1. Enzyme-linked Cell Surface Receptors 16 April 2007

2. Mechanism of PDGF signal transduction

3. Enzyme-linked Cell Surface Receptors Receptor Tyrosine kinases: phosphorylate specific tyrosines Receptor Tyrosine kinases: phosphorylate specific tyrosines Tyrosine kinase associated receptors: associate with intracellular proteins that have tyrosine kinase activity. Tyrosine kinase associated receptors: associate with intracellular proteins that have tyrosine kinase activity. Receptorlike tyrosine phosphatases: remove phosphate group Receptorlike tyrosine phosphatases: remove phosphate group Receptor Serine/ Threonine kinases: phosphorylate specific Serine/ Threonine Receptor Serine/ Threonine kinases: phosphorylate specific Serine/ Threonine Receptor guanylyl cyclases: directly catalyzes the production of cGMP Receptor guanylyl cyclases: directly catalyzes the production of cGMP Histidine kinase associated receptors: kinase phoshorylates itself on histidine and then transfers the phosphate to a second intracellular signaling protein. Histidine kinase associated receptors: kinase phoshorylates itself on histidine and then transfers the phosphate to a second intracellular signaling protein.

4. Receptor Tyrosine Kinases (RTKs) Intrinsic tyrosine kinase activity Intrinsic tyrosine kinase activity Soluble or membrane-bound ligands: Soluble or membrane-bound ligands: Nerve growth factor, NGF Nerve growth factor, NGF Platelet-derived growth factor, PDGF Platelet-derived growth factor, PDGF Fibroblast growth factor, EGF Fibroblast growth factor, EGF Epidermal growt factor, EGF Epidermal growt factor, EGF Insulin Insulin Downstream pathway activation: Downstream pathway activation: Ras-MAP kinase pathway Ras-MAP kinase pathway

5. 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

6. Functions include: Functions include: Cell proliferation, differentiation Cell proliferation, differentiation Cell survival Cell survival Cellular metabolism Cellular metabolism Some RTKs have been discovered in cancer research Some RTKs have been discovered in cancer research Her2, constitutively active form in breast cancer Her2, constitutively active form in breast cancer EGF-R overexpression in breast cancer EGF-R overexpression in breast cancer Other RTKs have been uncovered in studies of developmental mutations that block differentiation Other RTKs have been uncovered in studies of developmental mutations that block differentiation

7. Outline Activated RTKs transmit signal to Ras protein Activated RTKs transmit signal to Ras protein Ras transduces signal to downstream serine- threonine kinases Ras transduces signal to downstream serine- threonine kinases Ultimate activation of MAP kinase Ultimate activation of MAP kinase Activation of transcription factors Activation of transcription factors

8. Ligand binding to RTKs Most RTKs are monomeric Most RTKs are monomeric ligand binding to EC domain induces dimerization ligand binding to EC domain induces dimerization FGF binds to heparan sulfate enhancing its binding to receptor: dimeric receptor-ligand complex FGF binds to heparan sulfate enhancing its binding to receptor: dimeric receptor-ligand complex Some ligands are dimeric: direct dimerization of receptors Some ligands are dimeric: direct dimerization of receptors Insulin receptors occur naturally as a dimer Insulin receptors occur naturally as a dimer Activation is due to the conformational change of the receptor upon ligand binding Activation is due to the conformational change of the receptor upon ligand binding

9. Substrate + ATPSubstrate-P + ADP Protein Tyrosine Kinase Protein Tyrosine Phosphatase (PTP)

10. 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).

11. Subfamilies of Receptor Tyrosine Kinases

12. 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)

13. TABLE 15–4 Some Signaling Proteins That Act Via Receptor Tyrosine Kinases SIGNALING LIGAND RECEPTORS SOME RESPONSES Epidermal growth factor (EGF) EGF receptorstimulates proliferation of various cell types Insulin insulin receptor stimulates carbohydrate utilization and protein synthesis Insulin-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 factorsPDGF 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

14. 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

15. Receptor Dimerization and Kinase Activation From Hunter (2001) Nature 411,355.

17. Consequences of receptor dimerization Kinase in one subunit P* one or more tyrosine residues on the other Kinase in one subunit P* one or more tyrosine residues on the other Binding of ATP (insulin-R) or protein substrates (FGF-R) Binding of ATP (insulin-R) or protein substrates (FGF-R) Enhanced kinase activity: P* of other sites on the receptor Enhanced kinase activity: P* of other sites on the receptor P*-tyrosine residues become docking sites for adapter proteins P*-tyrosine residues become docking sites for adapter proteins Small proteins with SH2, PTB and SH3 domains, but without intrinsic enzymatic or signaling activities Small proteins with SH2, PTB and SH3 domains, but without intrinsic enzymatic or signaling activities Coupling activated RTKs to components of signaling pathways such as Ras Coupling activated RTKs to components of signaling pathways such as Ras

