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G Protein-Coupled Receptors Stuart C. Sealfon Major Classes of GPCRs N C C • Class I: rhodopsin-like C D R Y C C C • Class II: glucagon-like N C C C C C C • Class III: metabotropic glutamate-like C N C C C Class I: Rhodopsin-like • • • • • visual pigments (rhodopsin) neurotransmitter receptors peptide receptors glycoprotein hormone receptors protease activated receptors Class II: Glucagon-like • • • • • Calcitonin Corticotropin releasing factor (CRF) Glucagon Parathyroid hormone (PTH) Pituitary adenylate cycase-activating peptide (PACAP) Class III: mGlu-like • Calcium sensor • Gamma-aminobutyric acid type B (GABAB) • Metabotropic gluamate (mGlu) 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 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. 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 Mechanisms of ligand interaction • • • • Rhodopsin Neurotransmitter receptors Glycoprotein hormone receptors Protease activated receptors 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. • 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. Rigid body model • Rotation and displacement of cytoplasmic end of helix 6 Farrens D.L., et al. (1996). Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin. Science 274 (5288):768-770. 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. Coupling Promiscuity • Many GPCRs couple to more than one G protein subtype Signal Trafficking Drug 5HT2R activation IP AA Signaling responses 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. 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. Receptor RNA processing/Isoforms • D2 splice variants • 5HT2C editing • D4R and behavior GRKs/arrestin Heterologous PKA desensitization Homologous GRK desensitization NE GRK Homologous desensitization arrestin PKA Heterologous desensitization Non-heterotrimeric G protein coupling • • • • Regulation of Na/H exchanger/NHERF Arrestin/SRC ERK signaling Direct SRC ERK signaling ARF/RhoA signaling Dimerization • • • • • Assembly domains Chimeric receptor crosstalk Classical GnRH studies GABAB R1/R2 dimers PAR-3 cofactor for PAR-4 Functional D2R SSTR5 dimer somatostatin + S + SSTR5 R somatostatin + SSTR5 del C + D2 receptor somatostatin SSTR5 del C S X S Recovery of SS Signaling via D2R Biological Functions for RAMPS • Transport CRLR to the cell surface • Define its pharmacology • Determine its glycosylation state 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 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. 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.