Molecular basis of G protein-coupled receptor function

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SUMMARY GPCR homodimers: structure-function studies Among the various subclasses of GPCRs, the class A subfamily represents by far the largest subgroup of GPCRs, consisting of 670 members in human. Many studies have demonstrated that class A GPCRs are able to form dimeric or oligomeric complexes. Considerable evidence suggests that the formation of such complexes can affect various aspects of GPCR function, including (but not limited to) receptor-G protein coupling efficiency and selectivity. A proper understanding of how class A GPCRs function at the molecular level requires the identification of the structural elements governing the dimerization/oligomerization of this class of receptors. For nearly two decades, the M3 muscarinic acetylcholine receptor (M3R) has served as an excellent model system to explore various aspects of class A GPCR dimerization. Recently, we used a BRET approach, combined with systemic alanine substitution mutagenesis studies, to identify residues involved in M3R dimerization (McMillin et al. J Biol Chem 286, 28584-98, 2011). The outcome of this work, combined with molecular modeling studies, strongly suggested the existence of multiple, structurally distinct M3R dimers that are probably transient in nature. To provide more direct experimental support for this concept, we employed a disulfide cross-linking strategy to trap various M3R dimeric species present in a native lipid environment (transfected COS-7 cells). Disulfide cross-linking studies were carried out with many mutant M3Rs containing single cysteine (Cys) substitutions within different distinct cytoplasmic M3R regions. The pattern of cross-links that we obtained, in combination with molecular modeling studies, was consistent with the existence of multiple M3R/M3R interfaces. Disulfide cross-linking of helix 8 (H8) led to significant impairments in M3R-mediated G protein activation, suggesting that changes in the structural orientation of H8 are critical for efficient receptor-G protein coupling. These findings provide novel structural and functional insights into the mechanisms involved in M3R dimerization (oligomerization). Since the M3R shows a high degree of sequence similarity with many other class A GPCRs, our findings should be of considerable general interest. The work summarized in this paragraph is described in a manuscript that we recently submitted for publication (J. Hu, K. Hu, T. Liu, R. Mistry, R. A. J. Challiss, S. Costanzi and J. Wess). Structure-based docking approaches to identify novel muscarinic ligands Recently, we, in collaboration with Brian Kobilkas lab, published the first high-resolution X-ray structure of the M3R (Kruse et al. Nature 482, 552-6, 2012). At the same time, Haga et al. (Nature 482, 547-51, 2012) reported the X-ray structure of another muscarinic receptor subtype, the M2 muscarinic receptor (M2R). At present, ligands that can activate or block these two receptor subtypes with a high degree of selectivity are not available. However, M2R- or M3R-selective compounds are predicted to prove useful for the treatment of a number of human diseases. In an attempt to identify such agents, we, in collaboration with the labs of Brian Kobilka (Stanford University) and Brian Shoichet (UCSF) decided to employ structure-based docking against the M2R and M3R subtypes. About 3 million molecules were screened (in silico) for ligands with new physical properties, chemotypes, and receptor subtype selectivity. Interestingly, this screen led to the identification of several molecules that had both high affinity for the M2R subtype and represented new chemotypes. Moreover, we obtained several compounds that had significantly higher affinity for the M3R than for the M2R. Interestingly, one of these agents (compound 16) proved to be a partial agonist at the M3R but was completely inactive at the M2R. Consistent with this observation, compound 16 was able to stimulate insulin secretion from a mouse beta-cell line expressing endogenous M3Rs. These data strongly suggest that structure-based docking approaches will greatly facilitate the discovery of novel classes of receptor subtype-selective muscarinic ligands.