enzyme linked receptors lecture

5/23/2018 Enzyme Linked Receptors Lecture

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Enzyme Linked Cell

Surface Receptors


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Enzyme-linked Receptors include growth factor

receptors which signal complex cellular reactions

leading to proliferation, differentiation, or survival.

Responses to growth factors are usually slow.

Enzyme-linked receptors also mediate direct, rapid

effectors on the cytoskeleton, controlling cell

m o v e m e n t a n d s h a p e ( p h a g o c y t o s i s ) .

Abnormalities in signaling via enzyme-linked

receptors can lead to cancer. The largest class ofenzyme-linked receptors contain a cytoplasmic domain

which functions as a tyrosine protein kinase.


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Six known classes of enzyme-linked

receptors

1. receptor guanylyl cyclases

2. receptor tyrosine kinases

3. tyrosine-kinase associated

receptors

4. receptor tyrosine phosphatases

5. receptor serine/threonine kinases

6.Histidine-kinase-associated

receptors


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Examples of RTKs and their ligands

Nerve growth factor..NGFR

Platelet-derived growth factor .PDGFR

Fibroblast growth factorFGFR

InsulinIR

Epidermal growth factorEGFR?..Her2/Neu/ErbB2

Actions in cell:

Regulation of cell proliferationRegulation of cell differentiation

Regulation of cell motility

Promotion of cell survival

Modulation of cellular metabolism


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Seven subfamilies of RTKs


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General structure of receptor tyrosine kinases

These receptors usually have only one transmembrane segment (

helix). Signal molecules must cross-link, bring together more than

one receptor in order to induce a signal. No allosteric shape

changes.


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Receptor tyrosine kinases

Receptor itself possesses intrinsictyrosine kinase

activity

Once the ligand binds, the receptor can dimerize

and it become an active tyrosine kinase

It phosphorylates itself (autophosphorylation),causing:

1. Increase kinase activity

2. Increased affinity for other proteins

Once bound, these docking proteins can becomephosphorylated


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EGF EGF

Ligand binding usually causes dimerization of the receptor

P P

P P

Membrane

outside of the cell

inside of the cell


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The docking of intracellular signaling proteins onan activated RTK


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Phosphorylated tyrosines serve as docking

sites for proteins containing SH2 domains

The SH2 (src-homology) domain

P-tyr-Compact

-Modular

-Binds to

proteins

sequences

containing

phosphotyrosines

AA side chain

NH2 COOH

Three-D

structure of an

SH2 domain

Representation of PDGFR

Not shown: proteins with PTB

(phosphotyrosine-binding) domains can

also bind to activated receptors.


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Ligand-dependent autophosphorylation anddocking

Not shown: proteins with PTB

(phosphotyrosine-binding) domains can

also bind to activated receptors.


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GRB2

P-Tyr binds downstream regulators containing SH2 domains

PP

PP

PLC

PI3K

SH2 Domain

GAP

Ras pathway


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Scaffolding proteins help organize MAPKs


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Cell Division pathwayMammalian Ras activation

GRB2

PP

PP

PLC

PI3K

SH2 Domain

GAP

(DRK)

SOS

RasGTP

Downstream

pathways


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Grb-2Adaptor: SH2- DomainSOS is a Ras-GEF (guanine nucleotideexchange factor)Ras: GTP-binding Protein

(Onkogen detected in Rat Sarcoma)

Sos

Activation of Ras by activated RTK


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SH2 (Src-homology 2 domain)

-Protein interaction domains; interact with phosphotyrosines on activated RTK

SH3 domains

-protein interaction domains; interacts with SOS (son of sevenless). SOS

regulates Ras activity.

SH3SH2SH3

Grb-2 is composed only of SH2 and SH3 domains

Grb-2

Grb-2 is an adaptor protein that couples activated RTKs to

downstream signaling proteins such as Ras.


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Adaptor proteinscouple RTKs to

Ras

Adaptor

protein: Grb-2

GEF: SOS (son

of sevenless) -

promotes loss

of GDP from

Ras


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RAS is a monomeric GTPase

Ras-monomeric GTPase

-anchored at plasma

membrane

-switches between

active and inactive states

-regulated by GEF (guanine

exchange

factor)

and GAP (GTPase activatingprotein)

Mutant on forms of Ras

are associated with cancer

Ribbon structure of Ras/GDP


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GTP

GAP

GDP

GEF

ActiveInactive

Stimulus

Regulation of Ras activity

Ras superfamily of small GTPases help relay signals from RTKs

Downstream

Signaling

Pathways

Guanine nucleotide Exchange Factor

GTPase Activating Protein


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Cycling of Ras between active and inactiveforms

inactive active

4 steps:

1.GEF facilitates dissociation

of GDP from Ras2. GTP binds to Ras, GEF

dissociates

3. GAP binds to Ras/GTP

4. GTP is hydrolyzed, Ras is

inactive.


