector used in the subcloning procedure was pASl

(CLONTECH). It was digested with NdeI and Pst1 (BRL) at 37OC for 2 hrs and

subsequently isolated and purified fiom a 1% TBE agarose gel in 1X TBE buffer using a

QIAEX II gel extraction kit (Qiagen, CA). Purifieci mouse ROM1 C-terminal tail cDNA

fragments were ligated into 100 ng of vector for a 1 :2 molar ratio of vector to insert with

1 unit of T4 ligase (BRL) in 1X ligase buffer. The ligation reactions were incubated

overnight at 14OC. DHSa competent cells (BRL) were transformed with haif of the

liçation reaction and plated ont0 LB agar plates containing 100 pg/ml ampicillin. Plates

were incubated at 37°C overnight.

Plasmid DNA from cultured transformants were prepared using the Qiagen

Plasmid Miniprep kit (Qiagen, CA). Restriction enzyme digestions were perfonned to

identiQ positive transformants.

Sequencing

Sequencing reactions were perfonned with the T7 dideoxy DNA sequencing kit

(Pharmacia) and [ 3 ' ~ ] d ~ ~ ~ (NEN) according to the protocol designed by the

manufacturer. Reaction products were separateci on 6% polyacrylamide gels

(acrylamide/N'N/ -bis-methy lene-acrylamide ratio 3 6.51 1, 0.053% APS, 0.052% Temed) at

80 W for 2-4 hrs. Gels were fixed with 10% glacial acetic acid and 10% methanol

solution, dried for Ihr at 80°C and exposed to XAR film (Kodak). The oligonucleotide

primer used to sequence the two-hybnd bait construct was 5'

TCATCGGAAGAGAGTAG 3'.

Transformation o f Y 190 with bait construct

The yeast strain Y 190 (CLONTECH) was transforrned with the ROMl C-terminal

tail bait construct using a slightly modified version of the CLONTECH procedure. An

ovemight culture of Y 190 was grown at 30°C in synthetic dropout (SD) media lacking

uracil until an ODm of 0.5-0.8 was achieved. The cells were pelleted at 3000 rpm for 5

min, resuspended in 1 ml of sterile double deionized H20 (ddHZO) and transferred into

eppendorf tubes. The resuspended pellet was then centriiùged at 14000 rpm for 1 min.

The supernatant was cleared and the remaining pellet was resuspended in 5 volumes of

1 M lithium acetate (LIAOC) to make the cells competent. In separate eppendorf tubes,

the following components were added: 300 pl of PEG rnix (40% PEG/ 1 X TE/ 1 X

LiOAC), 10 pl of 5 mglml denatured, single-stranded salmon spenn (Pharmacia), 3-5 pl of

plasmid DNA, and 100 pl of competent Y 190 cells. The transformation mix was vortexed

for 30 sec, shaken at 300 rpm for 30 min at 30°C, and then heat-shocked at 42OC for 20

min. Ceils were centrifùged at 14,000 rpm for 1 min, resuspended in 100 pl of ddH20 and

plated ont0 appropriate selective plates. Transformations were incubated for two days at

30°C.

Preparation of yeast protein Iysates

Overnight 10 ml cultures of the ROMl bait and pVA3 positive control (p53/GAL4

DNA-binding domain hybnd) in Y190 were grown in SD selection medium lacking

tryptophan to an ODW of 0.5-0.8 units. The bait vector, pAS1, contains the TRPl gene

for selection in trp- auxotropic yeast strains. Cells were pelleted and resuspended in 1 ml

of sterile dd&û containing 10 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma). The

resuspension was transferred to an eppendorf tube and centrifùged at 14000 rpm for 1

min. The supernatant was cleared and the pellet was resuspended in 3 vol of 3X laemmli

sample buffer (Sambrook et al., 1989) with 10 mM PMSF and 1 vol of acid washed glass

beads (Sigma). The cells were lysed by vortexhg for 30 sec and cooling on ice for 1 min

(six times), boiling for 5 min, placing sarnples on ice for 5 min and then centrifughg for 5

min at room temperature. The supernatant was transferred to a fresh eppendorf tube and

stored at -20°C.

SDS PAGE

Yeast protein lysates were resolved using 5% stacking and 10% separating

acrylamide gels, 0.75 mm thick, in the Bio-Rad Minigel system. The amount of yeast

lysate loaded per lane was 5-10 pg. Gels were stained with either Coomasie blue

(Sambrook et al., 1989) or the proteins were transferred to nitrocellulose for Western blot

analysis.

Western blots

Proteins were electrophoretically transferred to Hybond C nitrocellulose. The

transfer was performed at 4OC for 1 hr at 100 V in transfer buffer which consisted of 25

mM Tris, 192 mM glycine and 20% rnethanol. M e r transfer, the nitrocellulose blot was

washed with Tris-buffered saline (20 rnM Tris-HCI, 0.5 M NaCl, pH 7.2) including 0.05%

Tween-20 (TBST). Non-specific background was elirninated by blocking the blot with

5% milk solution dissolved in TBST for 1 hr at room temperature, followed by one 5

minute wash at room temperature with TBST. The blot was then incubated ovemight at

room temperature with anti-GALA anti-mouse IgGh monoclonal antibody (Santa Cruz)

diluted in 1% milk in TBST (1500 dilution). Unbound pnmary antibody was removed by

three 30 min washes with TBST, at room temperature. The nitrocellulose blot was

subsequently incubated for 1 hr at room temperature with the secondary antibody anti-

mouse IgG (whole molecule) peroxidase conjugate (Sigma) diluted 15000 in TBST with

1% milk. Excess secondary antibody was removed by three 30 min washes with TBST

solution at room temperature. The electrochemical luminesence @CL) detection system

(Dupont) was employed to visualize the protein bands after exposure to film.

Beta-galactosidase assays

a) Filter assay for P-galactosidase

Whatman filter papers were layered over agar plates containing appropriate

selection medium. Colonies to be assayed were streaked in small patches directly ont0 the

filters. The plates were incubated at 30°C for one day to allow the cells to grow. The

filters were then lifted out and placed ceIl side up in liquid nitrogen in order to penneablize

the yeast cells. M e r 30 seconds of exposure to liquid nitrogen, the filters were placed on

Whatman 3 MM chromotography paper in unused petri dishes, soaked with a Z buffer/X-

gal solution. The Z buffer (pH 7.0) contained 16.lgL NaZHPOJH20 , 5.5 g/L

NaH$04*H20, 0.75 g/L KCI, and 0.246 g/L MgSO1m20. From a stock X-gal solution

of 50 mg/ml, 10 pL was added to 2.5 ml Z buffer. The filters were then placed at 30°C

between 30 min to overnight.

b) Liquid culture assay using ONPG (O-nitro P-D-galactopyranoside) as substrate

Ovemight cultures of 5 ml were prepared in liquid SD selection medium and

grown to saturation. The next day 2 ml of the overnight cultures were transferred to 8 ml

of YPD and incubated at 30°C for 3-5 hrs with shakuig (250 rpm) until the cells reached

an OD600 of 0.5-0.8 units. On the day of the experiment, ONPG (Sigma) was dissolved in

Z buffer to a concentration of 4 mg/ml by shaking for 1-2 hrs.

M e r reaching mid-log phase, the cells were hawested by placing 1.5 ml of each

culture into eppendorf tubes, centrifûged at 14,000 rpm for 1 min, decanted and then

resuspended in 1 .S ml of Z buffer. The cells were vortexed and centrifbged again. The

supernatant was removed and the pellets were resuspended in 300 pl of Z buffer. To a

fresh microcentrifùge tube, 0.1 ml of the ce11 suspensions were added. The tubes were

placed first in liquid nitrogen for 1 min and then in a 37OC water bath for 1 min to thaw

the cells. This process was repeated three times to ensure ceii fiactionation. A blank tube

was also set up containing only 0.1 ml of Z buffer. To 0.7 ml of Z buffer 1.9 pl of P- mercaptoethanol was added. The Z buffer/P-mercaptoethanol solution was dispersai into

each reaction tube, and 160 pl of ONPG in Z buffer was then added to each tube. The

time it took for the yellow colour to develop at 30°C was measured. Once the yellow

colour appeared 0.4 ml of 1 M Na2C03 was added to the tube and the timer was stopped

for each reaction and blank tube. The tubes were centrifbged for 10 min at 14,000 rpm.

The supernatants were transferred to clan cuvettes. Samples were then read in a

spectrophotometer (Beckman DU-65) at an OD of 420 nrn relative to the blank. The

following equation was used to detennine the amount of activation present (Miller, 1972):

P-galactosidase units = 1000 x ODud(t x 0.5 x ODa)

where: t is the elapsed time in min

Two-Hybrid sçreen

The bovine adult retinal oligo-dT primed two-hybnd library was a gift from Ching-

Hwa Sung of Corne11 University. The two-hybrid protocol followed was designed by D.

Gietz (Gietz and Schietl, 1995). A 10 ml ovemight culture of Y 190 cells with the bait

plasmid was begun in SD lacking tryptophan (trp) and uracil (ura). The following

morning the culture was diluted into 300 ml SD lacking trp and ura and grown until an

OD, reading of 0.6 was achieved the next day. Two flasks containing 250 ml YPD and

50 ml of 300 mg& adenine were warmed at 30°C. The yeast culture was diluted into the

YPD media (total volume 500 ml) to yield a starting O D a of 0.15 and then grown for

four hours or until the ODsW reached 0.6.

The cells were centrifùged at 5000 rpm for 5 min. To the pellet, 25 mls of sterile

water was added and the pellet was disrupted. Resuspended pellets were combined into

one GSA bottle. A lithium acetate mix (40 ml) was prepared containing 100 mM LiOAC

and iX TE, and added to the resuspended ceils. The cells were centrifùged at 1500 rpm

for 5 min. Finally, the pelîeted cells were resuspended in 5 mls LiOAC mix to achieve

cornpetence. As a control for the two-hybrid screen, 100 pl of the LiOAC treated cells

were removed and to each aliquot was added one of the following: i) no DNA plated

ont0 SD -leu, -trp, -ura; ii) pGAD plated ont0 SD -leu, -trp, -ura; and iii) pGAD plated

ont0 SD -leu, -trp, -ura, -his with 30 mM amino-triazole (Sigma). The plasmid pGAD

contains the full-length GAIA protein cloned into pASl (CLONTECH). To the remaining

cells were added LOO pg of the retinal target library DNA and 5 mg of denatured salmon

sperm (Pharmacia). Retinal denved cDNAs were subcloned into the p ACT11 vector which

contains the LEU2 gene for selection in leu- auxotropic yeast strains. CeUs (0.5 ml

aliquots) were then transferred into 10 round bottom polypropylene tubes and to each

tube 3 rnls of PEG mix (40% PEG/lX LiOAC/lX TE) was added; tubes were inverted to

rnix the contents. The transformation mix was incubated at 30°C for 30 min and then

heat-shocked at 42°C for 20 min. Cells were centrifùged at 1500 rpm for 3 min, decanted

and resuspended in 2 mis of sterile water.

In order to obtain 1x10' transformants per 150 mm plate, 0.65 mls of the library

transformation was added to each plate containing SD -leu, -trp, -his with 30 mM arnino-

triazole (Sigma), for a total of 30 plates. To determine the transformation efficiency a

I / 100 fold dilution of the bait plus library was made and plated as 20, 100 and 500 pl

aliquots ont0 3 separate SD - leu, -trp plates. Library plates were incubated for 5-7 days

at 30°C while the control plates were grown for 2 days at 30°C. Colonies which appeared

much larger than background colonies were selected and placed onto fiesh SD -leu, -trp,

-ura plates and then tested for lac2 expression by filter f&galactosidase assays.

isolation of two-hybrid targets

From the two-hybrid screen, yeast transformants that were capable of growth on

-his 7 3-AT plates and were Lac' were subject to additional analysis. For this purpose,

the targets from each independent positive were isolateci. A 3 ml ovemight of each

positive transformant was inoculated into SD liquid media lacking leucine. Cells were

centrifbged at 3000 rpm for 10 min. Each pellet was resuspended in 200 pl of sterile

water and transferred into 1.5 ml eppendorf tubes. To each tube, 0.2 ml of yeast lysis

solution (2% Triton X-100, 1% SDS, 100 mM NaCI, 10 mM Tns-HCI pH 8.0, and 1 .O

mM EDTA), 0.2 ml phenol/chloroform/isoamyl alcohol (2924: 1) and 0.3 g of acid-

washed glas beads were added. Each tube was vortexed for 3 min to lyse the cells and

then centrif'uged for 10 min at room temperature. Supematants were transferred into fresh

eppendorf tubes. DNA was precipitated by adding 1110 volume 3 M NaOAC and 2.5

volumes 100% ethanol. Tubes were centrifùged for 10 min at 4OC and then decanted.

DNA pellets were washed with 70% ethanol, centrifùged for 10 min at 4°C and then dried

under a vacuum for 10 min. Yeast minipreps were resuspended in 50 pi 10 mM Tns-HCI

pH 8.0.

