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En z yme- linked Cell Surface Receptors. 16 April 2007. Mechanism of PDGF signal transduction. En z yme- linked Cell Surface Receptors. Receptor Tyrosine kinases: phosphorylate specific tyrosines

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En z yme linked cell surface receptors

Enzyme-linked Cell Surface Receptors

16 April 2007



En z yme linked cell surface receptors1
Enzyme-linked Cell Surface Receptors

  • Receptor Tyrosine kinases: phosphorylate specific tyrosines

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

  • Receptorlike tyrosine phosphatases:remove phosphate group

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

  • 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.


Receptor tyrosine kinases rtks
Receptor Tyrosine Kinases (RTKs)

  • Intrinsic tyrosine kinase activity

  • Soluble or membrane-bound ligands:

    • Nerve growth factor, NGF

    • Platelet-derived growth factor, PDGF

    • Fibroblast growth factor, EGF

    • Epidermal growt factor, EGF

    • Insulin

  • Downstream pathway activation:

    • Ras-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:

    • Cell proliferation, differentiation

    • Cell survival

    • Cellular metabolism

  • Some RTKs have been discovered in cancer research

    • Her2, constitutively active form in breast cancer

    • EGF-R overexpression in breast cancer

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


Outline
Outline

  • Activated RTKs transmit signal to Ras protein

  • Ras transduces signal to downstream serine-threonine kinases

  • Ultimate activation of MAP kinase

  • Activation of transcription factors


Ligand binding to rtks
Ligand binding to RTKs

  • Most RTKs are monomeric

  • ligand binding to EC domain induces dimerization

  • FGF binds to heparan sulfate enhancing its binding to receptor: dimeric receptor-ligand complex

  • Some ligands are dimeric: direct dimerization of receptors

  • Insulin receptors occur naturally as a dimer

    • Activation is due to the conformational change of the receptor upon ligand binding


Protein Tyrosine Kinase

Substrate + ATP Substrate-P + ADP

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).



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 types

Insulin insulin receptor stimulates carbohydrate utilization and proteinsynthesis

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

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

factor (M-CSF)proliferationand differentiation

Fibroblast growth factors FGF receptors stimulate proliferation of various cell (FGF-(FGF1 to FGF-24) (FGF-R1–FGFR4) types; inhibit differentiation of someprecursor cells; inductivesignals 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 Tyrosine Kinases

  • 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 Tyrosine Kinases

    From Hunter (2001) Nature 411,355.


    Consequences of receptor dimerization
    Consequences of receptor dimerization Tyrosine Kinases

    • Kinase in one subunit P* one or more tyrosine residues on the other

      • Binding of ATP (insulin-R) or protein substrates (FGF-R)

      • Enhanced kinase activity: P* of other sites on the receptor

  • P*-tyrosine residues become docking sites for adapter proteins

    • 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


  • Ras upstream and downstream signaling
    Ras upstream and downstream signaling. Tyrosine Kinases

    • 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.

    Campbell and Der 2004


    Ras Tyrosine Kinases

    • Monomeric GTPase switch protein

    • Its activation is enhanced by GEF

      • GDP-GTP exchange

    • Deactivation of Ras-GTP complex requires GAP, which increases intrinsic GTPase activity 100 fold

    • Lifetime of Ras-GTP is higher than that of G

      • Ras is a small protein (170 aa. Vs 300 aa of G)

      • G has a domain that functions like GAP


    • Mutant ras proteins are associated with many cancers Tyrosine Kinases

    • 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)

      • This blocks the binding of GAP to ras, and prevents GTP hydrolysis.


    Linking ras to rtks
    Linking ras to RTKs Tyrosine Kinases

    • Experimental evidences

      • Fibroblasts were induced to proliferate with FGF and EGF

      • Anti-ras antibody microinjected: cell proliferation arrest

      • Injection of mutant ras proteins allows cell to proliferate in the absence of growth factors.

    • Ligand-bound RTKs activate ras! How?


    • Two cytosolic proteins are involved: GRB2, Sos Tyrosine Kinases

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

    • Two SH3 domains of GRB2 bind to and activate Sos

    • 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


    • Individual eyes of drosophila: ommatidia Tyrosine Kinases

    • 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

    • Flies with sevenless mutation lack R7 cells in their eyes




    • Protein domains of GRB2: resulted in the development of R7 cells

      • SH2, PTB: bind to phosphotyrosine residues

      • SH3 (2) : bind to proline rich sequences

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

        • Other residues determine binding-specificity

    • 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


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