GPCR homodimers: structure-function studies Most GPCRs belong to the class A subfamily of GPCRs which consists of 670 members in human. Class A GPCRs are able to form dimeric or oligomeric complexes. Accumulating evidence suggests that the formation of such complexes can modulate various aspects of GPCR function, including (but not limited to) receptor-G protein coupling efficiency and selectivity. Thus, in order to better understand how class A GPCRs function at the molecular level, it is critical to identify the structural elements governing the dimerization/oligomerization of this class of receptors. During the past few years, we have used the M3 muscarinic acetylcholine receptor (M3R) as a model system to explore various aspects of class A GPCR dimerization. Recently, we started to employ a disulfide cross-linking strategy to trap various M3R dimeric species present in a native lipid environment (membranes from transfected COS-7 cells). We carried out disulfide cross-linking studies with a large mutant M3Rs containing single cysteine (Cys) substitutions on the cytoplasmic surface of the M3R regions. The pattern of cross-links that we obtained, together with molecular modeling studies, strongly suggested the existence of multiple M3R/M3R interfaces. We also noted that disulfide cross-links involving helix 8 (H8) interfered with productive M3R-G protein coupling. This observation supports the concept that receptor-mediated G protein activation requires conformational changes that involve H8. The M3R shows a high degree of structural homology with most other class A GPCRs. For this reason, the findings summarized above should be of considerable general interest. (Hu J, et al. Novel structural and functional insights into M3 muscarinic receptor dimer/oligomer formation. J Biol Chem 288, 34777-90, 2013) Structure of the agonist-activated M2 muscarinic receptor (M2R) Recently, a multi-lab collaborative effort resulted in the determination of the structure of an agonist (iperoxo)-bound, active state of the human M2R. Iperoxo is an orthosteric agonist that displays very high potency at the M2R. The key features of the active conformation of the M2R are a significant outward displacement of the cytoplasmic end of TM6, together with a smaller outward movement of the C-terminal portion of TM5 and a rearrangement of the highly conserved NPXXY (TM7) and DRY (cytoplasmic end of TM3) motifs. Similar conformational changes have been reported for the active-state conformations of rhodopsin and the beta2-adrenergic receptor. Iperoxo binding to the M2R leads to striking changes in the structure of the orthosteric binding site. Specifically, iperoxo binding to the M2R results in a significant contraction of the orthosteric binding site, which completely occludes the agonist ligand from solvent. In the active M2R conformation, TM5, TM6, and TM7 move inward toward the iperoxo ligand and TM3 undergoes a slight rotation about its axis. Most importantly, the inward movement of TM6 allows the side chain of N4046.52 to form a hydrogen bond with the isoxazoline ring of iperoxo. The inward motion of the exofacial portion of TM6 leads to the formation of a hydrogen network between Y4036.51, Y1043.33, and Y4267.39, resulting in the closure of the tyrosine lid above the agonist ligand. With almost no exception, the M2R amino acid side chains that contact the agonist ligand also play important roles in the binding of QNB, a muscarinic antagonist/inverse agonist, to the inactive M2R. (Kruse AC, et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101-6, 2013) Mode of binding of an allosteric modulator to the M2R The M2R has served as an excellent model system for studying the regulation of GPCR function by small allosteric modulators. The inactive M2R, like the antagonist-bound M3R, features a large extracellular vestibule, which is predicted to be involved in the binding of allosteric muscarinic ligands. Strikingly, agonist activation of the M2R triggers a pronounced contraction of this outer cavity, primarily due to the inward movement of the extracellular portion of TM6. We solved the structure of the iperoxo-occupied M2R in complex with LY2119620, a positive allosteric. LY2119620 shows strong positive cooperativity with iperoxo and selectively enhances the affinity of the orthosteric agonist for the M2R. In the M2R-iperoxo-LY2119620 complex, the allosteric modulator is located directly above the orthosteric agonist. LY2119620 engages in extensive interactions with the extracellular vestibule. Such contacts include aromatic stacking, hydrogen bond, and chargecharge interactions. Interestingly, the structure of the M2RiperoxoLY2119620 complex is very similar to that of the M2iperoxo complex, indicating that the binding site for LY2119620 is largely pre-formed after binding of the orthosteric agonist iperoxo. The iperoxo-stabilized contraction of the extracellular vestibule enables LY2119620 to engage in far more extensive interactions with this outer receptor cavity, as compared to the inactive state of the M2R. These findings support the concept that LY2119620 and, most likely, other muscarinic positive allosteric modulators enhance the receptor affinity of orthosteric agonists by stabilizing the active conformation of the receptor and slowing agonist dissociation from the orthosteric binding pocket. These data provide the first structural view of how a drug-like allosteric ligand binds to a GPCR. This new structural information should guide the development of novel muscarinic agents endowed with a high degree of selectivity for distinct mAChR subtypes. (Kruse AC, et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101-6, 2013)
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