1. G-Protein-Coupled Receptor (GPCR): Structure and Function
Yun Huang

2. G-Protein-Coupled Receptor (GPCR)
The largest family of integral membrane protein involved in many biological process and pathologies;
50% of all modern drugs and 25% of the top 200 best selling drugs are estimated to target GPCRs;
Transduce the signals mediated by diverse signaling molecules, such as ions, peptides, lipids and photons, to induce different intracelluar function;
Bind their ligand and to activate different G proteins;

3. GPCR-Gα Fusion Protein
Upper: Schematic of a G-protein-coupled receptor (GPCR)-Gα fusion protein;
Left: G-protein cycling. Rate-limiting receptor-promoted GDP dissociation;
Nat Rev Drug Discov Jul;3(7):575, Review.

4. Three Families of GPCR
Ligands: neurotranmitters (dopamine and serotonin)
Three families of GPCR share the similar structure: Extracellular domain, 7 TM and intracellular C-tail. 7 TM proteins account for ~40% of the human genome.
Ligands: hormones (glucagon, secretin, PTH)
Example: mGluR, CaR
Nat Rev Drug Discov Jul;3(7):575, Review.

5. Family 1 GPCRs: Rhodopsin-like Receptors
Represent the predominant class of GPCRs;
Several highly conserved amino acid (red circles);
Disulfide bridge between first and second extracellular loops (ECLs), palmitoylated cys in C-tail;
The binding of small molecule ligands occures within the TM region;
Example: Rhodopsin, Dopamine receptor, Chemokine receptors;
Rhodopsin: Dopamine receptors: related to Parkinson’s disease and schizophrenia. There are several allosteric modulation: zinc, sodium and L-pro-L-Leu-L-gly. The three short peptide has been extensively modified for the potential treatment of Parkinson’s disease.
Chemokine: the function is related to leukocyte trafficking. Related to HIV-1 infection.
Nat Rev Drug Discov Jul;3(7):575, Review.

6. Rhodopsin
Rod cell specific visual pigment protein found in the vertebrate retina
Responsible for achromatic vision under dim light conditions
348 amino acids, 7-TM, 11-cis-retinaldehyde (chromophore, derived from Vit A)
Posttransliational modifications: acetylation (M1), N-Glycosylation (N2, N15), S-palmitoylation (C322, C323)
Prosthetic group retinaldehyde moiety covalently attached to K296
Functions as oligomers (dimers?) N 3 4 1 5 6 2 7 C
1U19
Current Opinion inStructural Biology, 2005, 15: ; Experimental Eye Research, 2005, 81:

7. Visual Signal Transduction
Initiated by rhodopsin in rod cells.
When light strikes 11-cis retinal and is absorbed, it isomerizes to all-trans retinal, changing the shape of the molecule and the receptor it is bound to.
Alters its interaction with transducin, the member of the G-protein gene family that is specific in its role in visual signal transduction.
Activation of transducin causes its alpha subunit to dissociate from the trimer and exchange bound GDP for GTP, activating in turn a membrane-bound cyclic-GMP specific phosphodiesterase that hydrolyzes cGMP.
Further leads to hyperpolarization and downstream action potential, conveying signal from sensor neuron to the CNS.

8. Family 2 GPCRs: Secretin-like Receptors
15 peptide-binding receptors in human;
Relatively long N-terminus (~ residues) and a juxtamembrane doamin of 7 membrane-spanning α-helices (J-domain);
Tertiary structure is stabilized by 3 disulfide bonds within 6 highly conserved Cys
Share little sequence homology with family 1 and 3 GPCRs;
Example: Corticotropin-releasing factor receptor, Glucagon receptor, PTH receptor;
Nat Rev Drug Discov Jul;3(7):575, Review.

9. Family 2 GPCRs: Secretin-like Receptors
Drug Discovery today, 2005: March;10(3): Review.

10. Corticotropin-Releasing Factor Receptor (CRF)
Related to human stress response;
NMR structure of ECD1-CRF-R2β;
Clustered in the cleft region between the tip of the first β-sheet and the edge of the “palm” of the second β-sheet;
The central core contains a salt bridge sandwiched between aromatic side chains;
PNAS, 2004;101:

11. Binding Model of Family 2 GPCRs
Peptide interaction (two-domain model)
Non-peptide interaction
Native peptide interaction with the isolated J-domain is extremely weaker (-100,000 fold) than the affinity for whole receptor. N-domain trap the peptide to increase the local concentration of peptide in the vicinity of J-domain, therefore, enable the interaction of the peptide to J-domain.
J-domain mediates the G-protein activation.
Drug Discovery today, 2005: March;10(3): Review.

