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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.