Crosslinking Analysis of the M3 Muscarinic Acetylcholine Receptor/G alpha q Interface in Both the Inactive and Active States

Abstract

The interaction of G protein‐coupled receptors (GPCRs) with heterotrimeric G proteins represents one of the most fundamental biological processes. Although detailed structural information is now available for several GPCRs and heterotrimeric G proteins, the molecular architecture of the GPCR/G protein complex remains poorly defined, primarily due to the lack of a high‐resolution X‐ray structure. In the present study, we applied a comprehensive GPCR/Gα chemical cross‐linking strategy to map a receptor/Gα interface, both prior to and after agonist‐induced receptor activation. By employing the M3 muscarinic acetylcholine receptor (M3R)/Gαq system as a model system, we examined the ability of ~250 combinations of Cys‐substituted M3R and Gαq proteins to undergo cross‐link formation. We identified many specific M3R/Gαq–contact sites, both in the inactive and the active receptor conformation. Co‐immunoprecipitation and mass spectrometry experiments confirmed the molecular identity of the M3R/Gαq complexes. The observed M3R/Gαq cross‐linking patterns yielded a sufficient number of structural constraints allowing us to generate the first evidenced‐based model of a GPCR/Gα complex. Since heterotrimeric G proteins as well as most GPCRs share a high degree of structural homology, our findings should be of great general relevance.