Recruitment of arrestin-2 to activated M2 muscarinic acetylcholine receptors. Roles of G protein-coupled receptor kinase 2 and Giα protein

BELTRAME, SABRINA; BAYONÉS, LUCAS; WALDNER, CLAUDIA; GOIN, JUAN CARLOS

Huerta Grande

Congreso; XXVII Congreso Anual de la Sociedad Argentina de Investigación en Neurociencias; 2012

Sociedad Argentina de Investigación en Neurociencias

Previous studies have explored the role of phosphorylation and arrestin (Arr) binding in M2 muscarinic acetylcholine receptor (M2R) desensitization and internalization. Here we studied the influence of G protein-coupled receptor kinase 2 (GRK2) and Gi alpha protein (Gi alpha) on the interaction between M2R and Arr-2 by bioluminescence resonance energy transfer (BRET). Titration assays on HEK 293 cells expressing various proportions of M2-RLuc and Arr-2-YFP demonstrated that agonist exposure (carbachol, 10 micromolar, 20 min) promotes the interaction between M2R and Arr-2 only in the presence of overexpressing GRK2. Agonist-mediated effects were time- and concentration-dependent (t1/2: 2.2 min, Emax: 26.1 mB, EC50: 2.2 micromolar). Cellular overexpression of kinase deficient mutant GRK2K220R prevented agonist-induced Arr-2 recruitment to the M2R, suggesting that this effect is mediated by receptor phosphorylation. Coexpression of increasing amounts of Gi alpha promoted a gradual decrease in agonist-mediated Arr-2 binding to M2R, supporting the classical notion that G alpha proteins and arrestins compete for binding to activated G protein-coupled receptors. We conclude that agonist-induced Arr-2 binding to the M2R requires receptor phosphorylation and that this effect is affected by the cellular complement of Gi alpha protein