TY - JOUR
T1 - Identification of Gβγ binding sites in the third intracellular loop of the M3-muscarinic receptor and their role in receptor regulation
AU - Wu, Guangyu
AU - Bogatkevich, Galina S.
AU - Mukhin, Yurii V.
AU - Benovic, Jeffrey L.
AU - Hildebrandt, John D.
AU - Lanier, Stephen M.
N1 - Copyright:
Copyright 2004 Elsevier Science B.V., Amsterdam. All rights reserved.
PY - 2000/3/24
Y1 - 2000/3/24
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.275.12.9026
DO - 10.1074/jbc.275.12.9026
M3 - Article
C2 - 10722752
AN - SCOPUS:0034708266
SN - 0021-9258
VL - 275
SP - 9026
EP - 9034
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 12
ER -