18. Ras upstream and downstream signaling. Through a variety of adaptor proteins, these signals cause guanine nucleotide exchange factors to replace the GDP-bound to inactive Ras with GTP. GAPs trigger the hydrolysis of GTP back to the inactive GDP-bound form. GTP-bound Ras binds to a plethora of downstream effector molecules to stimulate intracellular signaling of several pathways. Through a variety of adaptor proteins, these signals cause guanine nucleotide exchange factors to replace the GDP-bound to inactive Ras with GTP. GAPs trigger the hydrolysis of GTP back to the inactive GDP-bound form. GTP-bound Ras binds to a plethora of downstream effector molecules to stimulate intracellular signaling of several pathways. RTKs could activate Ras either by aactivating GEF or inhibiting GAP. RTKs could activate Ras either by aactivating GEF or inhibiting GAP. Campbell and Der 2004

19. Ras Monomeric GTPase switch protein Monomeric GTPase switch protein Its activation is enhanced by GEF Its activation is enhanced by GEF GDP-GTP exchange GDP-GTP exchange Deactivation of Ras-GTP complex requires GAP, which increases intrinsic GTPase activity 100 fold Deactivation of Ras-GTP complex requires GAP, which increases intrinsic GTPase activity 100 fold Lifetime of Ras-GTP is higher than that of G  Lifetime of Ras-GTP is higher than that of G  Ras is a small protein (170 aa. Vs 300 aa of G  ) Ras is a small protein (170 aa. Vs 300 aa of G  ) G  has a domain that functions like GAP G  has a domain that functions like GAP

20. Mutant ras proteins are associated with many cancers Mutant ras proteins are associated with many cancers Mutant ras can bind GTP but can not hydrolyze it, and thus remain constitutively in “on” state Mutant ras can bind GTP but can not hydrolyze it, and thus remain constitutively in “on” state Most oncogenic ras proteins contain a mutation in codon 12 (Gly) Most oncogenic ras proteins contain a mutation in codon 12 (Gly) This blocks the binding of GAP to ras, and prevents GTP hydrolysis. This blocks the binding of GAP to ras, and prevents GTP hydrolysis.

21. Linking ras to RTKs Experimental evidences Experimental evidences Fibroblasts were induced to proliferate with FGF and EGF Fibroblasts were induced to proliferate with FGF and EGF Anti-ras antibody microinjected: cell proliferation arrest Anti-ras antibody microinjected: cell proliferation arrest Injection of mutant ras proteins allows cell to proliferate in the absence of growth factors. Injection of mutant ras proteins allows cell to proliferate in the absence of growth factors. Ligand-bound RTKs activate ras! How? Ligand-bound RTKs activate ras! How?

22. Two cytosolic proteins are involved: GRB2, Sos Two cytosolic proteins are involved: GRB2, Sos SH2 domain in GRB2 binds to a P*-tyrosine residue in the activated receptor SH2 domain in GRB2 binds to a P*-tyrosine residue in the activated receptor Two SH3 domains of GRB2 bind to and activate Sos Two SH3 domains of GRB2 bind to and activate Sos Sos is GEF protein and convert inactive GDP-ras into active GTP-ras Sos is GEF protein and convert inactive GDP-ras into active GTP-ras Developmental studies elucidated the role of GRB2 and Sos in linking RTKs to ras activation Developmental studies elucidated the role of GRB2 and Sos in linking RTKs to ras activation

26. Individual eyes of drosophila: ommatidia Individual eyes of drosophila: ommatidia Each ommatidium consists of 22 cells, 8 of which are photosensitive neurons: retinula or R cells (R1-R8) Each ommatidium consists of 22 cells, 8 of which are photosensitive neurons: retinula or R cells (R1-R8) The RTK sevenless is dedicated to the regulation of R7 development The RTK sevenless is dedicated to the regulation of R7 development Flies with sevenless mutation lack R7 cells in their eyes Flies with sevenless mutation lack R7 cells in their eyes

30. R8 cells express Boss (bride of sevenless) on their surface which acts as a ligand for Sev RTK on R7 cells R8 cells express Boss (bride of sevenless) on their surface which acts as a ligand for Sev RTK on R7 cells Studies with temperature sensitive sev mutants allowed the discovery of downstream proteins in Drosophila Studies with temperature sensitive sev mutants allowed the discovery of downstream proteins in Drosophila SH2 containing GRB2 SH2 containing GRB2 Sos (GEF) Sos (GEF) Ras Ras

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

33. Protein domains of GRB2: Protein domains of GRB2: SH2, PTB: bind to phosphotyrosine residues SH2, PTB: bind to phosphotyrosine residues SH3 (2) : bind to proline rich sequences SH3 (2) : bind to proline rich sequences Proline residues: extended conformation, fit to binding pockets on SH3 domain Proline residues: extended conformation, fit to binding pockets on SH3 domain Other residues determine binding-specificity Other residues determine binding-specificity Upon RTK activation, Sos is recruited to membrane, near to its substrate, Ras. Upon RTK activation, Sos is recruited to membrane, near to its substrate, Ras. C-terminus of Sos inhibits its nucleotide exchange activiyt; binding of GRB2 relieves this inhibition C-terminus of Sos inhibits its nucleotide exchange activiyt; binding of GRB2 relieves this inhibition