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Activated Ras induces a kinasecascade that leads to cell proliferation

The tyrosine phosphorylations and the activation of Ras areshort-lived;

-phosphorylation events are reversed by tyrosine-specific

protein phosphatases.

To stimulate the cells to proliferate or to differentiate, the cellconverts the signal into a cascade of serine/threonine

phosphorylations. This cascade activatesMAP kinase.

RTK Ras MAP KinasePLC IP3 and DAG

Adaptor proteins/modifiers of Ras activity MAP Kinase Kinases

Protein Kinase C

GRB2 SOS RAF MEK

Mobilization of Ca++


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Steps from activated Ras to MAPkinase.

1.Activated Ras recruits Map kinase-kinase-kinase (MAPKKK)

to the cell membrane and induces a conformational change in

MAPKKKthat activates its ser/thr kinase activity.

-usually RAF.2. MAP Kinase-kinase -kinase phosphorylates MAP kinase-kinase

(MEK).

3. MAP-kinase-kinase catalyzes a phosphorylation event on

threonine and one on tyrosine to make MAP-kinasefully active.


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Ras activates

MAP-Kinase

Pathway

1- MAPKKK

2- MAPKK (MEK)

3- MAPK (Raf)

MAPK:

Mitogen-activated

Kinase

(there are three

MAP-Kinase cascades:

MEK/ERK

P38

JNK)


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1. Ras takes up GTP

2. Binding of GTP to Ras exposes

a binding site for Raf (Map KKK).

Releases Raf from inhibitory complexes.

3+ 4. GTP is hydrolyzed, activeRaf dissociates.

5. Raf binds to and phosphorylates MEK

(MAP KK) to activate it.

6. MEK phosphorylates MAP kinase

on a threonine and a tyrosine residue.

NOTE: MAP kinase is active only whenboth threonine and tyrosine are P

MEK

Map kinase

Map kinase

Map kinase

Raf

Raf

Raf

MEK


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The Ras-Raf-MAP kinase pathway

Tyr-P

Grb2

SH3 domains

Proline-rich regions (-PXXP-)

Sos Ras

(inactive)

GDP GTP

PiRas

(active)

Raf

MEK

MAP kinase

P

PP

DNA

Nucleus

MAP kinase

PPfos jun

PP

Increase gene expression

SH2 domain


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GRB2

PP

PP

PLC

PI3K

GAP

Other signaling pathways

Cell survival

Cell survival pathwaysRTKs can activate PI3-Kinase


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OH OH

OH

123

4

5 6

P O

O

OO

-

CH2

CH CH2

C OCH2

C O

O O

CH2

PhosphatidylInositol(PI)

PI3K

OH OH

OH

123

4

5 6

P O

O

OO

-

C

H2

CH CH2

C OCH2

C O

O O

CH2

PI(3)P

PI3 Kinase (PI3K) and cell survival pathways

PI3K

+

PI4KOH OH

OH

123

4

5 6

P O

O

OO

-

CH2

CH CH2

C OCH2

C O

O O

CH2

PI(3,4)P2

+PI5K

OH OH

OH

123

4

5 6

P O

O

OO

-

CH2

CH CH2

C OCH2

C O

O O

CH2

PI(3,4,5)P2

AKT

PH PH

PDK1

P

SER

BAD

ser

PI 3 Kinase Pathway and Survival


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PKB, PDK:(PDK: PI-dependent

kinase)

Ser/Thr kinases

PI-3 Kinase Pathway and Survival


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R7 photoreceptor development

Fruitfly (Drosophila melanogaster)

Compound eye (800 ommatidia)

Each ommatidium has 8 photoreceptorcells; each detects a different wavelength

of light


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R7 photoreceptor development

Photoreceptor cells recruited as an

undifferentiated precursor from epithelial

sheet of cells

Each photoreceptor develops in a specific

order beginning with R8 & ending with R7

(responds to ultraviolet light)


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The R7 photoreceptor developmental

pathway is a RTK-MAP kinase cascade


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Cytokines: Secreted proteins that stimulate cell migration, growth,differentiation,and/or survival of various blood cells including cells of the immune system.