Yeast minipreps were cleaned using the QIAEX II gel extraction kit. Each

purified miniprep (5 pl) was transformed into MH6 E. coli cells (gift from B. Andrews)

which are ampicillin sensitive and have a defect in the leuB gene that can be complemented

by the LEU2 encoded plasmid gene. Transformed cells were plated on LB agar plates

containing 100 pg/ml ampicillin. Colonies isolated the foUowing day after growth at 37OC

were patched ont0 M9 minimal plates (Sambrook et al., 1989) lacking leucine. Cells

capable of growth after incubation of plates ovemight at 37°C contained the target

plasmid only. Target DNA was then isolated by a standard alkaline lysis procedure

(Sarnbrook et al., 1989) and sequenced to identify the target capable of interacting with

the bait in yeast. Target DNA was sequenced with a primer upstream of the insert 5'

CTACAGGGATGTTTAATACC 3'.

RESULTS

Construction of the ROMl C-terminal tail bait

To investigate the role of ROMl in photoreceptor structure and function, a two-

hybrid screen was initiated. The ability of ROM1 to interact with RDS has been well

analyzed, however, the disk rim cornplex, to which they both belong to, is still poorly

characterized (Bascorn et al., 1992). Further insight into the fbnction of ROMl requires

identification of the proteins with which ROM1 associates. In order to identifL ROMI-

interacting partners a two-hybrid screen was initiated with the C-terminal tail of ROMl.

The DNA fragment encoding the C-temiinal tail of mouse ROMl was subcloned i n - h e

into the bah vector pAS1. Thus, the resulting bait fiision protein consisted of the ROMl

C-terminal tail and GALA bhding domain (Fig. 2- 1 a).

Analysis of ROMl Gtenninal bait expression in the two-hybrid system

Before proceeding with the two-hybrid screen, 1 optimized the screening

conditions by establishing expression of the bait in yeast celis and determining the ability

of the bait fusion aione to cause activation of the reporter genes. The bait consmict was

initially transformed into the yeast strain Y 190. This strain contains two reporter genes

GAL4 Binding Domain

HA epitope Mouse Rom1 C-ter tail - 22 arnino acids

Protein Markers ( k W

Figure 2-1. A. The mouse ROM1 C-terminal tail two-hybrid constmct, referred to as MRC. The constmct contains the mouse C-terminal tail (66 amino acids) fused in-frame to the GALA binding domain including the seven amino acid peptide present at the junction beween TM4 and the C-ter tail. An HA epitope separates the two polypeptides. B. Western blot of yeast lysates expressing the MRC bait Yeast lysates were separated on a 10% SDS-PAGE. Proteins were transferred ont0 Hybond C, probed with anti-GALA antibody and visualized using the ECL system. Lanes 1 and 2 show MRC fusion protein migrating at 34 kDa. Lane 3 shows the positive control, murine p53 (amino acids 72- 390) migrating at 54 kDa (see arrow) and lane 4 negative conml (Y 190 alone).

(HL93 and lad) used to analyze bait-target interactions identified in a two-hybrid screen.

The HIS3 gene is under the control of the native GALl UAS, while the locZ gene is under

the control of a synthetic UA& 17-mer consensus sequence (Bai and Elledge, 1996). The

upstream activation sequences are sites that the GALA binding domain can recognize. It is

important to note that the lac2 reporter gene was integrated into the Y190 yeast genome

along with a uracil marker (Bai and Elledge, 1996). Thus, maintenance of the lac2

reporter gene requires growing cells in the absence of uracil.

Western blot analysis of yeast lysates was performed in order to confirm that the

bait was appropriately expressed (Fig. 2-lb). Using an anti-GAL4 antibody, the mouse

ROMl C-terminal tail-GALA fusion protein (Fig. 2-1 b lanes 1 and 2), as well as a positive

control (GAL4 binding domain-p53) were readily detected in yeast lysates.

To determine whether the mouse ROMl C-terminai tail-GAZA fiision alone could

activate the two reporter genes, expression was assessed by examining the ability of the

bait strain to grow in media lacking histidine or by detecting P-galactosidase activity by

tilter or liquid assays. Conditions were optimized to identiQ the appropriate

concentration of 3-aminotriazole (3-AT) to be added to the agar plates lacking

tryptophan, histidine and uracil. The compound 3-aminotriazole inhibits the HIS3 gene

product, which is produced by low expression of the reporter gene in Y 190.

Various concentrations of 3-AT were added to plates, ranging from 15 m M to 50

rnM. Slight activation was observed with the ROMl C-terminal tail bait in the presence of

15, 20 and 25 m M 3-AT. However, this degree of activation was repressed by 30 mM 3-

AT. Thus, in order to perform the two-hybrid screen and reduce the number of false

positives identified, 30 rnM 3-AT was used in the medium of each library plate.

The extent to which the ROM 1 C-terminal bait activated the lacZ reporter gene

was also analyzed. Filter assays were used to test for the activation of P-galactosidase in a

qualitative assay. Functional B-galactosidase is capable of cleaving X-gal, causing the

yeast cells to produce a blue colour during filter incubation at 30°C. A qualitative P- galactosidase filter assay was perfomed with the ROMl C-terminal tail bait. Although

the bait was capable of activating of the IacZ reporter gene, this activation was very weak

compared to the positive control. To deterrnine the extent of the activation, a quantitative

B-galactosidase assay was perfonned (Fig. 2-2). The bait alone is capable of activating the

lm2 reporter gene (Fig. 2-2, MRC alone). in additon, the bait was tested in the presence

of a target protein which should not interact the ROM1 C-terminal tail. The ROMl C-

terminal bait with a negative control target plasmid pTDI, which encodes an SV40 large

T-antigenGAL4 activation domain fiision activated the lacZ reporter gene (Fig. 2-2,

MRC + pTD1). However, this degree of bait activation in cornparison to the positive

control, which contained an interacting bait and target, indicated that it is weak and

therefore managable in a two-hybnd screen (Fig. 2-2, pVA3 + pTD1).

Two-hy brid screen

Having determined that the ROMl C-terminal tail bait was expressed in Y190 and

having defined the appropriate concentration of 3-AT (30 mM) required to suppress

background activation of the HIS3 reporter gene, 1 performed a two-hybrid screen. The

oligo-dT primed adult bovine retinal library contains retina cDNAs cloned into the

p ACT11 target expression vector (EcoRI-XhoI). A total of 1 .5 million transformants were

screened using the methods designed by D. Getz (Gietz and Schietl, 1995).

The initial screen examined the expression of the HïS3 reporter gene. This step in

the two-hybrid system was accomplished by transforming the library DNA into Y190 cells

already containing the ROMl C-terminal tail bait and then plating the transformations

onto plates containing 30 mM AT and lacking leucine, tryptophan and histidine. Plates

were incubated at 30°C for 5-7 days until positive colonies were identified by their larger

sire in companson to background growth. From the total of 1.5 million clones screened,

233 HIS' transformants were isolated. Each yeast two-hybnd positive was picked and

streaked ont0 fresh plates which were incubated at 30°C for two days.

1 next exarnined the ability of the target protein to interact with the bait by

m e a s u ~ g activation of the lac2 reporter gene in P-galactosidase filter assays. The use of

a second reporter gene reduces the number of false positives by identifying those cDNA

encoded proteins which are capable of activating ody the HIS3 reporter gene. This

analysis led to the identification of 66 HIS' Lac' putative interactors.

Quantitative Liquid Beta-Gal Assay

Figure 2-2. Quantitative liquid beta-galactosidase assay. Samples were grown in appropnate yeast media and tested for expression of the lac2 marker. Quantitative vaiues were obtained using standard Müller uni6 shown on the y-axis. The negative control contained no plasmid DNA. The MRC bait was tested alone for autoactivation, with a non-specific bait (pTDl), and with each two-hybrid target isolated from the screen (l4a, 15q, 3 la, I 18, 233).

The target plasmid was isolated fkom the 66 HIS' Lac' yeast transfonnants.

Target isolation was accomplished by selecting for plasmids which express the LEU2

marker and can complement a defect in the leuf3 gene of bacterial cells (MH6).

After isolating each target cDNA, specificity tests were perfomed to identifL those

targets capable of interacting only with the ROMl C-terminal tail bait and not with other

baits. Three non-specific baits fused to the GALA binding domain were chosen. These

included the standard baits pVA3 and pLAM5' described above, and a pCHXlO bait

(provided by B. Muskat), which contains full-length CHXl O (Liu et al., 1994) excluding

the C-tenninal acidic domain. Each target cDNA was subsequently transfonned into

Y 190 cells expressing one of the three non-specific baits, which were previously show

not to cause activation of the reporter genes on their own, and, in parallel, into yeast cells

expressing the ROMl C-terminal tail bait. Bait specificity interaction was determined by

exarnining lac2 reporter gene activation using P-galactosidase filter assays. Of the 66

HIS' ~ a c ' putative interactors identified, only five were shown to interact specifically with

the ROMl C-terminal tail bait. The remaining 61 putative interactors activated l d gene

expression in the presence of each non-specific bait.

To quanti@ the interactions between the five specific targets and the ROMl C-

terminal tail bait, quantitative P-galactosidase liquid assays were performed. In the

presence of the ROMl C-terminal tail bait, al1 five specific targets were capable of

activating the lac2 reporter gene to a level at least 2-3 times greater than that of the

negative controls (Fig. 2-2, MRC + 14% 1 Sq, 3 la, 1 18, or 233).

Characterization of the ROMl interacting partners

To acquire information about the target proteins found to interact specifically with

the ROMl C-terminal tail bait, the cDNA insert of each of the five isolated cDNAs was

sequenced. Since the sequence coding for the GALA activation domain (AD) is known,

the frame of each target protein was determined by analyzing the GAL4 AD/target

sequence.

Once the sequence of the target proteins identified in the two-hybrid screen were

known, they were compared to each other and used to search the NCBI database

(Altschul et al., 1997). The cDNAs are refemd to by the number they were onginally

given when isolated fiom the library plates (14% 1 Sq, 3 la, 11 8, and 233) (Table 2-la-e).

The cDNA 159 encodes the smallest polypeptide, of 48 amino acids while the cDNA 233

encodes the largest polypeptide, of 126 amino acids (Table 2-lb and e). To assess the

possibility that the same cDNA was identified in the screen more than once, the five

isolated cDNAs were compared to each other using the Geneworks alignment program.

The result of the cDNA alignrnents revealed that each one was unique (results not show).

Thus, five different target proteins are putative interactors with the ROM1 C-terminal tail.

To gain further insight into the identity of the target proteins, proteins cornparisons were

performed using the database for each cDNA.

The results of the database protein comparisons are sumrnarized in Tables 2-1 a-e.

Al1 of the five cDNAs are novel based on protein and nucleotide searches. Database

searches performed with the nucleotide sequence of cDNA 15q revealed that it is

homologous to a human PAC clone 102G20 and is homologous to an uncharacterized

mRNA from a brain library. However, there are no retinal ESTs. An EST contains

partial nucleotide sequence of a randornly picked cDNA fiom a particular tissue library.

The protein sequence encoded by the cDNA 1Sq is novel, sharing 50% identity over 28

amino acids with ICB-1 (Table 2-1 b), a recently identified protein putatively involved in

cell matrix interactions (Treeck et al., 1998). The cDNA 1 18 encodes a 1 14 arnino acid

polypeptide that is 45% identical to a region of MCBP, a RNA-binding protein (Table 2-

Id) (Funke et al., 1996). The region showing homology between the predicted protein

encoded by cDNA 118 and MCBP, when used in a database search did not contain a

known motif currently present in the database. The cDNAs 14a and 233 encode two

different novel cDNAs each of which have some homology to different known proteins in

the database over a stretch of 37 arnino acids (Table 2-la and e). However, the regions of

homology do not contain any known domains, as determined either by using the database

or by analyzing published literature on the proteins identitied fiom the database. Finally,

cDNA 31a encodes a 68 amino acid novel polypeptide. Protein database andysis and

sequence examination revealed a 42 amino acid probe and histidine rich region (Table 2-

Protein Homology:

40% identity to retina derived POU-domain factor 1 (human) and 34% identity to secretin (porcine)

14a polypeptide sequence:

PACPLGDREI DGRDHPLTLP HPRCSWPGVW 30 ELVALGNLPL LLLDPKALRL GPWICRSWAE 60 SPCSLSWMPS RGPGSQLTVP LEGPGHLRVA 90 RHPLASLAVS PTVALMLPVQ GLX

Table 2-la. ROM1 C-terminal iail two-hybrid target 14a. Proteins homologous to the polypeptide encoded by 14a are listed, with the percent identity observed. The amino acids in the polypeptide showing homology are underlined.

Protein Homology:

50% identity to ICB-1 (human)

15q polypeptide sequence:

PVLASIEKTT CRAYLQVCLL PAIKTSSSTC PTLPASTPGF LLVVFWKLX

Table 24b, ROM1 C-terminal taii two-hybrid target 15q. Protein homologous to the polypeptide encoded by 15q is listed, with the percent identity observed. The amino acids in the polypeptide showing homology are underlined.

Protein Homology :

3 1 a polypeptide sequence:

EKGRKKSEGK PCCLFKCTEV LAQGPPTHHT 30 TTHHPHPQLE HSPPAPCSH IPPRGRDVEG 60 RQGRRAGPX 68

Table 2-1c. ROM 1 C-terminai tail two-hybnd interac ting target 3 1 a.

