G Protein-Coupled Receptors

Stuart C. Sealfon

Major Classes of GPCRs

• Class I: rhodopsin-like

• Class II: glucagon-like

• Class III: metabotropic

glutamate-like

CC

C

C

C

C

CC

N

C

DRY

CC

C

N

C

CC

N

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 ligandsin the binding pocket of the human serotonin 5-HT2A receptordetermines relative efficacy. Mol Pharmacol 63 (1):36-43.

• Terniary and extended terniary model– Accommodate activation in absence of agonist

(constitutive activity)

Spontaneous activity of WTand 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

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.

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

IP AA Signalingresponses

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.

Agonist-directed signaling

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):94-104.

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

Heterologousdesensitization

Homologousdesensitization

• 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

• GABAB R1/R2 dimers

• PAR-3 cofactor for PAR-4

Functional D2R SSTR5 dimer

GSSTR5 R

S

G

SSTR5 del C

somatostatin

+SSTR5 del C X

Ssomatostatin

+

Ssomatostatin

+

+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):1823-1826.

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.

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