- Examples:

Erythropoietin : - regulates erythropoiesis (red blood cell formation)- made AMGEN a ton of $$$$$

Interferons: - alpha, beta, and gamma forms- functions include resistance to viral infection

Interleukins: - over 20 distinct forms- stimulate various cells of the immune systemincluding B and T cells, megakaryocytes, neutrophils,macrophages

Cytokine signaling by the JAK-STAT pathway


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C t ki t C i t f i l b i l l


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Cytokine receptors:

-Signal through tyrosine phosphorylation, yet no tyrosine kinase domain insubunits conserved membrane-prox im al domain (shaded boxes) in one ormore subuni t is required for tyros ine phosphory lat ion and cel lu lar

responses

- Membrane-proximal domains stably interact withJanus

Kinases

Consist of one or more single-membrane-spanning molecules

EPO-R IL6-R INFg-RR1 R1

R2 R2

gp gp130 130


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Janus Kinases (JAKs)

Janus, the Roman God of Gates and Doors,has two facesone regarding what is behind

and the other looking toward what lies ahead.

PTKKL

Four vertebrate members (JAK1, JAK2, JAK3, TYK2) with similar structure.- PTK is functional protein-tyrosine kinase domain (act. loop autoP sites)

- KL is a :kinase-like domain. Not a functional catalytic domain butmay play an important regulatory role

N C

interacts with receptor

YYPP


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Receptor

cytokine

JAK/STAT pathway 1. Cytokine binding induces receptor conformation change


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cytokine

JAK JAKP P

JAK/STAT pathway 2. JAKs activated by t ransautophosphorylation.


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JAK JAK

P P

P P

JAK/STAT pathway 3. JAKs phosphorylate the receptors and STATs are recruited.

STAT = signal transducer and activator of transcription


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JAK JAK

P P

P P

P P

JAK/STAT pathway 4. JAKs phosphorylate the STATs , causing them to dissociate.

STATs then dimerize through reciprocal SH2 interactions.


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JAK JAK

P P

P P

P

P

JAK/STAT pathway 5. STAT dimers translocate to the nucleus.


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P

P

ISRE/GAS

Gene transcription

Nucleus

JAK/STAT pathway 6. STAT dimers bind to specific gene-regulatory elements a activate transcription.


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Type 1 (/) IFN signaling


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Ch i i f STAT i li


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Characteristics of STAT signaling

1. JAKs are constitutively associated with the receptor and are activated

upon receptor dimerization.

2. STAT pre-exists in the cytoplasm and is activated by tyrosine

phosphorylation and then translocates into the nucleus to activate

genes.

3. The STAT SH2 domain has dual roles: 1) binding to the receptor

2) forming homo- or heterodimers

4. STAT dimerization is mediated through SH2-phosphotyrosine

interactions. Dimerization is required for STAT to bind to DNA.

5. Specificity of the STAT signaling pathways can be achieved at

multiple steps.

Common Protein Kinases Regulating Signal


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Transduction


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BMP2 subfamily: Gastrulation, neurogenesis,chondrogenesis, interdigital apoptosis,mesoderm patterning

BMP5 subfamily: Development, neurogenesis

GDF5 subfamily: Chondrogenesis in developing limbs

BMP3 subfamily: Osteogenic differentiation, monocyte

chemotaxis

Activin subfamily: FSH production, erythroid celldifferentiation, mesoderm induction (frog)

TGF-beta subfamily: cell cycle arrest in epithelial and

hematopoietic cells, mesenchymal cellproliferation and differentiation, woundhealing, ECM production and modulation,angiogenesis, chemotaxis, invasion,immunosuppression, apoptosis


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From Massague, 1998. Ann Rev Biochem. 67:753-791.

SBE= SMAD binding element


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Roberts, et al., 2000. pp39-51. From: Signaling networks and cell cycle control. Ed. S.

Gutkind, Human Press, NJ.

SAD

All About Smads


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Miyazawa ( 2002) Genes to Cells 7: 1191-1204

All About Smads

R-Smads (Receptor-regulated Smads)

e.g. Smad2 and Smad3Activated by activin, nodal and TGF (Alk4,5,7)

Co-Smads (Common partner Smads)

R-Smad and Co-Smads interact with co-activatorsand co-repressors and transcription factors

I-Smads (Inhibitory Smads)

Associate with type I receptors and prevent theactivation of R-Smads (Smad 6 inhibits Smad 4)

TGF Pathway Modulation


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Herpin et al. ( 2004) Dev Comp Immunol. 28: 461-85.

y


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enzyme linked receptors lecture

Documents

rtk5232018 enzyme

cell5232018 enzyme

known classes of enzyme

receptor tyrosine kinases3

thanone receptor

docking proteins

tyrosine protein kinase

growth factorreceptors