Protein Homology:

45% identity to mCBP (mouse)

1 18 polypeptide sequence:

VSGVPDAIIL CVRQICAVIL ESPPKGATIP 30 YHPSLSLGTV LLSTNOGFSV GOYGTVTPA 60 EVTKLQQLSG HAVPFASPSM VPGLDPSTOT 90 SSQEFLVPND LIGCVIGRQG NKIKX 114

Tabte 2-Id. ROM1 C-terminal tail two-hybnd target 118. Protein homologous to the polypeptide encoded by 118 is listed, with the percent identity observed. The amino acids in the polypeptide showing homology are underlined.

Protein Homology:

48% identity to RRG4 (rat)

233 polypeptide sequence:

KRGRFPAAQH SDSGFELRSQ PWQSPRGPAQ 30 GKEPLGPRDA AVPARVPRRC RAVASSRPLP 60 GIPELVTEDH IQAPGSGHP TALSPEAGQA 90 ALSPSTIGAG HLKEAAFGPT DLVTLKEASF 120 PEVSHPX 126

Table 2-le. ROM1 C-terminal tail hvo-hybrid target 233. Protein homologous to the polypeptide encoded by 233 is listed, with the percent identity observed. The amho acids in the polypeptide showing homology are underlined.

1 c). Fifieen of the 42 amino acid present in the polypeptide encoded by 3 la is either a

proline or histidine nsidue.

DISCUSSION

ROMl was identified as an evolutionarily consented, abundant and retinal specific

cDNA in a diflerential hybridization screen (Bascom et al., 1992). Characterization of

ROM1 indicated that it shared overall structural similarity to RDS, the two proteins being

comparable in their cellular specificity, subcelluiar localization and membrane topology

(Bascom et al., 1992). Furthemore, at least 19 dierent proteins share the same topology

as ROMl and RDS, even though the primary sequences are not conserved (Wright and

Tomlinson, 1994).

Despite similanties between ROMl and RDS the two proteins are not functionally

redundant. For instance, lack of either murine RDS or ROMl causes photoreceptor ce11

death (Travis et al., 199 la; G. Clarke unpublished). The roles of ROMl and RDS in the

photoreceptor outer segment is currently unknown, but recent advances into the

biochemical roles for ROMl and RDS have been made. Boesze-Battaglia et al. (1998)

have shown that RDS is capable of mediating membrane fusion, at least in vitro,

suçgesting that RDS may be required for rod outer segment membrane fusion. The

authors have also determined that residues 3 13 to 327 in the RDS protein promote the

observed membrane fusion (Boesze-Battagiia et al., 1998). This fiisogenic region is

present in the C-terminal tail of RDS. Previous analysis performed on RDS have shown it

to be phosphorylated at the C-terminal tail, and have established, that the phosphorylated

form of RDS enhances membrane fusion (Boesze-Battaglia et al., t 997). Furthemore, the

phosphoqlation occured on a serine residue (BoeszeBattaglia et al., 1997). There are

two serine residues present in the C-terminal tail of RDS. The authors speculate that

ser321 located within the fùsogenic region is perhaps the serine that is phosphoiylated in

RDS thereby contnbuting to the fusogenic nature of the protein (Boesze-Battaglia et al.,

1 998).

The C-terminai tail of ROMl, used in the two hybrid screen, revealed that it

contains no serine residues. In fact, homology between ROMl and RDS is weakest at the

C-terminal tail. Although this difference in peptide sequence rnay suggest that ROMl is

not involved in disk morphogenesis, ~oml-' mice have longer disks in the outer segments

of photoreceptor cells (personal communication G. Clarke). Taken together t hese findings

indicate that ROMl does have a critical role in proper disk nm formation, however, the

differences highlighted above between ROMl and RDS rnay well in fact reflect dserent

protein associations the two partake in during disk morphogenesis.

To begin to identi@ ROMl interacting partners a two-hybrid screen was

performed using the C-terminal tail of ROMl. Findings fiom the analysis of the

photoreceptors fiom the ~ o m " mouse has revealed a role for ROMl in disk formation and

in maintaining outer segment structure. Thus, ROM1 interacting proteins identified from

the two-hybrid screen could have roles in disk biogenesis andor outer segment structure

and maintenance.

The two-hybrid screen performed using the ROMl C-terminal tail identified 66

HIS' Lac' transformants. Only 5 of these 66 HIS' Lac' transformants were found to be

capable of specificaily interacting with the ROMl C-terminal tail bait. Quantitative P- çalactosidase assays showed that the interaction between the predicted proteins encoded

by the cDNAs isolated and the ROMl C-terminal tail bait was weak (Fig. 2-2). These

weak interactions rnay be due to one of five possible situations. First, the weakness of the

interaction between the targets and the ROMl C-terminal tail is indicative of the in vivo

condition. Second, full-length ROMl is required to strengthen the interaction. Thus, the

other regions of ROMl rnay interact with the proteins isolated in the two-hybrid screen,

thereby stabilizing the binding. Third, the conformation of the C-temiinal tail rnay be

important for the interaction with the targets, and the only appropriate confonnation is

obtained wit h full-lengt h ROM 1. This qualification rnay also apply to the targets. Fourth,

other proteins rnay be needed to enhance the weak binding observed. Fah, none of these

interactions occun in vivo, and the weak nature of the two-hybnd result reflects the fact

that the interaction between the targets and the ROMl C-terminal tail is spunous.

Sequence analysis of the five cDNAs isolated indicated that none of the targets

were isolated more than once and that the predicted proteins encoded by the cDNAs were

al1 novel (Table 2-la-e). Considering that the only three known proteins at the disk rims

of photoreceptor ceils are ROMl, RDS and ABC& the identification of five novel cDNAs

from the two-hybrid screen is not surpnsing (Fig. 2-3). Assuming that the putative ROMl

interacting proteins do indeed associate with ROMl in vivo, possible insights into the

fùnctions of these proteins was investigated using protein and nucleotide sequence

cornparisons of each cDNA to known proteins. Although some of the predicted proteins

encoded by the cDNAs shared some homology with known proteins in the database

(Table 2-la-e), no recognizable motifs were identified in any of the protein sequences

encoded by the cDNA insert of the target plasmid.

Understanding the function of proteins present in the disk rim complex requires

their identification and examining their associations with known disk rim proteins such as

ROMI. Despite the lack of information available on the cDNAs isolated from the ROMl

C-terminal tail two-hybrid screen, it is conceivable that the proteins identified may be

involved in structural maintenance of the disk nms and photoreceptor outer segments.

Additiondly, as discussed earlier, a role for ROMl in disk morphogenesis has been

identified by the analysis of ~ o m l " mice. Perhaps some of the cDNAs isolated encode

proteins that are components of the myosin Wa-actin motor known to be responsible for

disk morphogenesis (Williams, 1992). Finally, proteins present at the disk rims do not

necessarily have to be required for the strutural integrity of the photoreceptor outer

segment but may in fact be necessary for outer segment function as is the case with

ABCR, a putative ATP transporter. It is possible that some of the proteins encoded by

cDNAs isolated in the ROMl C-terminal tail two-hybrid screen are involved in processes

such as light transduction which occurs largely in the outer segment.

Despite the interesting possible roles polypeptides encoded by the cDNAs isolated

may engage in, fùrther experiments are required in order to substantiate the interaction

obsexved between the targets and ROMl C-terminal tail bait. First, in situ hybndization

c ytoplasm

unknown disk - rim protein

ABCR

RDS RDS disuiphide l inked homodimer

@ R O M 1 linked ROM 1 homodimer disulphide heterotetramer RDS-ROM 1 non-covalent

Figure 2-3. Hypothetical mode1 of the rod disk rim. The only three known proteins at the disk rim are: ROM 1, RDS and ABCR. Rotein-protein interactions may be required to stabilize the disk rim curvature. This figure shows one possible interaction, between ROM1 and cable-like proteins extending between disks and from disks to the plasma membrane. Unidentificd disk rim proteins such as those identified in the MRC two-hybrid screen may associate with ROM 1.

studies are needed in order to dehe the spatial and temporal expression similarities

between the putative interactors and ROMI. Second, the interaction between the target

and bait must be shown using alternative methods such as a n i t y chromotography. The

advantage of affinity chromotography is that the protein-protein inteïactions are tested

directly without the possibility of another unknown protein mediating the detected

association first observed in yeast. Third, protein localkation studies using antibodies

raised to the ROM l interacting proteins can be perfonned. Addressing these points will

help to validate the interaction observed in the two-hybnd system between the isolatecl

targets and ROM 1.

CHAPTER 3

Molecular characterization of PHRl and identification of PHR1-interacting proteins using the yeast two-hybrid

system

The majority of the work presented in this chapter is mine. Jonathan Horsford and 1 performed the PHRI Ni situ hybridization on adult mouse retinal sections. The monoclonal antibodies were made by Greg Lee at the Centers of Excellence. The polyclonal antibodies were a gifi from S. Xu of Dr. Valle's group at Johns Hopkins University. The immunofluorescence was peformed by Danka Vidgen.

Abundant mRNAs of a differentiated ceIl often encode molecules that are required

for the differentiated cellular phenotype (Bascom et al., 1992). Evolutionarily consewed

genes that are expressed abundantly in the retina are likely to have an important role in its

structure, function, and development. Such genes, when mutated, are also likely to

represent candidate genes for diseases Secting the eye. The isolation of specific,

abundant, and conserved (SAC) or abundant and conserved (AC) cDNAs by differential

hybridization is an effective strategy for identifjmg genes required for retina structure,

function, and development. Two important rethal abundant and conserved genes isolated

by differential hybridization were ROM1 and CHXlO (Bascom et al., 1992). ROM1 is ah

important disk rim protein required for photoreceptor outer segment function and

structure (Bascom et al., 1992; G. Clarke unpublished) whereas CM10 is a homedomain

protein involved in the function and development of the neuroretina and inner nuclear

layer (Liu et al., 1994). The PHRl retinal AC cDNA was also identified by differential

hybridization, and may therefore to encode a protein important for retinai biology.

Mer the cloning of the PHRl cDNA, its expression was detennined to be

restricted to the retina, brain, kidney, liver, and lung by Northern blot analysis (Girami,

1994), with highest expression found in the retina. PHRl protein sequence analysis

revealed that it has regions of similarity to several proteins including dynamin, Akt,

oxysterol binding protein, and h o , dl of which contain a pleckstrin homology (PH)

domain. The region of hornology between these proteins and PHRl was restncted to

sequences in their respective PH domains, indicating that PHRl contains a PH domain.

PH domains have been identified in over 100 signalhg (Ma et al., 1997). The PH

domain is a loosely conserved 100-120 amino acid motif, with the only invariant amino

acid being a tryptophan residue present in the C-terminal region of the domain. Despite

the low degree of identity (generally 10-20%) beiween pleckstrin homology domains of

different proteins, it has been detennined, by NMR spectroscopy and X-ray

crystallography, that the six PH domains which have been solved to date ali share the same

basic fold. Each domain consists of a P-sandwich formed by two neariy orthogonal

antiparallel fbsheets of four and three strands, respectively. An amphipathic C-terminal a

helix closes off one end of the sandwich (Lemmon et al., 1997).

Based on structural similarities between the PH domain and retinol-binding

proteins, it was thought that the PH domain rnay be capable of binding to lipophilic

molecules (Jakoby et ai., 1993; Harlan et al., 1994). in vitro assays using radioactively

labelled phosphatidylinositol phosphates (PIPs) have show that the PH domain is capable

of interacting with phosphoinositides and that this association is required for recruitrnent

of PH domain-containing proteins to membranes (Harlan et al., 1994). The interaction

between PIPs and PH domains of different proteins is also specinc (Lernmon et al., 1997).

Thus, PLC& interacts most strongly to phosphatidylinositol-1,4,5-triphosphate, while

pleckstrin specifically associates with phosphatidylinositol-4,5-biphosphate (Harlan et al.,

1994; Ferguson et al., 1995).

The participation of many PH domain-containhg proteins in signalling cascades

has suggested that PH domains may act as effkctors of G proteins or PKC in signai

transduction pathways (Cohen et al., 1995). The PH domain-containing proteins such as

Btk, rasGAP, PARK, PLCy, Akt, P-spectrin, oxysterol binding protein, and IRS-1 have al1

been shown to interact with the Gpy subunits of G proteins (Touhara et al., 1994; Jiang et

al., 1998). Interactions between the PH domains of Btk and RAC-protein kinase and PKC

have also been demonstrated in vitro and in vivo (Yao et al., 1994; Yao et al., 1997;

Konishi et al., 1996). PKC is also important for regulating the cytoskeletal reorganization

functions of PH domain-containing proteins such as pleckstrin (Abrams et al., 1995) and

ARNO (Frank et al., 19%). Identification of protein-protein interactions involving the PH

domains of different proteins has been determined by anity chromatography and CO-

irnmunoprecipitation expenments. The results corn these experiments have indicated that

the C-terminal region of the PH domain and amino acids immediately C-terminal to the PH

domain (-30-35 amino acids) are required for interactions with G proteins whereas the N-

terminal betaZbeta3 strands of the PH domain are required for interactions with PKC.

Other proteins capable of interacting with the PH domain alone have been

identified for the PH domains of IRS-1 and IRS-2 using the yeast two-hybnd system

(Table 3-A). The PH domains of iRS-1 and IRS-2 have been shown to bind to the acidic

motifs present in Lon protease, myeloblast protein, and nucleolin (Table 3-A).