12. Drug Design Based on Protein Structure
Comprise a central core: a “top” side chain and a “bottom” aromatic ring;
Developed by Janssen Pharmaceuticals NY & Neurocrine Biosciences;
Phase II clinical trials for treating depression and anxiety
Drug Discovery today, 2005: March;10(3): Review.

13. Family 3 GPCRs: mGluR-like Receptors
Very long N-terminus;
Ligand-binding domain is located in N-terminus and is thought to resemble a Venus fly trap (VFT) that can open and close with the agonist bound inside;
Cys residues are conserved, the tertiary structures are likely highly conserved;
Share similar ligands and downstream signaling pathway;
Constitutive homo- or heterodimers: mGluR (homo), GAGAβ(hetero);
Example: metabotropic glutamate receptor (mGluR), CaR, GABAβ
Two agonists can active homodimer; heterodimer is different;
GAGAB: GABAb1 binds a agonist but cannot activate the G protein, whereas GABAb2 is responsible for G protein activation.
Glutamate and other L-amino acid activate CaRs and high [Ca]o activate mGluR
Nat Rev Drug Discov Jul;3(7):575, Review.

14. Structure of mGluR and CaR
Glutamate is a neurotransmitter in CNS and functions in long-term potentiation, learning and memory;
Metabotropic glutamate receptor (mGluR) mediate excitatory transmission on the cellular surface through initial binding of glutamate
Calcium sensing receptor (CaR) shares 27% sequence identity with (mGluR);
Response to [Ca2+]o, L-amino acids, polyamines, ionic strength and pH;
Parathyroid CaR plays a central role in systemic PTH level and Ca2+ homeostasis;
Nature 2006, 407: ; Current Opinion in Neurobiology 2003, 13:

15. Structure of mGluR and CaR?
CaR
Nature 2006, 407: ; Current Opinion in Neurobiology 2003, 13:

16. Extracellular Calcium Sensing Receptor (CaR)
CaR related signaling pathway
PTH: key endocrine factor in systemic Ca2+ homeostasis and involve moving Ca2+ between bone and blood; Stimulate net bone loss or formation;
Cell calcium : Review;

17. Disease Related Natural-Occurring Mutations on CaR
Familial hypocalciuric hypercalcemia (FHH),
Neonatal severe hyperparathyroidism (NSHPT),
Autosomal dominant hypoparathyroidism (ADH)

18. Drug Design Based on Protein Structure
Calcimimetics:
Mimic or potentiate the action of extracellular Ca2+ at the Ca2+ receptor;
Agonists or allosteric activators of the receptor;
Calcilytics:
Ca2+ receptor antagonists;
Selective and does not affect the activity of mGluRs;
Increase [Ca2+]i and inhibits PTH secretion;
Treatment of HPTH;
Drug Discovery today, 2005: March;10(3): Review.

19. Big questions remain for GPCRs at the structure/function interface
Determine the structures corresponding to the various states of GPCRs, especially in the active state;
Understand how GPCRs interact with and activate heterotrimeric G proteins;
Gain a molecular understanding of the mechanism of action of allosteric modulators, therefore, leads for drug development;
Nat Rev Drug Discov Jul;3(7):575, Review.

20. Reference
The state of GPCR research in Nat Rev Drug Discov Jul;3(7):575,
Structure of rhodopsin and the metarhodopsin I photointermediate. Curr Opin Struct Biol Aug;15(4):
Mechanisms of peptide and nonpeptide ligand binding to Class B G-protein-coupled receptors. Drug Discov Today Mar 15;10(6):
NMR structure and peptide hormone binding site of the first extracellular domain of a type B1 G protein-coupled receptor. Proc Natl Acad Sci U S A Aug 31;101(35):
Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Nature Oct 26;407(6807):971-7.
Extracellular Ca2+-sensing receptors--an overview. Cell Calcium Mar;35(3):