Recruitment of nonvisual arrestins to the activated M2 muscarinic acetylcholine receptor. Facilitation of receptor/arrestin interaction by GRK2

LAURA CAMILA CARRERA PÁEZ ; SABRINA BELTRAME; JUAN CARLOS GOIN; CLAUDIA WALDNER

Buenos aires

Congreso; 2º Congreso Internacional FALAN; 2016

Sociedada argentina de investigacion en neurociencias, FALAN

Although homologous short term regulation of the M2 muscarinic acetylcholine receptor (M2R) can be facilitated by a synergistic action of receptor phosphorylation and arrestin binding, the ability of receptor phosphorylation to facilitate arrestin binding to the activated M2R remains controversial. Here we examined the influence of overexpressing G protein-coupled receptor kinase-2 (GRK2) on carbachol-induced arrestin-2 (Arr-2) and arrestin-3 (Arr-3) recruitment to the M2R by bioluminescence resonance energy transfer (BRET) in intact HEK 293T cells. Titration assays on cells coexpressing various proportions of M2-RLuc and Arr-2-YFP or Arr-3-YFP demonstrated that agonist exposure promotes the interaction between M2R and either arrestin only in the presence of overexpressing GRK2. Agonist-mediated effects were dependent on incubation time, agonist concentration and expression levels of GRK2. Cellular overexpression of kinase deficient mutant GRK2K220R strongly inhibited agonist-induced arrestin/receptor interaction, suggesting that arrestin recruitment is facilitated by receptor phosphorylation. These results support previous in vitro findings showing that agonist-induced receptor phosphorylation facilitates the binding of nonvisual arrestins to the activated M2R