PH domain-containing proteins such as IRS-1, IRS-2, BO1 1, BOIZ, hGrb 1 Oy, and

Trio have also been used in their entirety as baits in the yeast two-hybrid system

successfully (Table 3-A). In al1 these examples, the proteins identified in the yeast two-

hybrid system interacted with other regions of the baits outside or including the PH

domain. PHRI, for example, contains other possible sites for protein-protein interaction

besides its PH domain, including a cluster of prolines at the N-terminus and a cluster of

tyrosines C-terminal to the PH domain (Fig. 16). SH3 domains, for exarnple, are known

to bind proline rich seqences (Cohen et al., 1995) while clusters of tyrosine residues are

potential sites of phosphorylation (Songyang et al., 1995). In addition, the yeast two-

hybnd system has also been successful in identifying PH domain-containing proteins

capable of interacting with baits such as Syk, HAPI, rho, racl, BCR, insulin receptor (IR),

MEK 1, phosphatidylinositol3-kinase, and SHP-2 (Table 3-A).

As an initial step in defining the role of PHRl in photoreceptor and neuronal

biology we have exarnined the location of PHRl in adult mouse retina and have

undertaken a two-hybnd screen. In collaboration with S. Xu, we have recently

detedned the ability of PHRl to bind to the py-subunits of the G protein transducin

(personal communication S. Xu). Furthermore, the localization of PHRl to the outer

segment of photoreceptors, the site of phototransduction, suggests that PHRl may

fùnction as a previously unrecognized modulator of the light transduction pathway.

The two-hybrid screen was performed using full-length PHRl as the bait. The

PHRl two-hybrid screen has generated 50 potential PHRl interactors, 36 of which

interacted strongly with PHRl while the remaining 14 interacted weakly. Two dBerent

PHRl specific interacting proteins were isolated twice in the two-hybrid screen. Both of

these potential PHR1-interacting proteins are novel, based on sequence analysis, with one

containing a protein-protein interaction motif cded a RING hger domain. Only two of

the fourteen weak PHR1-interacting proteins were specific in their interaction with PHRl.

Both of these potential PHR1-interacting proteins are daerent and novel.

Table 3-A. PH domain containing proteins used or identifiai using the yeast two-hybnd system. The only PH domain containhg protein interacton capable of interacting with oniy the PH domain were found for IRS- 1 and IRS-2.

PH domain containing proteins used in the two- hybrid system

, (sait) Insulin receptor substrate- 1 (ES- 1) IRS-1 PH domain

'Y contains SH2 and

only Insulin receptor substrate-2 (IRS-2) IRS-2 PH domain O ~ Y

BOX1 and BO12

PH domains

-

containing proteins identified in a two- hybrid screen

nucleotide exchange factor) contains a Dbl hornology @H and

PH domain -Duo which contains a GEF

PH domain containing protein interactors identified from the yeast two-hybrid system

-Cytoplasmic portion of the insulin receptôr (R) -Acidic motif of nucleolin

-Cytoplasmic portion of the insulin receptor (IR) -Acidic motifs of Lon protease, myeloblast protein, and nucleolin -Beml~ binds to the SH3 domai; of BO11 and BOi.2. -The Rho-type GTPase Cdc-42p binds to a segment of BO11 which contains its PH domain -hGrblOgarnma bound to a segment of hGrb 10 w hich included the insert between the PH and SH2 domains (IPS) and the SH2 domain -Interaction between the N- terminal region of hGrb logamma involves the IPS/SH2 region and the PH domain of a second hGrb 1 Ogamrna -GEF domain- 1 of Trio interacts with actin binding protein füamin

Syk tyrosine kinase

HAPl (Huntingtin- associated protein 1)

References

Sawka-Verhelle et al., 1996 Burks et al..

et al., 1996 Burks et al..

Bender et. al., 1996

Dong et al., 1998

Bellanger et al., 1998

Deckert et al., 1998

Colomer et ai., 1998

Table 3-A.

PH domain containing proteins used in the two- hybnd system (Baitl

PH domain containing proteins identified in a two- hybrid screen

-citron which contains a C6N2 zinc finger, PH domain, a long coiled-coil forming region including 4 leucine zippers, and the rho 1 rac binding site -APS adaptor molecule which contains PH and SH2 domains

-PSM which is pro- rich and contains PH, and SH2 domains -GrblO which contains a pro-nch region and a central PH domain and a C-terminal SH2 domain -Gab 1 an insulin receptor substrate with a PH domain

-ffirb 14 adaptor protein SH2 and PH domains

PH domain containing protein interactors identined from the yeast two-hybnd system

GTP-bound forrns of rho and rac 1 (GTPases)

Associates with BCR, and potentiaily with the immuuoreceptor tyrosine- based activation motif (ITAM), Lnlc signal transducer which links T- cell receptor to PLCgamma, Grb2, and phosphatidy . . linositol3- kinase. Cytoplasmic fragment of the insulin rece3or (IR)

The MEKI kinase interacts with the SH2 domain of Grb 10

Tyrosine phosphorylated Gabl interacts with phosphatidylinositol3- kinase and SHP-2 The SH2 domain and the region between the PH and SH2 domain mediate the interaction with insulin receDtor (IR)

References

Madade et al., 1995

Yokouchi et al., 1998

Riedel et al., 1997

Nantel et al., 1998

Rocchi et ai., 1998

Kasus-Jacobi et al., 1998

EXPERIMENTAL PROCEDURES

RT-PCR

RNA from mouse retina, brain, kidney and liver were isolated using the ~ ~ l z o l ~

Reagent (GIBCO BRL). Mouse retina, brain, kidney, and liver single-stranded cDNA was

made by adding 5 pg of total RNA to 0.5 vg of oligo d(T) (1 2- 18 T's - GIBCO BRL) and

9 pl of diethylpyrocarbonate (DEPC) treated water. The mix was incubated at 70°C for

10 min and then chilled on ice for at least one minute. During ice incubation, the PCR

machine was set for one cycle with the following conditions: 42°C for 1 hr, 70°C for IS

min and then to the 4°C soak temperature. To each reaction tube was added 4 pl 5x first

strand buffer (GIBCO BRL), 2 pL 0.1M DTT (GIBCO BRL) and 1 pl 1OmM dNTP

(Pharmacia). The components were mixed and incubated initially at 42OC for 5 min and

subsequently, 1 pl of Superscript U RT (GIBCO BRL) (200 U/pl) was added to each tube

and incubated for a further 50 min at 42T. The reaction was terminated by incubation at

70°C for 15 min and then treating each tube with 1 pl RNase H (3.8 U/pI) (GIBCO BRL)

for 20 min at 37OC.

The entire coding region of PHRl was amplified by PCR for each reverse

transcribed RNA sample. The PCR reaction perfomed consisted of the primer set 5'

GTTCCACCCGACTCCATC 3' and 5' CCAAGAGCAGCACCCGTG 3'. The

components used in the amplification were: 10 rnM Tris, pH 8.0, 50 mM KCI, 1.5 rnM

MgCl*, 0.01% gelatin, 10 pM of each dNTP, 50 ng of each primer, and 1 pl of reverse

transcribed RNA. The PCR machine was set with an initial denaturation temperature of

98OC for 5 min. Following denaturation, PFU polymerase (2.5 U/p1, Stratagene) was

added to each reaction and the PCR machine was set for 30 cycles of amplification. Each

PCR cycle was carried out at a denaturation temperature of 95OC for 1 min, an annealing

temperature of 59OC for 2 min and an extension setting of 72OC for 2 min. The same

conditions were applied to plasmid DNA containing full-length human PHRl except that

the primer set used consisted of 5' GGAATTCCATATGAGCCCTWAGCCCCG 3' and

5' ACGCGTCGACGGCTCAGAACCAGCAGGG 3'.

In situ Hybridization

A 624 bp PstI-BamHI fiagnient fiom the coding region of mouse PHRI

subcloned into pBluescript SK (Stratagene) was used as a template to generate [a-

3 3 ~ ] ~ ~ ~ (DuPont-N'EN) labelled riboprobes. Sense probe was made from the T7

promoter by in vitro transcription of the BanilII linearized template and antisense probe

was in vitro transcribed fiom the T3 promoter using EçoRI digested template.

Riboprobes were generated using the Stratagene in vitro transcription kit according to the

instructions of the manufacturer.

Mouse adult retina tissue slides purchased fiom Novagen were hybridized at 5S°C

oveniight with 150 pl of hybridization solution containing 3 3 ~ labelled probe at a

concentration of 1 x 105 cpm/pl. The washing protocol was as follows (Millen and Hui,

1 994):

a) 20 min at 6S°C in shaking water bath with a solution containing 50%

formamide, 2X SSC, and 0.1% P-mercaptoethanol @Me) (wash buffer 2).

b) Three washes of 10 min each with prewarmed (37'C) 0.5M NaCI, lOmM Tris-

HCl pH 8.0, and 5mM EDTA at 37OC (wash buffer 3).

c) Treat with 20 ~<g/ml RNAse in prewarmed wash buffer 3 at 37OC for 30 min.

d) Rinse in wash buffer 3 at 37OC for 15 min.

e) Repeat above wash with wash buffer 2 at 65 OC for 20 min.

f ) Rinse in 2X SSC, the 0.1X SSC, 15 min each at 37OC.

g) The sections were dehydrated by quickly processing through 30%, 60%, 80%,

95% ethanol made with 0.3M ammonium acetate, and hnce through 100%

ethanol.

The sections were allowed to dry and then dipped in Eastman Kodak NTB-2

Nuclear emulsion. Sections were exposed at 4OC for 3-5 days. Each section was

developed, counterstained with toluduine blue and mounted with permount (Milien and

Hui, 1994). Dark field and bright field pictures were taken with the Leica Leitz DMRB

microscope using Kodak Elite ï I Ektachrome 100 film.

Transient expression of PBRl in manmalian celb

To transfect mammalian ceUs with PHRI, tùll-length PHRl cDNA was subcloned

into the pcDNA 3.1 + A marnmalian expression vector (Invitrogen). FulClength PHRI

was arnplified Rom plasmid DNA by PCR with PFU polymerase. The primer set used in

the PCR reaction was as follows: 5' CCGGAATTCCGGATGAGCCCTGCAGCCCCG 3'

which includes an EcoRl site and 5' GCTCTAGAGCGAACCAGCAGGGCGACCA 3'

which contains a XbaI restriction site. The PCR reaction was performed as outlined in the

RT-PCR section above. The PCR product was purified by the Qiagen PCR purification

kit. The amplified product and vector were then digested with EcoRI and XbaI at 37OC

for 2 hrs, mn on a 1% agarose gel and purified by the Qiagen gel extraction kit following

manufacturer's instructions. The PHRl PCR product was subsequently subcloned into the

vector using the rapid ligation kit (BoeMnger-Mannheim). Ligation products were

confirmed to be correct by sequencing using the Tt primer site present in the vector. The

entire PHRl insert subcloned into the pcDNA 3.1 + A vector was sequenced to confimi

the absence of any PCR induced errors.

The following standard protocol was used for transient transfection of adherent

HEK293 cells (ATCC) in 60-mm dishes (supe&ectm, Qiagen). The day before

transfection cells were seeded to a concentration of 2-8 x 10' cells per 60-mm dish in 5 ml

of DMEM @ulbecco's Modified Eagle Media) growth medium containing 10% serum

(fetal calf senim) and incubated ovemight at 37OC with 5% CO2. Transfmion was

performed with no DNA, pcDNA vector only and the PHRl expression constmct. DNA

was prepared using the Qiagen Maxi prep. A total of 10 pg of DNA was diluted in 300 pl

of growth medium without senun. A reporter constnia containing the lacZ gene (5 pg)

was also included in the reactions to confimi efficient transfe*ion by Western blot analysis

of cell lysates with anti-P-galactosidase antibodies (Promega). To the DNA solutions was

added 30 pl of superfectThi transfection reagent (Qiagen). The reaction was mixed by

trituration. Sarnples were incubated for 10 min at room temperature to allow the

superfectTM transfection ragent to complex with the plasmid DNA. During complex

formation, growth media was removed from the petri dishes by gentle aspiration and cells

were washed with 4 ml of 1X PBS (phosphate buffered saline). M e r the 10 min

incubation period, 3 mls of growth medium with serum was added to each reaction tube

containing transfection complexes and mixed by trituration. The total volume was

inunediately transferred to the ceils in 60-mm dishes. Cells were incubated at 37°C for

two-three hours with the complexes. After the incubation period medium was removed by

gentle aspiration and cells were washed with 4 ml of 1X PBS. New cell growth medium +

serum was added. Cells were incubated for 24 hrs.

CeU lysate preparation

Once cells were incubated for 24 hrs at 37OC, they were removed h m the

incubator and placed on ice. Growth medium was removed by gentle aspiration and cells

were washed with cold 1X PBS ( 5 ml). Cells were lysed with 1X RIPA buffer (Sambrook

et al., 1989) which contains: 15 mM NaCl, 50 mM Tris-CI pH 8.0, 0.1% triton X-100,

0.5% deoxycholic acid (Sigma), 0.1% SDS, 1 m M PMSF, 1 m g h l STF (serum fetal calf

trypsinase inhibitor). Lysis was performed for 15 min with 1X RIPA buffer (2 d60-mm

petri dish). Lysates were collected and centnfbged at 4°C for 15 minutes. Supernatant

was transferred to a new tube and stored at -20°C.

