Identification of Gβγ binding sites in the third intracellular loop of the M3-muscarinic receptor and their role in receptor regulation

G beta gamma binds directly to the third intracellular (i3) loop subdomain of the M-3-muscarinic receptor (RIPE), In this report, we identified the G beta gamma binding motif and G-protein-coupled receptor kinase (GRK2) phosphorylation sites in the M-3-MR i3 loop via a strategy of deletional and site-directed mutagenesis. The G beta gamma binding domain was localized to Cys(289)-His(330) within the M-3-MR-Arg(252)Gin(490) i3 loop, and the binding properties (affinity, influence of ionic strength) of the M-3-MR-Cys(289)-Ris(330) i3 loop subdomain were similar to those observed for the entire i3 loop. Site-directed mutagenesis of the M-3-MR-Cys(289)-His(330) i3 loop subdomain indicated that Phe(312) Phe(314) and a negatively charged region (Glu(324)-Asp(329)) were required for interaction with G beta gamma, Generation of the full-length M-3-MR-Arg(252)-Gln(490) i3 peptides containing the F312A mutation mere also deficient in G beta gamma binding and exhibited a reduced capacity for phosphorylation by GRK2. A similar, parallel strategy resulted in identification of major residues ((SSS333)-S-331 (318)SASS(351)) phosphorylated by GRK2, which were just downstream of the G beta gamma binding motif, Full-length M-3-MR constructs lacking the 42-amino acid G beta gamma binding domain (Cys(289)-His(330)), containing the F312A mutation exhibited ligand recognition properties similar to wild type receptor and also effectively mediated agonist-induced increases in intracellular calcium following receptor expression in Chinese hamster ovary and/or COS 7 cells. However, the M-3-MR Delta Cys(289)-His(330) and M-3-MR(F312A) constructs mere deficient in agonist-induced sequestration, indicating a key role for the G beta gamma-MR i3 loop interaction in receptor regulation and signal processing.