Conservation of the phosphate-sensitive elements in the arrestin family of proteins

Abstract

Arrestins play a key role in the homologous desensitization of G protein-coupled receptors (GPCRs). These cytosolic proteins selectively bind to the agonist-activated and GPCR kinase-phosphorylated forms of the GPCR, precluding its further interaction with the G protein. Certain mutations in visual arrestin yield "constitutively active" proteins that bind with high affinity to the light-activated form of rhodopsin without requiring phosphorylation. The crystal structure of visual arrestin shows that these activating mutations perturb two groups of intramolecular interactions that keep arrestin in its basal (inactive) state. Here we introduced homologous mutations into arrestin2 and arrestin3 and found that the resulting mutants bind to the β2-adrenoreceptor in vitro in a phosphorylation-independent fashion. The same mutants effectively desensitize both the β2-adrenergic and δ-opioid receptors in the absence of receptor phosphorylation in Xenopus oocytes. Moreover, the arrestin mutants also desensitize the truncated δ-opioid receptor from which the C terminus, containing critical phosphorylation sites, has been removed. Conservation of the phosphate-sensitive hot spots in nonvisual arrestins suggests that the overall fold is similar to that of visual arrestin and that the mechanisms whereby receptor-attached phosphates drive arrestin transition into the active binding competent state are conserved throughout the arrestin family of proteins.