Protein electrophoresis and immunoblotting

Tissue and ceIl protein lysates were resolved using 5% stacking and 10%

separating, 0.75 mm thick, acrylamide gels in the Bio-Rad Minigel system. The amount of

tissue and cell lysate loaded per lane was 1 pg. The amount of purified his-tagged PHRl

loaded was 0.2 pg. Gels were either stained with Coomasie blue (Sambrook et al., 1989)

or the proteins were transferred to Ntrocellulose for Western blot analysis.

Proteins were electrophoretically transferred to Hybond C nitrocellulose as

outiined in Chapter 2. Non-specific background was elhinated by blocking the filter with

5% milk solution dissolved in TBST for 1 hr at room temperature, foliowed by one 5 min

wash at n o m temperature with TBST. The filter was then incubated overnight at room

temperature with anti-PHR1 rabbit IgG polyclonal antibody diiuted in 1% miik in TBST

(1 : 5000 dilution). The following day fiesh primary antibody was added to the filter at a

dilution of 1: 1000 and incubated at room temperature for 2-3 hrs. Unbound prirnary

antibody was removed by washing 3X 10 min with TBST, at room temperature. The

nitrocellulose filter was subsequently incubated for 1 hr at room temperature with the

secondary antibody anti-rabbit IgG (whole molecule) peroxidase conjugate (Sigma),

diluted 1 : 5000 in TBST with 1 % milk. Excess secondary antibody was removed by 4X 1 O

min washes with TBST solution at room temperature. The enhanced cherniluminesence

detection system (Dupont) was employed to visualite the protein bands after exposure to

film.

Immunofluorescent Iabelling of PHRl in the adult mouse retina

Wild type mice were sacrificed by ceMcal dislocation and adult mouse eyes were

quickly enucleated and transferred into cold 4% paraformaldehyde in phosphate buEered

saline (PBS) for ovemight fixation at 4°C. M e r PBS washes eyes were stored in 30%

(w/v) sucrose PBS overnight. Tissue was embedded in OCT compound (Tissue-TEK)

and quickly frozen in isopentane over iiquid nitrogen. 7-14 Pm cryostat sections were

collected on Sigma, ~ i l a n e - ~ r e ~ ~ ~ slides and stored at -20°C until use.

Cryostat sections used for immunfiuorescent staining were rehydrated in PB S pH

7.3 for 15 min. Retinal sections were blocked in 10% goat serum (Sigma) in PBS for 30

min and then permeablizied with 10% goat serum with 1% triton X-LOO in PBS for 10

min. Primary antibodies were fieshly diluted and spun down at 3000 rpm for 3 min.

Monoclonal antibody (clone 6) was diluted to either 150 or 1:200 while polyclonal

antibodies (2605 and 2606, provideci by S. Xu) were diluted to 1500. Incubation with

primary antibodies took place overnîght at 4OC. After washing with 3X 10 min PBS,

sections were treated with fluorescein-conjugated F(ab')Z fiagrnent secondary antibody

fieshly diluted in PBS. For the monoclonal antibody (clone 6), goat anti-mouse FITC

1: 100 was used, whereas for the polyclonal antibodies goat anti-rabbit FITC 1:70 was

used. Sections were incubated for 1 hr at room temperature with secondary antibodies,

and then were washed with 3X 10 min PBS and rinsed with double deionized HzO

(ddH20). S lides were mounted with Imrnunofloor (ICN Biomedical). Sections were

Mewed with a confocal microscope Zeiss LSM 4 10 invert. The extemal laser used was set

at Ar 488m while the intemal laser used was set at HeNe 543 nm and 633 m.

Two hybrid screen

The procedure for the two hybrid screen has been outlined in Chapter 2 (see

experimental procedures). The bait constnicted for the human PHRl screen included full

length PHRl subcloned into the PAS 1 vector. The primer set used to ampli@ full length

PHRl fiom plasmid DNA was 5'

GGAATTCCATATGAGCCCAGCAGCCCCGGTCCCGCCT 3', which contained a NdeI

restriction site and 5' AACTGCAGAACCAATGCATTGGTCAGAACCAGCAGGGCGA

3', which contained a Pst1 restriction site.

An oligo dT prirned adult bovine retinal two hybnd library was used in the screen.

The yeast strain used in the two hybnd screen was a gift fiom D. Gietz and was referred to

as KGY 3 7.

RESULTS

PHRl alternative splicing

PHRl was originally identified in a difEerential hybridization screen and was

initially referred to as clone 198L. The insert isolated fiom clone 198L was used to screen

a human adult retinal cDNA library to isolate the full-length cDNA. Three overlapping

clones were identified. Sequence analysis indicated that the PHRI gene exhibits

alternative splicing due to the absence of a 105 bp coding segment in two of the three

isolated clones. Analysis of the PHRI genornic structure and the sequences of the three

overlapping clones revealed that exon 7 was identical to the 105 bp fiagrnent removed by

alternative splicing. This exon encodes 35 amino acids C-terminal to the pleckstnn

homology domain of PHRI.

To directly determine the involvement of alternative splicing in the expression of

PHRI, RT-PCR was pedonned with mouse retina, brain, kidney and liver RNA. Each

PCR reaction used a primer set which amplifieci the entire coding region of PHRI. The

results from the RT-PCR conf'irmed the existence of two transcripts for PHRI, one

containing nill-length coding sequence and the other lacking exon 7 (Fig. 3-1). As

expected fiom the sequence of PHRI, the full-length PHRI coding region PCR product is

approximately 730 bp while the alternatively spliced version of PHRl is approximately

620 bp (Fig. 3-1). To determine when the increase in PHRI PCR products was linear,

samples from each RT-PCR reaction were separated by gel electrophoresis &er 10, 15,

20, and 27 cycles of amplification (data not shown). The relative amount of the two

PHRl PCR products could be compared since both amplified products from each PCR

reaction were made with the same primer set and cornparisons were made while the

increase in amount of products was linear. With respect to the retina, full length PHRl is

expressed twice as abundantly as the altematively spliced product, whereas in the other

tissues tested the altematively spliced product is more than four times as abundant as full

length PHRI. In addition, dunng the linear amplification of the PHRI coding products by

RT-PCR, it was noted that the abundance of the coding PHRI message was highest in the

retina, then brain, kidney, and liver as expected based on the Northem blot for PHRI (data

not shown).

To confimi that the alternatively transcribed product from the RT-PCR was the

outcome of the removai of exon 7, the two fragments produced by the RT-PCR were

digested with an enzyme (Pvu II) whose restriction site is only present in exon 7. An RT-

PCR was performed with mouse retina RNA (Fig. 3-2 lane 4). When digested with Pvu

II, only the top band corresponding to fuli-length PHRl was digested to produce two

bands, and the sizes of the two bands together equded that of full-length PHRI. Plasrnid

DNA containing full-length human PHRl was also amplilied to produce a 750 bp band

(Fig. 3-2 lane 6). The full-length product amplified from plasrnid DNA when digested

with Pvu II produced the expected two bands (Fig. 3-2 lane 9, the sizes of which together

equalled the undigested product (lane 6). To c o n t h that the alternatively spliced product

did not contain exon 7, the 620 bp product fiom the RT-PCR with mouse retinal RNA

Figure 3-1. Alternative splicing of PHRl in the retina, brain, kidney, and liver. PHRl RT-PCR with RNA from retina, brain, kidney and liver. The PCR reactions were perfomed with the same set of pnmers and for 30 rounds of amplification. Lane 1 shows the positive control PCR with plasmid DNA containing full-length coding product of PHRl (-730 bp). Lane 2 shows the negative control PCR lacking a DNA template. Lanes 3,4,5 and 6 are RT-PCR results with RNA from rnouse retina, brain, kidney and liver, respectively. In the retina, the full-length coding message of PHRl is twice as abundant as the alternatively spliced coding product (-620 bp). In contrast, the altematively spliced coding product of PHRl is more than four times as abundant as the full-length coding product of PHRI. Marker sizes are shown to the left of the figure.

Figure 3-2. Alternative splicing of exon 7 of PHRI. PHRI RT-PCR with RNA from mouse retina was digested with Pvu II (lane 2), a restriction enzyme whose recognition site is only present in exon 7. Only the full-length PHRl DNA product was digested by Pvu II producing two bands whose sizes together equalled that of full-length PHRI. Lane 3, undigested PHRl RT-PCR products. Lane 5 contains the full-length PHRl PCR product amplified from plasmid DNA containing full-length coding human PHR I . The PHRI product from lane 5 was digested with Pvu II, producing two bands (lane 4) whose sizes together equalled the full-length coding PHRI product. Lane 6 contains only the altematively spliced product (- 620 bp) purified from a RT-PCR reaction with adult mouse retinal RNA. The PHRl coding altematively spliced product from lane 6 was not digested with Pvu II. Lane 1 shows the lambda marker with the size of the fragments on the left, in nucleotides (nt).

was purified and digested with Pvu 11. No digestion was observed with Pw II providing

further evidence for the lack of exon 7 in the PHRI aitematively spliced product (Fig. 3-2

compare lanes 7 and 8).

Expression of endogenous PHRl in retinal and bnio lysates

To establish the expression and apparent mass of the PHRl protein in retina and

brain lysates, Westem blot analysis was performed with polyclonal antibodies raised

against full-length PHR1. Transient transfections were pediormed in HEK293 cells with

either the pcDNA 3.1 mammalian expression vector only as the negative control or with

the full-length PHRl cDNA subcloned into pcDNA 3.1. Lysates were prepared from

independent duplicate transfection experiments with cells expressing PHRl and cells

containhg vector only. Expression of P-galactosidase was tested by Westem blot analysis

in order to confirm proper transfection efficiency. Lysates fiom both PHRl expressing

cells and from cells transfected with the empty vector expressed the P-galactosidase

marker at similar levels indicating that al1 cells were transfected correctly (data not

show).

Once PHRl expression was confirmed by Western blot analysis in HEK293 cells

transfected with the PHRI rnamrnaiian expression constmct, positive and negative control

cell lysates were electrophoresed with lysates from mouse retina and brain in a 12.5%

SDS-PAGE. Proteins were ~ansferred to a nitroce~lulose fiher and probed with the

polyclonal antibody 2605 raised against full-length PHRl. His-tagged PHRl purified from

bacteria served as a positive control for the Westem.

The size of the bacterially puriîïed his-tagged PHRl was -25 kDa (Fig. 3-3 lane 8),

as predicted from the open reading of the iÙU-length cDNA. Expression of PHRl in the

HEU93 ce11 lysates, however, produced two major bands not observed in the negative

controls, one migrating at -37 kDa and the other migrating at -33 kDa Fig. 3-3 lanes 2

and 4). The srnalier band is believed to be a major degradation product. In the retina

lysate a doublet migrated at -37 kDa and -35 kDa. The larger band of the doublet in the

retina migrated at the same rate as the PHRl protein transiently expressed in the HEK293

cells, aithough it was less abundant (Fig. 3-3b compare lanes 2 and 4 with lane 6). The

Figure 3-3. PHR 1 expression in HEK293 cells, and mouse retinal and brain lysates. Two independent transfections were perfonned in HEK293 cells with full-length PHRI subcloned into pcDNA. An immunoblot, exposed for 10 sec panel A or 1 min panel B, of an SDS-PAGE with lysates from HEK293 cells transfected with the PHRl expression construct (3 v g ) (lanes 2 and 4), HEK293 cells transfected with vector only (3 pg) (lanes 3 and 5), mouse retina (1 pg) (lane 6), and rnouse brain (3 pg) (lane 7), probed with polyclonal antibodies to PHR 1. Full-length PHR 1 migrates at 37 kDa in both HEK293 cells (lanes 2 arrow and 4) and retina (panel B, lane 6 arrowhead). A major degradation band migrates at 33 kDa in the HEK293 ceIl lysates with hill-length PHR 1 (lanes 2 white arrowhead and 4). The altematively spliced PHRl prduct may be migrating at 35 kDa (panel B, lane 6 arrow). The 40 kDa band in panel A lane 7 (arrow) may represent full-length PHR 1 in the brain. As a control, his-tagged bacterially purified full-length PHRl migrates at 25 kDa (lane 8). Lane 1 shows the marker bands, the sizes for which are on the left (in kDa) for each panel A and B.

73

lower product of the doublet in the retina migrateci at -35 kDa and may be the result of

the translation of the altematively spliced product, since the expecteù size of the protein

produced by translation of the altematively spliced transcript would be 2-3 kDa smaller

than that of full-length PHRI.

The expression of PHRl in the brain differs fiom its expression in other tissues in

that transcription cm begin either at exon one or in intron two. The use of an alternative

promoter in intron IWO results in the expression of two brain-specific transcripts, one

containing exons 3-9 and the other containing exons 3-9 but lacking exon 7 as a result of

alternative splicing (Fig. 1-5) (personal communication S. Xu). The imunoblot of the

brain lysate probed with PHRl polyclonal antibodies differed fiom the others in that four

prominent bands were detected at 4 0 kDa, -28 kDa, -26 kDa and -25 ma. These

bands do not correspond to bands detected in the PHRl expressing HEK293 ce11 lysates

or retina lysate. Thus, it is difficult to determine which bands in the brain lysate

correspond to full-length PHRl or PHRl isofonns. The fact that PHRl migrates at a

larger than expected size (-25 kDa) based on its amino acid composition in HEK.293 cells,

retina and perhaps brain (-40 kDa) suggests that some form of post-translational

modification is taking place.

