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G Protein-Coupled Receptors Stuart C. Sealfon

G Protein-Coupled Receptors Stuart C. Sealfon. Major Classes of GPCRs. Class I: rhodopsin-like Class II: glucagon-like Class III: metabotropic glutamate-like. Class I: Rhodopsin-like. visual pigments (rhodopsin) neurotransmitter receptors peptide receptors

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G Protein-Coupled Receptors Stuart C. Sealfon

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  1. G Protein-Coupled ReceptorsStuart C. Sealfon

  2. Major Classes of GPCRs • Class I: rhodopsin-like • Class II: glucagon-like • Class III: metabotropic glutamate-like

  3. Class I: Rhodopsin-like • visual pigments (rhodopsin) • neurotransmitter receptors • peptide receptors • glycoprotein hormone receptors • protease activated receptors

  4. Class II: Glucagon-like • Calcitonin • Corticotropin releasing factor (CRF) • Glucagon • Parathyroid hormone (PTH) • Pituitary adenylate cycase-activating peptide (PACAP)

  5. Class III: mGlu-like • Calcium sensor • Gamma-aminobutyric acid type B (GABAB) • Metabotropic gluamate (mGlu)

  6. Various experimental approaches to study GPCR structure • Site directed mutagenesis • Chimeras/deletions • Homology modeling • Ligand and helix-helix cross linking • Cys side chain accessibility • Straight jacketed receptor • Electron spin resonance • X-ray crystallography

  7. Rhodopsin Crystal Structure • Why is it upside down? • 7 TM helices • 8th cytoplasmic helix • Cysteine bridges • N linked glycosylation Palczewski, K., T. Kumasaka, et al. (2000). Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289 (5480):739-745.

  8. Post-translational Modifications • Glycosylation • Contributes to stability, ligand affinity, signaling • Palmitoylation • Forms fourth intracellular loop • Modulates internalization, desensitization • Contributes to ERK coupling of endothelin R • Phosphorylation

  9. Mechanisms of ligand interaction • Rhodopsin • Neurotransmitter receptors • Glycoprotein hormone receptors • Protease activated receptors

  10. Neurotransmitter binding • Within helix bundle • Ionic dock to helix 3 Ebersole, B.J., et al. (2003). Molecular basis of partial agonism: orientation of indoleamine ligands in the binding pocket of the human serotonin 5-HT2A receptor determines relative efficacy. Mol Pharmacol 63 (1):36-43.

  11. Terniary and extended terniary model • Accommodate activation in absence of agonist (constitutive activity) Spontaneous activity of WT and mutant 5HT2C receptors Inverse agonist effects Agonist effects Rosendorff A., et al. (2000). Conserved helix 7 tyrosine functions as an activation relay in the serotonin 5HT(2C) receptor. Mol Brain Res. 84 (1-2):90-96.

  12. Rotation and displacement of cytoplasmic end of helix 6 Rigid body model Farrens D.L., et al. (1996). Requirement of rigid-body motion of transmembrane helices for lightactivation of rhodopsin. Science 274 (5288):768-770.

  13. Rigid Body Model:Straight jacketed receptor Rhodopsin still activates with bridges connecting the cytoplasmic ends of helices 1 & 7, and 3 & 5, and the extracellular ends of helices 3 & 4, and 5 & 6. Struthers M, Yu H, and Oprian DD, (2000). G protein-coupled receptor activation: analysis of a highly constrained, "straitjacketed" rhodopsin. Biochemistry 39 (27):7938-7942.

  14. Coupling Promiscuity • Many GPCRs couple to more than one G protein subtype

  15. Drug 5HT2R activation Signaling responses IP AA Signal Trafficking Berg K.A., et al. (1998). Effector pathway-dependent relative efficacy at serotonin type 2A and 2C receptors: evidence for agonist-directed trafficking of receptor stimulus. Mol Pharmacol 54 (1):94-104.

  16. Agonist-directed signaling Figure 1 Drug B Drug A R*A R*B Ga Figure 1 - Berg K.A., et al. (1998). Effector pathway-dependent relative efficacy at serotonin type 2A and 2C receptors: evidence for agonist-directed trafficking of receptor stimulus. Mol Pharmacol 54 (1):94-104.

  17. Receptor RNA processing/Isoforms • D2 splice variants • 5HT2C editing • D4R and behavior

  18. PKA NE GRKs/arrestin • Heterologous PKA desensitization • Homologous GRK desensitization Heterologous desensitization GRK arrestin Homologous desensitization

  19. Non-heterotrimeric G protein coupling • Regulation of Na/H exchanger/NHERF • Arrestin/SRC ERK signaling • Direct SRC ERK signaling • ARF/RhoA signaling

  20. Dimerization • Assembly domains • Chimeric receptor crosstalk • Classical GnRH studies • GABAB R1/R2 dimers • PAR-3 cofactor for PAR-4

  21. Ga Ga somatostatin S + X SSTR5 del C Functional D2R SSTR5 dimer somatostatin S + SSTR5 R somatostatin S + Recovery of SS Signaling via D2R SSTR5 del C + D2 receptor

  22. Biological Functions for RAMPS • Transport CRLR to the cell surface • Define its pharmacology • Determine its glycosylation state

  23. GPCRs and disease • Nephrogenic diabetes insipides V2 vasopressin receptor • Precocious puberty LH receptor • Kaposi's sarcoma KSHV encoded vGPCR • Retinitis pigmentosa/congenital night blindness Rhodopsin • Virus entry: HIV/CCR7R, JCV/5HT2 R • Familial gestational hyperthyroidism Thyrotropin receptor

  24. Thyroxine and Thyrotropin Concentrations during Patient's Pregnancy. Rodien P., et al. (1998) Familial gestational hyperthyroidism caused by a mutant thyrotropin receptor hypersensetive to human chorionic gonadtropin. N Engl J Med 339 (25):1823-1826.

  25. cAMP production by thyrotropin in cells transfected with WT or mutant thyrotropin receptor cAMP production by chorionic gonadotropin in cells transfected with WT or mutant thyrotropin receptor Rodien P., et al. (1998) Familial gestational hyperthyroidism caused by a mutant thyrotropin receptor hypersensitive to human chorionic gonadtropin. N Engl J Med 339 (25):1823-1826.

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