Enzyme-linked Cell Surface Receptors

16 April 200716 April 2007

C E L LG R O W TH

Pras

MAPK MAPK P

phosphorlyates multipletarget proteins

P D G F

d im e rize &ph os p ho ry la te e ac ho th e r

Mechanism of PDGF signal transduction

EnEnzzyme-yme-linked Cell Surface linked Cell Surface ReceptorsReceptors

Receptor Tyrosine kinases:Receptor Tyrosine kinases: phosphorylate specific phosphorylate specific tyrosinestyrosines

Tyrosine kinase associated receptors:Tyrosine kinase associated receptors: associate with associate with intracellular proteins that have tyrosine kinase activity.intracellular proteins that have tyrosine kinase activity.

Receptorlike tyrosine phosphatases:Receptorlike tyrosine phosphatases: remove remove phosphate groupphosphate group

Receptor Serine/ Threonine kinases:Receptor Serine/ Threonine kinases: phosphorylate phosphorylate specific Serine/ Threonine specific Serine/ Threonine

Receptor guanylyl cyclases:Receptor guanylyl cyclases: directly catalyzes the directly catalyzes the production of cGMPproduction of cGMP

Histidine kinase associated receptors:Histidine kinase associated receptors: kinase kinase phoshorylates itself on histidine and then transfers the phoshorylates itself on histidine and then transfers the phosphate to a second intracellular signaling protein.phosphate to a second intracellular signaling protein.

Receptor Tyrosine Kinases Receptor Tyrosine Kinases (RTKs)(RTKs)

Intrinsic tyrosine kinase activityIntrinsic tyrosine kinase activity Soluble or membrane-bound ligands:Soluble or membrane-bound ligands:

Nerve growth factor, NGFNerve growth factor, NGF Platelet-derived growth factor, PDGFPlatelet-derived growth factor, PDGF Fibroblast growth factor, EGFFibroblast growth factor, EGF Epidermal growt factor, EGFEpidermal growt factor, EGF InsulinInsulin

Downstream pathway activation:Downstream pathway activation: Ras-MAP kinase pathwayRas-MAP kinase pathway

TYROSINE KINASE RECEPTORS

• these receptors traverse the membrane only once

• respond exclusively to protein stimuli– cytokines– mitogenic growth factors:

• platelet derived growth factor

• epidermal growth factor

Functions include:Functions include: Cell proliferation, differentiationCell proliferation, differentiation Cell survivalCell survival Cellular metabolismCellular metabolism

Some RTKs have been discovered in cancer Some RTKs have been discovered in cancer researchresearch Her2, constitutively active form in breast cancerHer2, constitutively active form in breast cancer EGF-R overexpression in breast cancerEGF-R overexpression in breast cancer

Other RTKs have been uncovered in studies of Other RTKs have been uncovered in studies of developmental mutations that block developmental mutations that block differentiation differentiation

OutlineOutline

Activated RTKs transmit signal to Activated RTKs transmit signal to Ras proteinRas protein

Ras transduces signal to Ras transduces signal to downstream serine-threonine downstream serine-threonine kinaseskinases

Ultimate activation of MAP kinaseUltimate activation of MAP kinase Activation of transcription factorsActivation of transcription factors

Ligand binding to RTKsLigand binding to RTKs Most RTKs are monomericMost RTKs are monomeric ligand binding to EC domain induces ligand binding to EC domain induces

dimerizationdimerization FGF binds to heparan sulfate enhancing its FGF binds to heparan sulfate enhancing its

binding to receptor: dimeric receptor-ligand binding to receptor: dimeric receptor-ligand complexcomplex

Some ligands are dimeric: direct dimerization Some ligands are dimeric: direct dimerization of receptorsof receptors

Insulin receptors occur naturally as a dimerInsulin receptors occur naturally as a dimer Activation is due to the conformational change of Activation is due to the conformational change of

the receptor upon ligand bindingthe receptor upon ligand binding

Substrate + ATP Substrate-P + ADP

Protein Tyrosine Kinase

Protein Tyrosine Phosphatase (PTP)

Tyrosine Protein Phosphorylation

• Eukaryotic cells coordinate functions through environmental signals -

soluble factors, extracellular matrix, neighboring cells.

• Membrane receptors receive these cues and transduce signals into the

cell for appropriate response.

• Tyrosine kinase signalling is the major mechanism for receptor signal

transduction.

