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 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 (GABA B ) 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):

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):

Rigid Body Model: Straight jacketed receptor Struthers M, Yu H, and Oprian DD, (2000). G protein-coupled receptor activation: analysis of a highly constrained, "straitjacketed" rhodopsin. Biochemistry 39 (27): 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.

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

5HT2R activation Drug IPAA Signaling responses 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):

Agonist-directed signaling GG Drug A R* A R* B Drug B 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): Figure 1

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

GRKs/arrestin  Heterologous PKA desensitization  Homologous GRK desensitization NE PKA GRK arrestin Heterologous desensitization Homologous desensitization

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

Dimerization Assembly domains Chimeric receptor crosstalk Classical GnRH studies GABA B R1/R2 dimers PAR-3 cofactor for PAR-4

Functional D2R SSTR5 dimer GG SSTR5 R S GG SSTR5 del C somatostatin + SSTR5 del C X S somatostatin + S + + D2 receptor 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):

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): 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