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

Guangyu Wu, Galina S. Bogatkevich, Yurii V. Mukhin, Jeffrey L. Benovic, John D. Hildebrandt, Stephen M. Lanier

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)9026-9034
Number of pages9
JournalJournal of Biological Chemistry
Volume275
Issue number12
DOIs
StatePublished - Mar 24 2000

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Muscarinic M3 Receptors
Binding Sites
Mutagenesis
Phosphorylation
Site-Directed Mutagenesis
G-Protein-Coupled Receptor Kinases
Calcium-Sensing Receptors
Mutation
COS Cells
Muscarinic Receptors
Cricetulus
Ionic strength
Osmolar Concentration
Ovary
Signal processing
Ligands
Amino Acids
Peptides

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Identification of Gβγ binding sites in the third intracellular loop of the M3-muscarinic receptor and their role in receptor regulation. / Wu, Guangyu; Bogatkevich, Galina S.; Mukhin, Yurii V.; Benovic, Jeffrey L.; Hildebrandt, John D.; Lanier, Stephen M.

In: Journal of Biological Chemistry, Vol. 275, No. 12, 24.03.2000, p. 9026-9034.

Research output: Contribution to journalArticle

Wu, Guangyu ; Bogatkevich, Galina S. ; Mukhin, Yurii V. ; Benovic, Jeffrey L. ; Hildebrandt, John D. ; Lanier, Stephen M. / Identification of Gβγ binding sites in the third intracellular loop of the M3-muscarinic receptor and their role in receptor regulation. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 12. pp. 9026-9034.
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AU - Hildebrandt, John D.

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