PHRl expression and localization in the neuroretina

1'1 situ hybridization of mouse adult retinal sections with 3 3 ~ labelled probe was

performed to determine if the PHRl message is expressed throughout the neuroretina or if

it is cell type specific (Fig. 3-4a). PHRI expression was seen most abundantly in the imer

segments of the photoreceptor cells (Fig. 3-4b). It was also detected in the outer

plexiform layer, and in the ganglion cetl layer (Fig. 3-4b). No significant labelling was

found with the sense probe (Fig. 3 4 ) .

To determine the cellular and subcellular localization of PHR1, monoclonal and

polyclonal antibodies to full length PHRl were used on cryostat sections of mouse retina.

Immunofluorescent staining showed that PHRl abundantly localizes to the outer segments

of the photoreceptor ceUs in the outer nuclear layer (Fig. 305% and b). in addition, both

monoclonal and polyclonal antibodies detected PHRl in the ganglion cell layer (Fig. 3-Sa,

Figure 3-4. In situ hybridization of mouse PHRl in adult mouse retina. Sections were probed with 3 3 ~ labelled PHRl (nt 8-632). A. Bright field of the mouse retina stained with toluduine blue. B. Dark field picture of hybridization with the antisense probe. C. Dark field picture of hybridization with control sense probe. PHRI is expressed abundantly in the photoreceptor inner segment (1s) and at lower levels in the outer plexiform layer (OPL) and ganglion ce11 layer (GCL) as shown by the white signal (panel B). INL = inner nuclear layer, IPL = inner plexiform layer, ONL = outer nuclear layer and OS = outer segment.

Figure 3-5. PHRl is localized to the photoreceptor outer segment and to the ganglion cells of mouse adult retina. Panels A and B show micrographs of adult mouse retinal sections labelled by immunofluorescence with PHRl monoclonal or polyclonal antibodies, respectively. C. higher magnification of the ganglion cells stained with monoclonal antibodies to PHRl. PHRl staining is localized in ganglion cells to the plasma rnembrane(arrow).

and b). At higher magnifications on thinner sextions, PHRl is localized to the plasma

membrane of the ganglion ceUs (Fig. 30%). This finding is in agreement with in vitro

studies which have shown that PHRl is capable of associating with bovine rod outer

segment membrane fractions (personal commwication S. Xu).

Presence o f a putative Gterminal trrinsmembrane domain in PBRl

To determine the presence of transmembrane domains in PHRI, a hydropathy plot

was performed on full-length PHRl using the Kyte-Doolittle algorithm (1 982). The

hydropathy profile of full-length PHRl shows that PHRl contains a single putative

transmembrane domain at the C-temiinal end of the protein (Fig. 3-6). The putative

rnembrane-spanning domain consists of 22 residues and has an average hydrophobicity of

approximately +1.6 (Fig. 3-6). Thus, the only two known protein domains present in

PHRl are the C-terminal TM domain and the N-terminal PH domain.

Analysis of PHRl bait expression in the two-hybrid system

To identi@ proteins capable of interacting with PHRl in the retina, a two hybrid

screen was performed. The entire coding sequence for PHRl was subcloned into the

PAS l bait vector (Fig. 3-7a). As for the ROMl C-terminal tail screen, appropriate control

experiments were performed to establish that correct expression of the PHRI bait

occumed and to cietennine whether the bait was capable of autoactivating the reporter

genes.

The yeast strain used in the PHRl two hybrid screen was KGY37 (provided by D.

Gietz). Unlike the Y 190 strain used in the ROMl C-terminal tail screen, KGY37 does not

produce as many background colonies during the screening procedure and does not

spontaneously lose the IacZ reporter gene unless grown on SD media lacking uracil, the

marker used to integrate the lac2 gene into the yeast genorne (Gietz and Schiestl, 1995).

The P m 1 bait construct was transfonned into KGY37 afler it was demonstrated not to

contai. any PCR induced artefacts by sequencing analysis.

Before beglluiing the PHRl two-hybnd screen, expression of the PHRl bait in

KGY37 was evaluated by Western blot analysis. Fustt, yeast protein lysates were prepared

Figure 3-6. PHRl protein sequence analysis. Kyte-Doolittle (1982) hydropathy plot of PHR 1. The last 20 arnino acids code for the putative transmembrane domain. PHRl profde was plotted as a mean hydrophobie index against amino acid number, using a moving window of 19 aa with a one amino acid interval.

A GALA Binding Domain

+H3N COO'

- 22 amino acids

Protein Markers W a )

HA epitope PHRl

Figure 3-7. PHRl two-hybrid bait. A. Human PHRl two-hybrid constnict. The constnict contains full-length PHRl (243 arnino acids) fused in hune to the GALA binding domain. An HA epitope separates the two polypeptides. B. Western blot of yeast lysates expressing the PHRl bait. The PHRl construct was transformed into KGY37 cells. Lane 1 shows the PHRl fusion protein migrating at 50 Wla. Lane 2 shows the positive control, murine p53 (72-390) and lane 3 negative con001 (KGY37 alone).

from cells containing either a positive control plasmid (pVA3), the PHRl bait, or no

plasmid. The lysates were separated in a protein gel and subsequently transferred to a

nitrocellulose filter for Western blot analysis. An anti-GAIA binâing domain antibody was

used to detect PHRl bait expression (Fig. 3-7b). As predicted, a band rnigrating at -50

kDa was detected, indicating expression of the PHRl bait in KGY37 cells.

To evaluate the autoactivating potential of the PHRl bait, the expression of both

the HIS3 and lac2 reporter genes were analyzed. No P-galactosidase activity was

detected by filter assays, demonstrating that the PHRl bait did not autoactivate the lad

reporter gene. To determine if the PHRl bait autoactivated the HE3 reporter gene,

KGY37 cells expressing the PHRl bait were plated on SD media lacking histidine, but

containing various concentrations of 3-AT ranging fiom 15 mM - 50 rnM. Frorn this

analysis, it was determined that the addition of 15 rnM 3-AT was sufficient to minirnize

background growth to levels observed with the negative control.

The PBRl two-hybrid screen

The same target library was used as that described in Chapter 2. H'S3 reporter

gene expression was evaluated initialiy by transforming the library DNA into KGY37 cells

already containing the PHRl bait and then plating the transformations ont0 plates

containing 15 m M AT and lacking leucine, tryptophan and histidine (Gietz and Schiestl,

1995). Plates were incubated at 30°C between 5-7 days until positive colonies were

identified based on their larger size compared to background growth. From the total of

1.45 million clones screened, 90 HIS' transfomants were isolated. Each positive

transformant was picked and streaked ont0 fiesh plates (Leu-, Trp; His-, 15 mM 3-AT)

which were incubated at 30°C for two days.

The second phase of the screen in the two-hybrid system qualitatively examined

the ability of the target protein to interact with the bait and activate the lac2 reporter gene

(Bai and Elledge, 1996). To test for lac2 expression, B-galactosidase filter assays were

perfomed on ail 90 HIS' transfomüuits. From the examination of lac2 expression, 50

HIS' Lac' transfomants were identifid and were, subsequently, subdivided Uito 36 strong

and 14 weak HIS' ~ a c ' PHRl putative interactors. 1 first analyzed the 36 strong positive

cDNAs. The cDNAs encoding the PHRl putative interacting proteins were isolated fiom

the 36 HIS' ~ a c ' transformants by first selecting for yeast containing the target plasmid.

The target plasmids express the LEU2 marker and can therefore grow, by

complementation, in bacterial cells (MH6) that contain a defect in their leuB gene.

M e r isolating each target clone, specificity tests were perfonned to identify those

proteins encoded by the cDNAs capable of interacting only with the PHRl bait and not

with non-specific baits. The three non-specific baits chosen have been described in

Chapter 2. Each target was subsequently transformed into KGY37 cells expressing one of

the three non-specific baits, which were known not to cause autoactivation. Each target

plasmid was also transformed into yeast cells expressing the PHRl bait in order to re-

confirm the interaction originally identified fiom the PHRl two-hybrid screen. Specificity

was evaluated by determinhg the ability of the targets to activate the lac2 reporter gene

(P-galactosidase filter assays) in the presence of the PHRl bait only. Of the 36 HIS' Lac'

putative interactors identified, 9 were shown to interact specifically with the PHRl bait

(Tables 3-la-i).

Each cDNA was sequenced in order to identify the protein or nucleotide encoded

by the target construct. Database protein and nucleotide cornparisons were then

performed with each target cDNA (Tables 3-la-i). The first two sets of novel cDNAs

encoding PHR1-interacting proteins isolated fiom the screen are most interesting because

each one was identified twice and they each contain interesting protein homologies (Tables

3- 1 a-d).

The cDNAs of 1-3 and 29-1 encode the same protein target with the open reading

frame of the former cDNA being much larger than the latter (Fig. 3-8a) (Table 3-lc and

d). Cornparison of the proteins in the database with the proteins predicted by the open

reading m e s of cDNAs 1-3 and 29-1 revealed that they both contain a C6HC1 zinc-

binding motif, otherwise known as a RING finger domain. The RING finger domain is

considered to be required for protein-protein interactions (Borden and Freemont, 1996).

No other regions of the predicted proteins encoded by the cDNAs 1-3 and 29-1 showed

homology to known proteins in the database, indicating that they are novel molecules.

1 Protein Homology:

100% identical to the polypeptide encoded by cDNA 25-2 from amino acids 1-98 (Table 3- 1 b)

1 -4 polypeptide sequence (1 17 amino acids):

NRCSPRLPLVPGCAADHPRILQHGGRSSRTPIPP LHAPRWLPAQQDAAQSAPLFLWPPLLPLHRSL HPPQATSLWPSTSLYHQLLYHVPDILCTVQPSSR EGNPSRTESHSVPAWPEX

Table 3- la. PHRl two-hybrid target 1-4. Protein homologous to the polypeptide encoded by 1-4 is Iisted, with the percent identity observed. The amino acids in the polypeptide showing homology are in boldface.

1 Protein Hamology:

168% identical to Diff33 (rat)

25-2 polypeptide sequence (3 1 1 arnino acids):

NRCSPRLPLVPGCAADHPRILQHGGRSSRTPIPPLHA , PRWLPAQQDAAQSAPLFLWPPLLPLHRSLHPPQATS LWPSTSLYHQLLYHVPDILCTVQPSPERVILQGQNH TLCLPGLSKMESHTPDTGLTVMSAGIMYACVLFAC NEAPNLAEVFGPLWTVKVY SYEFQKPSLCFCCPETG

, EPEEGECQVRIRPRGVAARPADQETSPAPPVQVQ QLSYSYSAFHFVFFLASLYVMVTLTNWFSYEG AELEKTFITGSWATFWVKVASCWACVLLYLGL LLTPFCWSPIPDPQHPILRRHCHRVLPNDKYPIX

Table 34b. PHRl two-hybrid target 25-2. Protein homologous to the polypeptide encoded by 25-2 is listed, with the percent identity observed. The amino acids in the polypeptide showing homology are in boldface.

Protein Homology:

28% identical to AR1 (Drosophila) Contains a C6HC 1 RING finger domain

3- 1 polypeptide sequence (3 13 arnino acids):

GKSELSCMEGSCTCSFPASELEKVLPQTILYKYYER KAEEEVAAAYADELVRCPSCSFPALLDSDVKRFS CPNPRCRKETCRKCQGLWKEHNGLTCEELAEK DDIKYRTSIEEKMTAARIRKCHKCGTSLIKSEGCNR MSCRCGAQMCYLCRVSINGYDHFCQHPRSPGAPCQ ECSRCSLWTDPTEDDEKLIEEIQKEAEEEQKRKNGE NTFKRIGPPLEKPPEKVQRTEALPRPVPQNLHQPQIP XY AFVHPPFPLPPVRPVFNNFPLNMGPIPAPY VPALP NMRRQLRICPHPPAPGTQPAHALWPPASASX

Table 3- lc. PHR 1 two-hybnd target 3- 1. Proteins homologous to the polypeptide encoded by 3-1 are listed, with the percent identity obsewed. The amino acids in the polypeptide showing homology are in boldface.

Protein Homology :

28% identical to AR1 (Drosophila) 100% identical to the polypeptide encoded by cDNA 3-1 fiom arnino acids 1 - 1 18

Contains a C6HC1 RING finger domain

29- 1 polypeptide sequence (1 32 amino acids):

GKSELSCMEGSCTCSFPASELEKVLPQTILY KYYER KAEEEVAAAYADELVRCPSCSFPALLDSDVKRFS CPNPRCRKETCRKCQGLWKEHNGLTCEELAEK DDIKYRTSIEEKMTAAALGNATSVGPAYOIX

Table 3- Id. PHRl two-hybrid target 29- 1. Proteins homologous to the polypeptide encoded by 29-1 are listed, with the percent identity observed. The amino acids in the polypeptide showing homology are in boldface.

Protein Homology:

( Novel I 10-2 polypeptide sequence (23 amino acids):

PPAGSRHPAELPTRRCPGPGHAPX

Table 3- 1 e. PHRl two-hybrid target 10-2. The polypeptide sequence for 10-2 is novel.

Protein Homology:

b

21-6 polypeptide sequence (23 amino acids):

1 LAOYGISSPWTPRPRTPLLRCPPX

Table 3- 1 f. PHRl two-hybrid target 2 1-6. The polypeptide sequence for 2 1-6 is novel.

