Role of receptor-attached phosphates in binding of visual and non-visual arrestins to G protein-coupled receptors.

ABSTRACT: Arrestins are a small family of proteins that regulate G protein-coupled receptors (GPCRs). Arrestins specifically bind to phosphorylated active receptors, terminating G protein coupling, targeting receptors to endocytic vesicles, and initiating G protein-independent signaling. The interaction of rhodopsin-attached phosphates with Lys-14 and Lys-15 in ?-strand I was shown to disrupt the interaction of ?-helix I, ?-strand I, and the C-tail of visual arrestin-1, facilitating its transition into an active receptor-binding state. Here we tested the role of conserved lysines in homologous positions of non-visual arrestins by generating K2A mutants in which both lysines were replaced with alanines. K2A mutations in arrestin-1, -2, and -3 significantly reduced their binding to active phosphorhodopsin in vitro. The interaction of arrestins with several GPCRs in intact cells was monitored by a bioluminescence resonance energy transfer (BRET)-based assay. BRET data confirmed the role of Lys-14 and Lys-15 in arrestin-1 binding to non-cognate receptors. However, this was not the case for non-visual arrestins in which the K2A mutations had little effect on net BRET(max) values for the M2 muscarinic acetylcholine (M2R), ?(2)-adrenergic (?(2)AR), or D2 dopamine receptors. Moreover, a phosphorylation-deficient mutant of M2R interacted with wild type non-visual arrestins normally, whereas phosphorylation-deficient ?(2)AR mutants bound arrestins at 20-50% of the level of wild type ?(2)AR. Thus, the contribution of receptor-attached phosphates to arrestin binding varies depending on the receptor-arrestin pair. Although arrestin-1 always depends on receptor phosphorylation, its role in the recruitment of arrestin-2 and -3 is much greater in the case of ?(2)AR than M2R and D2 dopamine receptor.

SUBMITTER: Gimenez LE 

PROVIDER: S-EPMC3308753 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature