Direct Physical Scaffolding of Muscarinic M3 Receptor Signal Transduction Pathways

There is growing recognition that G protein‐coupled receptors (GPCRs) can be pre‐coupled to signal transduction partners to serve a specific and efficient signaling function. We previously found that the third intracellular loop (ICL3) of muscarinic M3 receptor (M3R), expressed as a purified protein fused with glutathione‐S‐transferase (GST), bound purified Gαq, Gβ 1 γ 2 , and phospholipase Cβ3 (PLCβ3). PLCβ3 binding to full length M3R in intact cells was only partially dependent on amino acids 275–470 of ICL3, suggesting that the M3R‐PLCβ3 binding interface is comprised of multiple contacts. Further definition of the PLCβ3 binding surface on M3R using purified GST fusion constructs confirmed the multiplicity of contacts with PLCβ3. Contacts close to the membrane interface were found at distinct amino and carboxyl terminal regions of ICL3, ICL2, and C terminal tail. This suggested that rather than being simply tethered to M3R, PLCβ binds to a broad intracellular surface of M3R proximal to the Gαq activation site. M3R binding sites were also defined for Gα q and Gβ 1 γ 2 , which were distinct from those for PLCβ3 and from sites involved in direct coupling to the Gα q Gβγ heterotrimer. Mapping and functional characterization of the PLCβ3 binding surface in the intact M3R will be presented. We propose that the observed receptor‐effector direct scaffolding represents an additional level of fine‐tuning GPCR signaling output.