• Tyrosine protein phosphorylation is rare (1%) relative to

serine/threonine phosphorylation.

• TK pathways mediate cell growth, differentiation, host defense, and

metabolic regulation.

• Protein tyrosine phosphorylation is the net effect of protein tyrosine

kinases (TKs) and protein tyrosine phosphatases (PTPs).

Subfamilies of Receptor Tyrosine Kinases

Protein Tyrosine Kinases (TKs)

Receptor tyrosine kinases (RTK)

– insulin receptor

– EGF receptor

– PDGF receptor

– TrkA

Non-receptor tyrosine kinases (NRTK)

– c-Src

– Janus kinases (Jak)

– Csk (C-terminal src kinase)

– Focal adhesion kinase (FAK)

TABLE 15–4 Some Signaling Proteins That Act Via Receptor Tyrosine Kinases

SIGNALING LIGAND RECEPTORS SOME RESPONSES

Epidermal growth factor (EGF) EGF receptor stimulates proliferation of various cell typesInsulin insulin receptor stimulates carbohydrate utilization

and protein synthesisInsulin-like growth factors IGF receptor-1 stimulate cell growth and survival

(IGF-1 and IGF-2)

Nerve growth factor (NGF) Trk A stimulates survival and growth of some neurons

Platelet-derived growth factors PDGF receptors stimulate survival, growth, and proliferation of various cell types

Macrophage-colony-stimulating M-CSF receptor stimulates monocyte/macrophage

factor (M-CSF) proliferation and differentiation

Fibroblast growth factors FGF receptors stimulate proliferation of various cell (FGF-(FGF1 to FGF-24) (FGF-R1–FGFR4) types; inhibit differentiation of some precursor cells; inductive signals in

development

Vascular endothelial growth VEGF receptor stimulates angiogenesis

factor (VEGF)

Ephrins (A and B types) Eph receptors (A and B) stimulate angiogenesis; guide cell and axon migration

Signaling from tyrosine kinase receptors

• Ligand induced dimerization• Autophosphorylation

• Phosphorylation in the catalytic domain increase the kinase activity

• Phosphorylation outside the catalytic domain creates specific binding for other proteins.

• Autophosphorylated receptors bind to signaling proteins that have SH2 (phosphotyrosine residues) domains

Receptor Dimerization and Kinase Activation

From Hunter (2001) Nature 411,355.

Consequences of receptor Consequences of receptor dimerizationdimerization

Kinase in one subunit P* one or more tyrosine Kinase in one subunit P* one or more tyrosine residues on the otherresidues on the other Binding of ATP (insulin-R) or protein substrates Binding of ATP (insulin-R) or protein substrates

(FGF-R)(FGF-R) Enhanced kinase activity: P* of other sites on the Enhanced kinase activity: P* of other sites on the

receptorreceptor P*-tyrosine residues become docking P*-tyrosine residues become docking

sites for adapter proteinssites for adapter proteins Small proteins with SH2, PTB and SH3 Small proteins with SH2, PTB and SH3

domains, but without intrinsic enzymatic or domains, but without intrinsic enzymatic or signaling activitiessignaling activities Coupling activated RTKs to components of Coupling activated RTKs to components of

signaling pathways such as Rassignaling pathways such as Ras

Ras upstream and downstream Ras upstream and downstream signaling. signaling.

Through a variety of adaptor Through a variety of adaptor proteins, these signals cause proteins, these signals cause guanine nucleotide guanine nucleotide exchange factors to replace exchange factors to replace the GDP-bound to inactive the GDP-bound to inactive Ras with GTP. GAPs trigger Ras with GTP. GAPs trigger the hydrolysis of GTP back the hydrolysis of GTP back to the inactive GDP-bound to the inactive GDP-bound form. GTP-bound Ras binds form. GTP-bound Ras binds to a plethora of downstream to a plethora of downstream effector molecules to effector molecules to stimulate intracellular stimulate intracellular signaling of several signaling of several pathways. pathways.