Protein Homology:

97% identical to 0TX2 (human and murine) from amino acids 1-98 of 0TX2

6- 1 polypeptide sequence (1 34 amino acids):

SNPGEARPSGWVPRIWADFAPPNNLSMMSYLKQP PYAVNGLSLTTSGMDLLHPSVGYPATPRKQRR ERTTFTRAQLDVLEALFAKTRYPDIFMREEVAL KINLPESRVQVWFKNRRAKCRNKQQATGRTEV KTKX

Table 3- 1 g. PHR 1 two-hybrid target 6- 1. Protein homologous to the polypeptide encoded by 6-1 is listed, with the percent identity observed. The amino acids in the polypeptide showing homology are in boldface.

Nucleotide Homology :

86% identical to the mRNA for Spm RNA binding protein

1-3 nucleotide sequence (440 nucleotides):

GCANAGGGTGTNGTGAATACAGCTGNGTCCGCAGCAGTCC AAGCTGTTCGGGGCAGAGGAAGAGGAACTCTAACAAGGG GGGCTTTTGTTGGGGCCACAGCTGCTCCCGGCTACATAGC TCCAGGCTATGGAACACCCTATGGTTACAGCACAGCTGCCCC TGCCTATGGTTTACCCAAGAGAATGGTTCTG'ITACCCGTTATGA AATTCCCAACATATCCTGTTCCCCACTACTCArlTCTTTTAGCAAA TGACAGAAGCTAA'ITCCTATTCCAACAACAACCCAGTACATACA GAATGTTAGCGAAAAAGCCTTïTïATCCTGCTITCTTTGAACAC ATACTTGATCAAAATTAmGTAAAGAACATc-rrCCTACm T G A T T T T A A C A A A T G C A A A T T T A G T T C T C T A A A C A AACAAAAAAGAAA

Table 3- 1 h. PHR 1 two-hybnd target 1-3. Nucleotide sequence of 1-3 is homologous to the mRNA coding for Spnr RNA binding protein, the percent identity is shown. The nucleotides in 1-3 showing homology are in boldface.

Nucleotide Homology:

pACTII vector

13- 1 nucleotide sequence (440 nucleotides):

CCTTNAAANTTGATACTCGTTTGATGTATATAACTATCTA TTCGATGATGAAGATACCCCACCAAACCCAAAAAAAGAG ATCTCTATGGCTTACCCATACGATGTTCCAGATTACGCTA GCTTGGGTGGTCATATGGCCATGGAGGCCCCGGGGATCCG AATTTTTTAAATGACTAGAATTAATGCCCATCTTTTTTTT GGACCTAAATTCTTCATGAAAATATATTACGAGGGCTTAT TCAGAAGCTTTGGACTTCTTCGCCAGAGGTTTGGTCAAGT CTCCAATCAAGGTTGTCGGCTTGTCTACCTTGCCAGAAAT TTACGAAAAGATGGAAAAGGGTCAAATCGTTGGTAGATA CGTTGTTGACACTTCTAAATAAGCGAATTTCTTATGATTT ATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTG TATACAAATTTT

Table 3- li. PHRl two-hybrid target 13-1. Nucleotide sequence of 13-1 is identical to pACTII vector. The entire sequence of 13-1 listed is in boldface because it is 100% identical to pACTa vector sequence.

A RING Finger domain

CC CCCCHC

homology to 1 Diff33 311

Figure 3-8. Strong PHRl putative interactors identified in the two-hybrid screen. A. The proteins encoded by cDNAsl-3 and 29-1. Shown is the RING finger domain highlighted by the blue and orange bars which consists of 7 Cys residues (C) and 1 His residue (H). B. The proteins encoded by cDNAs 1-4 and 25-2. The polypeptide encoded by cDNA 25-2 is 68% identical to Diff33 over 66 arnino acids. This region of homology with DiM 3 is not present in the polypeptide encoded by cDNA 1-4. The proteins encoded by cDNAs 29-1 and 1-4 greatly restrict the site of the region interacting with PHRl. The open regions at the end of the polypeptides 29- 1 and 1-4 represent amino acids not present in 1-3 or 25-2, respective1 y.

The cDNA 25-2 encodes a 311 amino acid protein which contains a region of

homology, at its C-terminus, to Diff33 (Table 3-lb). Dm3 is an uncharacterized protein,

but is expressed during oligodendroblast to oligodendrocyte differentiation (Pfeiffer,

S. S.E., unpublished). The predicted protein encoded by the cDNA 1-4 is 1 17 amino acids

(Table Ma). Cornparison between the proteins predicted by the open reading frame of

the cDNAs 1-4 and 25-2 revealed that they encode the same protein, with the open

reading frame of the cDNA of the latter being much larger than the former. Analysis of

the predicted protein encoded by 1-4 also revealed that it lacked the homologous region to

Difi33 present in the predicted protein encoded by 25-2 (Fig. 3-8b). No other regions of

homology are known in the proteins encoded by the cDNAs 25-2 and 1 4 making them, as

in the case of the cDNAs 1-3 and 29- 1, novel putative PHR1-interacting proteins.

The proteins encoded by the cDNAs 1-4 and 29-1 greatly restrict the region

interacting with PHRI. It should also be noted that there are 19 amino acids at the C-

terminal end of 1-4 and 14 amino acids at the C-terminal end of 29-1 which are not

present in 25-2 or 1-3, respectively (Fig. 3-8). Because of the absence of these amino

acids in 25-2 or 1-3 both of which can interact with PHRI, it is unlikely that these amino

acids in 1-4 or 29-1 are required for the interaction of PHRI.

The final two novel cDNAs, 10-2 and 21-6, isolated in the PHRl two-hybrid

screen encode different novel polypeptides, with the sizes of each encoded protein being

small, 23 amino acids for both 10-2 and 21-6 (Tables 3-le and 0. Moreover, neither

cDNA contains a domain or motif known in the database. Because of the srnall open

reading h e s , cDNAs 21-6 and 10-2 may not encode biologically relevant PHRI-

interacting proteins.

The final three targets that are specific in their interaction with the PHRl bait

likely represent false associations. The cDNA 13-1 consists only of the pACTII vector

(Table 34 ) , and that fiom cDNA 1-3 (Table 3-lh) encodes a RNA binding protein Spnr.

Both of these cDNAs comonly yield fdse positives in two-hybnd screens. In addition,

the protein predicted by the open reading fhne of the cDNA of bovine target clone 6-1 is

97% identical to murine O W , a homeodomain containhg transcription factor (Table 3-

lg). This protein is unükely to be a physiologicaüy relevant interactor of PHRl because it

Predicted Protein Sequence

VLCCVCRALG VPWGFQVVPI 20 LSYLGTFHPP SPTALAALPA 40 APSSRRALPP ASEPGSGLLA 60 QYVQRNX 66

PPAGSRHPAE LPTRRGPGPG 20 HAPQPRPCLP LDAPALPVPA 40 LRAPGHWLLV TLPQSARAGP 60 AVAGRPLRGG WAPAGPAQAV 80 CCPVLGAAPD QATCGAGSCT 100 WWRPX 1 04

Table 3-2. The weak targets specifc in their interaction with the PHRl bait. The proteins predicted by the open reading frame of the novel cDNAs 8-1 and 24-3 are shown.

is a nuclear transcription factor whereas PHRl localizes to the plasma membrane of

ganglion cells and to the outer segments of photoreceptor cells.

The 14 weak PHRl potential interacting targets were also tested for their ability to

interact with PHRl specifically. Only 2 of the 14 weak PHRl putative interactors were

specific in their association with the PHRl bait. The cDNAs of 8-1 and 24-3 were

sequenced and compared to each other, to the strong PHR1-interacting targets, and to the

database. From these comparisons, the polypeptides encoded by cDNAs 8-1 and 24-3

were detedned to be novel. The open reading hunes of 8-1 and 24-3 encode predicted

novel polypeptides of 66 and 104 amino acids, respectively (TaMe 3-2).

The remaining 39 non-specific interacting proteins were not considered

physiologically relevant for two reasons. First, each non-specific PHR 1 -interacting

protein autoactivated transcription alone in the absence of PHRl. Second, each non-

specific PHRI-interacting protein activated transcription of the reporter gene in the

presence of three different non-specific baits to the same degree as in the presence of the

PHRl bait.

DISCUSSION

Definition of the molecular role of PHRl in the retina requires detailed expression

studies, analysis of fùnctional domains and the identification of physiologically relevant

protein associations. Cornparison of the predicted PHRl protein based on the open

reading fiame of the full-length PHRI cDNA with proteins in the database revealed the

presence of a putative pleckstrin homology (PH) domain. PH domain-containing proteins

are usuaîiy involved in signalling transduction pathways and the cytoskeleton. The

expression of PHRl in the retina occurs in the photoreceptor and ganglion celis. Both

polyclonal and monoclonal antibodies localize PHRl to the photoreceptor outer segment

and ganglion cell layer. Closer examination of the localization of PHRl in the ganglion

cells revealed that PHRl is present at the plasma membrane. The ability of PHRl to

associate with membrane components has been shown directly by its retention in

membrane fiactions prepared ftom bovine rod outer segments (personal communication S.

Xu). The regions in PHRl which may be involved in membrane association are a C-

terminal trammembrane domain, and a N-terminal PH domain. PH domaùis of several

proteins, including PLCG, (Yagisawa et al., 1998), Akt (Frech et al., 1997), and pleckstrin

(Harlan et al., 1994), have been show to interact with specific phosphatidylinositol

phosphates, indicating that at least one fiinction of this domain is to target proteins to

membranes.

PHRI is alternatively transcribed, but its expression in the retina seems to differ

from other tissues. The full-length coding PHRI product is composed of al1 nine exons

whereas exon 7, which is immediately 3' to the exons encoding the PH domain, is not

present in the altematively spliced product. The full-length product is twice as abundant

as the altematively spliced product in the retina. In the brain, kidney, and liver the

altematively spliced product is more than four times as abundant as full-length PHRI.

Further analysis is required to detedne the significance of the difference in expression

between full-length and altematively spliced PHRI. Alternative splicing is common in

genes encoding PH domain-containhg proteins such as Nltersecti~, GRBIO, Sosl,2, and

BIR (Guipponi et al., 1998; Frantz et al., 1997; Fukuda et al., 1996). Generally, the

alternative splicing events that occur for genes encoding PH domain-containing proteins

removes exons coding for part or al1 of the PH domain or sequences upstream or

downstream of the PH domain. Thus, there is no common alternative splicing pattern for

genes encoding PH domain-containing proteins.

To determine the expression and apparent mass of PHRl in the retina, Western

blot analysis was performed with lysates prepared fiom PHRl expressing HEK293 cells

and retina using PHRl polyclonal antibodies. Full-length PHRl migrated at a much

slower rate than expected (-37 Da) , based on the sue of PHRl calculated fiom its amino

acid composition (-25 kDa), in the retina and when transiently expressed in HEK293 cells,

suggesting some fonn of post-translational modification.

Identification of the specific post-translational modifications of PHRl will

contribute to Our understanding of the fùnction of the protein, and also possibly the

regdation of its function. One attractive possibüity is that PHRl is phosphorylated, since

some other PH domain-containing proteins are phosphorylated such as Akt, PLCy, and

LRS-1 (Sable et al., 1998; Falasca et al., 1998; Myers et ai., 1995). The presence of

multiple tyrosine residues in the C-terminus of PHRl is ais0 supportive of this suggestion,

and phosphorylation of tyrosine residues has been demonstrateci in the PH domain-

containing proteins PLCy, and IRS-1 (Falasca et al., 1998; Myers et al., 1995). In

addition, phosphorylation of PHRl may be required to activate PHRI, or alter its

conformation to allow the molecule to perform its function. Phosphorylation is a common

form of post-translational modification required to activate proteins containing PH

domains including pleckstrin, IRS-1, and Akt (Ma et al., 1997; Voliovitch et al., 1995;

Andjelkovic et al., 1 997). Moreover, phosphory lation generally occurs N-terminai or C-

terminal to the PH domain and can involve senne, threonine a d o r tyrosine residues.

Another possibility is that the phosphorylation state of PHRl determines or enhances

protein-protein or protein-lipid interactions it partakes in. The role of phosphorylation in

detennining or enhancing protein-protein or protein-lipid interactions has been show for

IRS-1, Akt, and PLCy (Myers et al., 1995; Sable et al., 1998; Falasca et al., 1998).

Confirming the phosphorylation of PHRl as a possible form of post-translational

modification and identifjmg the residues phosphorylated is therefore important.

Although the function of PHRl in the retina, and particularly the photoreceptor

and ganglion cell, remains to be established, it has been show in vitro that PHRl is

capable of binding to the Gay subunits of transducin and that this interaction requires the

PH domain of PHRl (personal communication S. Xu). The G protein transducin is a

GTP-binding protein which mediates the light activation signal fiom photolyzed rhodopsin

to phosphodiesterase. The association of PHRl with a molenile involved in the light

transduction pathway would be strong direct evidence that PHRl is a component of the

visual transduction cascade.

