Identification of Gβγ Binding Sites in the Third Intracellular Loop of the M3-muscarinic Receptor and Their Role in Receptor Regulation*

By Guangyu Wu, G. Bogatkevich, Y. Mukhin, J. Benovic, J. Hildebrandt, S. Lanier. Journal of Biological Chemistry, 2000

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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-Arg252–Gln490 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 and348SASS351) 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 Gβγ-M3-MR i3 loop interaction in receptor regulation and signal processing.

Subfields: Cellular and Molecular Neuroscience, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/b355fdb038f30b21071a3e2f14a0e00dcda7681f