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Receptor Tyrosine Kinases.docx

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Biology 2382B
Jessica Kelly

Lecture 17 – Receptor Tyrosine Kinases (RTK) Receptor Tyrosine Kinases (RTKs) • N-terminal Extracellular (ligand-binding) domain o Ligands include growth factors (i.e. NGF, PDGF, FGF, EGF) and insulin (in some cases) which trigger cell growth, proliferation, & differentiation • Single transmembrane a-helix (unlike multi-membrane spanning GPCR’s) • C-terminal cytoplasmic domain with intrinsic tyrosine kinase activity which is stimulated by ligand binding  binding causes receptor dimerization • Receptor dimerization results in autophosphorylation which activates kinase activity  other tyrosine residues in cytosolic domain are also phosphorylated • Tyrosine phosphorylation recruits adaptor proteins that mediate interactions with Ras • Ras acts as a GTPase switch protein to signal further “downstream” kinases • Aberrant signaling  inovled in many types of human cancers These receptor proteins are involved in signalling pathways that result in cell growth proliferation, and differentiation…thus disruptions in these signalling pathways often result in cancer & are therefore a key interest of studying in cellular signalling… 1 Activation of RTK’s  RTK’s usually exist monomerically when not bound to their respective ligand (growth factor) • Ligand Binding  causes dimerization of monomeric RTK’s by causing a conformational change in the receptor • Dimerization  results in activation of the cytosolic portion of the protein  activates intrinsic kinase activity of the protein (RTK) = trans-autophosphorylation of cytoplasmic domains (tyrosine’s) • Initial phosphorylation on the activation lip (key event) o Acts a trigger to increase kinase activity o RTK phosphorylates tyrosine residues on other portions of the cytoplasmic domain Phosphotyrosines serve as docking sites for downstream signal-transduction proteins containing SH2 or PTB domains (adapter proteins)… 2 Adapter Proteins Adapter proteins contains unique domains that recognize specific sequences… Common Adapter Domains: o SH2 -src homology 2 domain (Src is an acronym for sarcoma) o PTB – Phosphotyrosine-binding domain IRS-1- Insulin receptor substrate protein SH2 domains present in at least 100 human proteins…many of which are signalling proteins… • Phosphotyrosine residues recruit specific adapter proteins (often specificity provided by specific residues adjacent to the phosphotyrosine) • Adapter proteins have SH2 domains that recognize phosphotyrosines • PTB domains also specifically bind to phosphotyrosine residues • SH2 and PTB domains  essential for recognition of the RTK by adapter proteins Adapter proteins act to mediate interactions between RTK and other signalling proteins (i.e. Ras)…  Phosphorylation of adapter proteins by RTK  can act to recruit others  results in the signal being transmitted 3 Discovery of First Oncogene  In 1911 Rous injected a cell free extract from a chicken tumor (sarcoma) into healthy chickens - all developed tumors  suspected cancer was transmissible  Cell-free transmissible agent  determined to be a retrovirus (Rous Sarcoma Virus) which produced the oncogenic effect  One of the genes (src) within the virus encodes a tyrosine kinase  The src gene is highly conserved and even a version found in humans c-Src – normal version of src (regulated & not oncogenic) v-Src – viral version of src (non-regulated & oncogenic) Viral src  lacks C-terminus relative to human version that acts as an inhibitory region (via phosphorylation)…thus viral src activity is un-regulated… • SH2 & SH3 (src homology 2 & 3)  highly conserved domains involved with RTK’s 4 Ras - a GTPase Switch Protein Ras – a lipid anchored monomeric G-protein (GTPase superfamily)…similar to Gab u smaller, lower GTPase activity & is not directly linked to cell-surface receptors Guanine Nucleotide Exchange Factor (GEF)  proteins that aid Ras in dissociating from GDP to allow binding of GTP (activation/turn on) GTPase Activating Protein (GAP)  increase the GTPase activity of Ras (Ras does not have very good/strong intrinsic GTPase activity (hydrolysis of GTP to GDP = inactivation/turning off) Ras activity is regulated by interactions between Ras & GEFs and GAPs… 20-30% Human cancers: Ras bindD GTP but no hydrolysis, mutation of glycine-12 prevents binding of GAPs (Ras protein)…this makes Ras a proto-oncogene… Ras - a constitutively active Ras mutant protein mutant 5 EGF-Induced Ras Activation: Step 1  Ras is initially in inactive state (i.e. GDP- bound)  anchored to the membrane by Prenylation by farnesyl) Epidermal Growth Factor (EGF) binds EGF Receptor (EGFR) which triggers dimerization  promotes trans- autophosphorylation/activation of kinase activity…  Autophosphorylation  sets up docking scaffold for adapter proteins containing SH2 and SH3 domains (bind to phosphotyrosines) Growth Factor Receptor Bound Protein (GRB) - an adapter protein with SH2 domain that binds phosphotyrosines on cytosolic domain of RTK Sos “son of sevenless” - a guanine nucleotide exchange factor (GEF) that binds the SH3 domains on GRB2 adapter proteins 6 EGF-Induced Ras Activation: Step 2
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