RTKs could activate Ras RTKs could activate Ras either by aactivating GEF or either by aactivating GEF or inhibiting GAP.inhibiting GAP. Campbell and Der 2004

RasRas

Monomeric GTPase switch proteinMonomeric GTPase switch protein Its activation is enhanced by GEFIts activation is enhanced by GEF

GDP-GTP exchangeGDP-GTP exchange Deactivation of Ras-GTP complex requires Deactivation of Ras-GTP complex requires

GAP, which increases intrinsic GTPase GAP, which increases intrinsic GTPase activity 100 foldactivity 100 fold

Lifetime of Ras-GTP is higher than that of Lifetime of Ras-GTP is higher than that of GG Ras is a small protein (170 aa. Vs 300 aa of GRas is a small protein (170 aa. Vs 300 aa of G)) GG has a domain that functions like GAP has a domain that functions like GAP

Mutant ras proteins are associated Mutant ras proteins are associated with many cancerswith many cancers

Mutant ras can bind GTP but can not Mutant ras can bind GTP but can not hydrolyze it, and thus remain hydrolyze it, and thus remain constitutively in “on” stateconstitutively in “on” state

Most oncogenic ras proteins contain Most oncogenic ras proteins contain a mutation in codon 12 (Gly)a mutation in codon 12 (Gly) This blocks the binding of GAP to ras, This blocks the binding of GAP to ras,

and prevents GTP hydrolysis.and prevents GTP hydrolysis.

Linking ras to RTKsLinking ras to RTKs

Experimental evidencesExperimental evidences Fibroblasts were induced to proliferate Fibroblasts were induced to proliferate

with FGF and EGFwith FGF and EGF Anti-ras antibody microinjected: cell Anti-ras antibody microinjected: cell

proliferation arrestproliferation arrest Injection of mutant ras proteins allows Injection of mutant ras proteins allows

cell to proliferate in the absence of cell to proliferate in the absence of growth factors.growth factors.

Ligand-bound RTKs activate ras! How?Ligand-bound RTKs activate ras! How?

Two cytosolic proteins are involved: GRB2, Two cytosolic proteins are involved: GRB2, SosSos

SH2 domain in GRB2 binds to a P*-tyrosine SH2 domain in GRB2 binds to a P*-tyrosine residue in the activated receptorresidue in the activated receptor

Two SH3 domains of GRB2 bind to and Two SH3 domains of GRB2 bind to and activate Sosactivate Sos

Sos is GEF protein and convert inactive Sos is GEF protein and convert inactive GDP-ras into active GTP-rasGDP-ras into active GTP-ras

Developmental studies elucidated the role of Developmental studies elucidated the role of GRB2 and Sos in linking RTKs to ras GRB2 and Sos in linking RTKs to ras activationactivation

Individual eyes of drosophila: Individual eyes of drosophila: ommatidiaommatidia

Each ommatidium consists of 22 cells, Each ommatidium consists of 22 cells, 8 of which are photosensitive 8 of which are photosensitive neurons: retinula or R cells (R1-R8)neurons: retinula or R cells (R1-R8)

The RTK sevenless is dedicated to the The RTK sevenless is dedicated to the regulation of R7 developmentregulation of R7 development

Flies with sevenless mutation lack R7 Flies with sevenless mutation lack R7 cells in their eyescells in their eyes

R8 cells express Boss (bride of R8 cells express Boss (bride of sevenless) on their surface which acts sevenless) on their surface which acts as a ligand for Sev RTK on R7 cellsas a ligand for Sev RTK on R7 cells

Studies with temperature sensitive Studies with temperature sensitive sev mutants allowed the discovery of sev mutants allowed the discovery of downstream proteins in Drosophiladownstream proteins in Drosophila SH2 containing GRB2SH2 containing GRB2 Sos (GEF)Sos (GEF) RasRas

Introduction of mutant Ras proteins Introduction of mutant Ras proteins into sev- mutant flies resulted in the into sev- mutant flies resulted in the development of R7 cellsdevelopment of R7 cells

Protein domains of GRB2:Protein domains of GRB2: SH2, PTB: bind to phosphotyrosine residuesSH2, PTB: bind to phosphotyrosine residues SH3 (2) : bind to proline rich sequencesSH3 (2) : bind to proline rich sequences

Proline residues: extended conformation, fit to Proline residues: extended conformation, fit to binding pockets on SH3 domainbinding pockets on SH3 domain

Other residues determine binding-specificityOther residues determine binding-specificity Upon RTK activation, Sos is recruited to Upon RTK activation, Sos is recruited to

membrane, near to its substrate, Ras.membrane, near to its substrate, Ras. C-terminus of Sos inhibits its nucleotide C-terminus of Sos inhibits its nucleotide

exchange activiyt; binding of GRB2 relieves exchange activiyt; binding of GRB2 relieves this inhibitionthis inhibition