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

Gβγ binds directly to the third intracellular (i3) loop subdomain of the M₃-muscarinic receptor (MR). In this report, we identified the Gβγ binding motif and G-protein-coupled receptor kinase (GRK2) phosphorylation sites in the M₃-MR i3 loop via a strategy of deletional and site-directed mutagenesis. The Gβγ binding domain was localized to Cys²⁸⁹-His³³⁰ within the M₃-MR-Arg²⁵²Gln⁴⁹⁰ i3 loop, and the binding properties (affinity, influence of ionic strength) of the M₃-MR-Cys²⁸⁹-His³³⁰ i3 loop subdomain were similar to those observed for the entire i3 loop. Site- directed mutagenesis of the M₃-MR-Cys²⁸⁹-His³³⁰ i3 loop subdomain indicated that Phe³¹², Phe³¹⁴, and a negatively charged region (Glu³²⁴-Asp³²⁹) were required for interaction with Gβγ. Generation of the full-length M₃-MR-Arg²⁵²-Gln⁴⁹⁰ i3 peptides containing the F312A mutation were also deficient in Gβγ binding and exhibited a reduced capacity for phosphorylation by GRK2. A similar, parallel strategy resulted in identification of major residues (³³¹SSS³³³ and ³⁴⁸SASS³⁵¹) phosphorylated by GRK2, which were just downstream of the Gβγ binding motif. Full-length M₃-MR constructs lacking the 42-amino acid Gβγ binding domain (Cys²⁸⁹-His³³⁰ or 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 M₃-MRΔCys²⁸⁹-His³³⁰ and M₃-MR(F312A) constructs were deficient in agonist-induced sequestration, indicating a key role for the GβγM₃-MR i3 loop interaction in receptor regulation and signal processing.