The PH domains of severai proteins, including Akt, p-spectrin, PARK, and IRS-1,

have been shown to bind to GPy subunits of G proteins (Touhara et al., 1994). This

association involves the C-terminal portion of the PH domain, and most signincantly in

terms of the differentiai splicing of exon 7 of PHRI, amho acids immediately C-terminal

to the PH domain. Recently, it has been shown that the 36 amho acids in the BM (Btk

motif) domain which are located immediately C-terminal to the PH domains of Btk and

GAPlm, are required for the interactions of these proteins with the Ga12 wbunit (Jiang et

al., 1998). The association of G a l l and Btk has functional significance, since it leads to an

increase in the kinase activity of Btk (Jiang et al., 1998). Although PHRl does not

contain a BM domain C-terminal to its PH domain, it does contain a stretch of 35 amino

acids encoded by exon 7 which is alternatively spliced. The removal of these amino acids

immediately C-terminal to the PH domain of PHRl may, therefore, have regulatory and

functional consequences for PHRI.

To begin to understand the role of PHRL in the retina, a two-hybrid screen was

performed using fùll-length PHRl as a b i t with an adult bovine retinal cDNA target

library. Four potential PHRI-interacting proteins were identified (1-4 and 25-2, 3-1 and

29- 1, 8- 1, 24-3). The two PHR1 -interacting candidates which interacted strongly with

PHRl were identified twice in the two-hybnd screen. Both cDNAs 1-4 and 25-2 encode

the sarne novel polypeptides, with cDNA 25-2 encoding a protein that is larger than the

protein encoded by cDNA 1-4 (Fig. 3-8b). In addition, the protein encoded by cDNA 25-

2 has hornology to an uncharacterized protein DiE33, which is expressed d u ~ g the

transition fiom oligodendroblast to oligodendrocyte differentiation (Table 3-1 b) (Pfeiffer,

S.S.E., unpublished). This region of homology to Diff33 is not present in cDNA 1-4. The

protein encoded by cDNA 1-4, however, greatly restricts the size of the region interacting

with PHRl in cornparison to the protein encoded by cDNA 25-2. Both cDNAs 3-1 and

29-1 encode the same novel polypeptides, with cDNA 3-1 encoding a protein that is larger

than the protein encoded by cDNA 29-1 (Fig. 3-8a). Thus, the protein encoded by cDNA

29- 1 restricts the size of the region interacting with PHRI . Interestingly, both proteins

encoded by cDNAs 3-1 and 29-1 contain a zinc binding motif referred to as the RING

finger dornain (Fig. 3-Sa). The RING finger domain present in 3-1 and 29-1 consists of 7

cysteine residues and 1 histidine residue, and is 28% identical to the RING finger domain

present in AR1 (Tables 3-lc and d). ARI is an uncharacterized Drosophila proteh. The

only information available on ARI fiom the database is that its RING fhger motif is

involved in axonal path-finding in the central nemous system of Drosophila (Oliveros, M.,

Apilera, M., Barbas, J. A., Martinez, I., Torroja, L., and Ferrus, A., unpublished). To

begin to define an axonal path-hding role for PHRI, the expression of PHRl in

Drosophila should be determined. If present, Drosophila PHRi mutants could be made

and exarnined for defects in axonal path-finding.

The RING finger mots is characterized by eight conserveci cysteine and histidine

residues which fom two 2n2+ binding sites (Brzovic et al., 1998). There are currently 80-

90 RING finger domain-containing proteins (personal communication A. Ballabio). RING

finger domain-containing proteins are found in organisms fiom yeast to humans. In

addition, proteins containing RING finger domains are involved in diverse cellular

functions and pathways including: 1) signalling pathways: TRAF2 (Cheng and Baltimore,

1996), and TRAe (Song and Donner, 199S), 2) gene transcription: SNLTRF (Moilanen et

al., 1998), and MSL proteins (Copp et al., 1998), 3) protein transpon: Vpsl lp and

Vps 1 8p (Rieder and Emr, 1997), 4) secretory pathways: RMA 1 (Matsuda and Nakano,

1998), 5) apoptosis: DIAPI (Oeda et al., 1998), and 6) embryonic patterning: XNF7

(Borden et al., 1995). The RING finger domain-containing proteins TRAF2, SNURF,

Vpsl lp, and Vpsl8p were identified using the yeast two-hybrid system to interact with

membrane associated proteins such as CD40, androgen receptor (AR), Vpsl6p and

Vps33p, respectively to fom protein complexes (Cheng and Baltimore, 1996; Moilanen et

al., 1998; Rieder and Emr, 1997). In these examples, the RING finger motif was show to

be essential for the protein-protein associations identified. Thus, it seems that the RING

finger domain is involved in protein-protein oligomerizations, acting as a convenient

scaffold for the assembly of protein complexes (Borden and Freemont, 1996). The

identification of a RING finger domain-containing protein as a candidate PHRl interacting

protein, suggests that PHRl may be a component of a membrane-associated protein

complex.

The two other cDNAs identined in the two-hybrid screen encode novel putative

PHR 1 -interacting proteins which interact weakly with PHRl (8- 1 and 24-3) (Table 3-2).

The weak association of these two different polypeptide targets with the PHRl bait may

indicate that: 1) an interaction between these proteins and PHRl occurs in vivo, but is

weak, 2) other proteins may be required to increase the strength of the interaction, 3) an

incorrect PHRl conformation allowed a spunous weak interaction, and 4) the fuiMength

target protein is required for a strong interaction with PHRI. Determinhg the expression

pattern and localization of ail four cDNAs by in situ hybridization and confirming the

interaction of PHRl and its potential interacting partners using other methods such as

affinity chromatopphy is required. The information gained from these analyses may

provide more insight into the biological role of PHRl in the retina.

CHAPTER 4

Concluding Remarks

FUTURE DIRECTIONS

The two-hybrid system was used to identlfy putative ROMI- and PHRI-

interacting proteins. From the two-hybrid screen perfomed with the ROM1 C-terminai

tail, five putative ROM 1 ginteracting proteins were identified. The PHRl two-hybnd

screen identified 11 putative PHRI-interacting proteins, two of which were each

independently isolated twice. To determine which of the ROM 1 - and PHR 1 ginteracting

proteins are capable of associating with ROMl or PHRl in vivo, the retinal expression of

the transcnpt encoding each ROMl - and PHR1 -interacting protein has to be examined. In

addition, the interaction between ROM 1 and each ROM 1 ginteracting protein and PHRl

and each PHR1 -interacting protein has to be confinned using a direct binding method such

as afEnity chromatography.

Localuing expression of PHRl and ROMl putative inteneton in the retina

To determine if the location of expression of the genes encoding PHRl and ROMl

putative interactors in the retina coincide with the expression of PHRI and ROMI,

respectively, in situ hybndization experiments have to be perfomed on adult bovine

retinal sections. Adult bovine retinal sections will be used in the in situ hybndization

experiments because the two-hybrid library used in the screens for both PHRl and ROMl

was made from adult bovine retinas. The PHRl gene is expressed in the photoreceptor

and ganglion ce11 layers. Those genes encoding PHRl putative interactors which are

expressed in the photoreceptor and/or ganglion ce11 layers will be examined further

because they are more likely to encode PHRl interactors than if they were not expressed

in the sarne regions in the retina as P H . 1 . The ROMI gene is expressed only in the

photoreceptor ce11 layer. Thus, genes encoding ROMl putative interacton which are

expressed in the photoreceptor layer wül be exarnined further for their ability to associate

with ROMl.

Using aninity chromatography to con- PBRl and ROMl interacton

To establish that the interaction of the PHRI- or ROM1-interacting proteins and

PHRl or ROMl, respectively are not artefacts of the two-hybrid system, affinity

chromatography will be used to confin the results fiom the PHRl and ROMl two-hybrid

screens. Attinity chromatography is an in vitro system which directly tests an interaction

between two proteins.

The genes encoding putative PHRl or ROMl interactors which are expressed in

the layen of retina that express PHRI or ROM, respectively will be exarnined as strong

potential PHR 1 - or ROM 1 -interacting candidates. The cDNAs encoding PHRl and

ROMl interactors will be subcloned into GSî expression vectors. Columns will be

assembled with the GST-PHR 1 or GST-ROM 1 interacting fusion proteins associated with

glutathione sepharose beads. Ce11 lysates from mamrnalian cells transiently expressing

PHRl or ROMl will be passed through the GST fusion protein columns and colurnns

containing GST alone. The association of PHRl or ROM1 with GST fused PHRl or

ROM1 interactors, resepectively can be detected by Western blot analysis with antibodies

raised against PHRl or ROM, after eluting bound proteins from the columns.

Identifying PHRl and ROMl interacting proteins using aClinity chromatography

Aanity chromatography has four advantages over the two-hybrid system to detect

protein interactions. First, affinity chromatography is a direct method in identifjmg

protein interactions whereas the two-hybrid system takes place in vivo where yeast host

proteins may interfere with protein-protein associations. Second, protein complexes,

present in the protein lysate, which bind to the protein of interest may be detected using

affinity chromatography. In the yeast two-hybrid system, interactions between protein

complexes and the bait can oniy be detected if al1 cDNAs coding for the molecules

forming the complex are present and expressed with the bait. The fact that the cDNAs

encoding the Gpy subunits of transducin were not identified in the PHRl two-hybrid

screen is not surprishg because the likelihood of both cDNAs, encoding each member of

the heterodimeric GPy subunit, would be transformed into the same yeast ceii with the bait

is low. Third, the procedure for Wty chromatography is less tirne consuming than the

screening of a yeast two-hybrid library against a protein of interest. Fourth, the abüity to

detect interacting proteins at low amounts (picograms) after atnnity chromatography is

much easier with ment technological advances in mass spectrornetry (personal

communication J. Ingles). Thus, the increase in the saisitivity and efficiency of afnnity

chromatography offers another technique to detect ROMI- and PHR1-interacting proteins

in retinal protein lysates which may not be identified in the two-hybrid system.

Future perspectives for PERl

To define the function and importance of PHRl in the retina and other tissues will

require detailed genetic and biochemical analyses. Understanding the role of PHRl in

areas of expression including the retina cm be examineci by targeting the gene in mice.

Eliminating expression of PHRl in transgenic mice may lead to an ocular phenotype. For

example, it is possible that there will be photoreceptor andlor ganglion ceIl morphological

and functional abnomalities in phri" rnice. Thus, characterizing the phenotype of phri'

mice may give insight into the biological role of PHRI.

The post-translational modification of PHRl observed in HEK293 cells transiently

expressing PHRl and in retina and brain protein lysates can be assessed by determining,

for example, the phosphorylation state of PHRl (Fig. 3-3). As discussed previously in

Chapter 3, there are clusters of tyrosine residues in PHRl located C-terrninal to the PH

domain which could be possible sites of phosphorylation. To determine if PHRl is

tyrosine phosphorylated, the protein cm be immunoprecipitated fiom HEK293 cells wit h

monoclonal or polyclonal antibodies raiseci against full-length PHRI. The

irnrnunoprecipitated PHRl can be separated by SDS-PAGE, transferred to a nitrocellulose

filter, and phosphotyrosine residues cm be detected by Western blot analysis with anit-

phosphotyrosine antibodies. To identify the tyrosine residues phosphorylated in PHRl,

the irnmunoprecipitated PHRl fkom HEK293 ceils can be cleaved into smaller peptides

and then separated by reverse phase-high performance liquid chrornatography.

Phosphorylated peptides isolated by this procedure can be microsequenced to identify the

phosphotyrosine residues in PHRI.

Future perspectives for ROM1

To idente more ROMl putative interacting proteins, other hydrophilic regions of

ROMl can be used as baits in two-hybrid screens. The two remainhg cytoplasmic regions

of ROMl, besicles the C-terminal tail, are the N-terminal tail (-23 amino acids) and the

loop between the second and third transmembrane domains (-18 arnino acids). Baits cm

be constructed with each of these short cytoplasmic regions of ROMl and used in two-

hybrid screens. One problem with using a short region of a protein in a two-hybrid screen

is that the polypeptide fused to the GALA binding domain may not fold properly. Thus, it

is possible that the two-hybrid screen will only generate cDNAs encoding spunous

interacting proteins. An alternative method to detect ROMl putative interactors is to use

affïnity chromatography. The cytoplasmic regions of ROMl mentioned above cm be

fused to GST and bound to glutathione sepharose beads. Retinai protein lysates can then

be passed through the GST fusion protein columns, and ROM1-interacting proteins can be

eluted, separated by SDS-PAGE, and identified by silver staining. ROM1-interacting

proteins will be identified by microsequencing. Non-specific binding proteins will be

identified fiom the negative control GST only column.

To begin to establish a biochemical role for ROMl in disk morphogenesis as

suggested by the formation of longer disks in the photoreceptor outer segment of ~ o m l "

mice, its involvement in membrane fision can be examined. A role for RDS in membrane

fusion has been established by the protocol designed by Boesze-Battaglia et al (1997). It

was determined that phosphorylation of serine residues in the C-terminai tail of RDS is

required for membrane fùsion (Boesze-Battaglia et al., 1997). ROM1 , however, does not

contain any senne residues in its C-terminal tail. It is possible that ROMl plays no role in

membrane fusion because of the lack of serine phosphorylation sites in the ROM1 C-

terminal tail. However, the role of ROMl in membrane fision could dEer nom RDS or is

required to support the fùnction of RDS and is, therefore, dependent on the presence of

RDS. Membrane fusion experiments designed for RDS cm be used to examine the role of

ROMl in the fusion between retinal rod outer segment membranes and mode1 membranes

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