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Two receptor classes

Two receptor classes. Receptor tyrosine kinases (RTKs) Ligand induced dimerization Autophosphorylation Substrate phosphorylation Adapter proteins G-Protein coupled receptors (GPCRs) Ligand induced activation Guanine exchange factor (GEF) Second messenger cascade. Nucleotide cyclases.

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Two receptor classes

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  1. Two receptor classes • Receptor tyrosine kinases (RTKs) • Ligand induced dimerization • Autophosphorylation • Substrate phosphorylation • Adapter proteins • G-Protein coupled receptors (GPCRs) • Ligand induced activation • Guanine exchange factor (GEF) • Second messenger cascade

  2. Nucleotide cyclases Kinases Phospholipases Hierarchical overview Signaling molecule G-Protein coupled receptor Receptor tyrosine kinase Second messengers Effector kinase cascades Gene expression Protein activity Phenotypic behavior

  3. eg: Insulin • Generated by pancreas • Acts on muscle & other tissues • Multiple mechanisms • Multiplicative mechanisms IR IRS-1 Shc Glut4 translocation GRB2 PI3-K PKC Protein synthesis Raf mTOR Akt MEK Gene transcription Elk-1 MAPK

  4. eg: Prostaglandin E2 • Locally generated • Inflammatory mediator • Labor EPR Gq Gi L-type Ca2+ Potentiation AC PLC PKC In/Decreased Contractility Raf CaM Ca2+ MEK Gene transcription Elk-1 MAPK

  5. Receptor tyrosine kinases • Single pass transmembrane protein • Ligand induces dimerization • Kinase activity • Autophosphorylation • Complex formation FGF receptor binding FGF FGF Cytoplasmic

  6. Phosphotyrosine binding • Phospho-Tyrosine Binding (PTB) • Src homology (SH2, SH3) domain • Common amino acid motif • Phosphotyrosine binding pocket • Phosphorylation dependent association Interaction with both pY and nearby residues Sh2 domain from Itk PDB:2etz xnnletyewy nksisrdkae kllldtgkeg afmvrdsrtp gtytvsvftk aiisenpcik hyhiketnds pkryyvaeky vfdsiplliq yhqynggglv trlrypvcg

  7. Phosphotyrosine binding • Recruit biologically active molecules • Phospholipases, PI3-K • GTPase modulators (Sos, DOCK180) • Adapter proteins (Grb-2, Shc, Nck, Crk) • Increase effective availability of substrate • Membrane phospholipids • Other pY-bound proteins • Increase biological activity • Phosphorylation dependent activation • pY-binding dependent activation

  8. eg: FGFR phosphorylation FGF DNA Synthesis cdc related kinase Crk Y463 Y583 Shc Y653 PLC Y730 GRB2 Y766 IP3+DAG Ca2+, motility Sos MAPK growth

  9. Modulation & Termination • Modulation • Receptor antagonists • Combinatorial control • Termination • Protein Tyrosine Phosphatases (PTPs) • Internalization • Ubiquitinylation

  10. G-Protein Coupled Receptor • GPCR are 7 pass transmembrane proteins • Rhodopsin/b-adrenergic • Secretin/vasointestinal peptide • Metabotropic glutamate • Ga Guanine exchange factor (GEF) • Heterotrimeric G-Protein • Ga - Gbg binding • Can function monomeric • Also dimerize

  11. GPCR • Receptor ligation catalyzes GDP-GTP exchange on Ga • GTP bound Ga dissociates from Gbg • Ga modulates secondary signaling • Gbg may also modulate secondary signaling Unligated Receptor Bound Receptor Ga-GDP Ga-GTP Gbg Gbg

  12. G-a mediated signaling • Acylated, membrane bound Ga and target • Ga allosterically regulated by GTP • Target allosterically regulated by Ga • Membrane association decreases diffusion distance Gas Adenylate cyclase GTP Substrate ATP

  13. G Protein Classes Metabotropic neurotransmitter receptors are all GPCRs

  14. General Scheme • Agonist binding triggers nucleotide exchange • G subunits dissociate • Ga binds effectors • GTP hydrolysis restores inactive state • Effector may be a GAP

  15. eg: Synaptic remodeling • Rearrangement of neural networks • Synaptic reinforcement • Long term potentiation • Remodeling of dendritic spines • Calcium dependent cell motility Stimulation of cultured neuron with NMDA results in rapid development of a new dendritic spine Goldin, et al., 2001

  16. Glutamate - Ga12/13 • Metabotropic glutamate receptor 1 is Ga12 coupled GPCR • Ga12 is a Rho-GEF • RhoA small GTPases • RhoA, Rac1 and Cdc42 • Subcellular transport • Cytoskeletal remodeling • Actin filament growth(mDia) • Stress fiber anchorage (ROCK) Kinectin ROCK mDia

  17. Receptor regulation, negative feedback • Activity depends on association of intracellular loops • Rapid desensitization • G-protein coupled Receptor Kinases (GRK) • PKA, PKC • Internalization • Arrestin • Clathrin/caveolae • Long-term desensitization • Downregulation • G-Protein deactivation

  18. G-Protein regulation • In vivo signaling much faster than reconstituted systems • Regulator of G-protein Signaling (RGS) • Ga GAP • Esp Gi, Gq • PLCb, RhoGEF are RGS • Kinetics • Ga: minutes • Ga-RGS: tenths of second

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