Molecular determinants underlying the formation of stable intracellular G protein-coupled receptor-beta-arrestin complexes after receptor endocytosis

beta -Arrestins bind agonist-activated G protein-coupled receptors (GPCRs) and mediate their desensitization and internalization. Although beta -arres tins dissociate from some receptors at the plasma membrane, such as the bet a2 adrenergic receptor, they remain associated with other GPCRs and interna lize with them into endocytic vesicles. Formation of stable receptor-beta - arrestin complexes that persist inside the cell impedes receptor resensitiz ation, and the aberrant formation of these complexes may play a role in GPC R-based diseases (Barak, L, S., Oakley, R, H,, Laporte, S. A., and Caron, M . G. (2001) Proc. Natl. Acad, Sci, U, S, A. 98, 93-98), Here, we investigat e the molecular determinants responsible for sustained receptor/beta -arres tin interactions. We show in real time and in live human embryonic kidney ( HEK-293) cells that a beta -arrestin-2-green fluorescent protein conjugate internalizes into endocytic vesicles with agonist-activated neurotensin-1 r eceptor, oxytocin receptor, angiotensin II type 1A receptor, and substance P receptor. Using receptor mutagenesis, we demonstrate that the ability of beta -arrestin to remain associated with these receptors is mediated by spe cific clusters of serine and threonine residues located in the receptor car boxyl-terminal tail. These clusters are remarkably conserved in their posit ion within the carboxyl-terminal domain and serve as primary sites of agoni st-dependent receptor phosphorylation. In addition, we identify a beta -arr estin mutant with enhanced affinity for the agonist-activated beta -adrener gic receptor that traffics into endocytic vesicles with receptors that lack serine/threonine clusters and normally dissociate from wild-type beta -arr estin at the plasma membrane, By identifying receptor and beta -arrestin re sidues critical for the formation of stable receptor-beta -arrestin complex es, these studies provide novel targets for regulating GPCR responsiveness and treating diseases resulting from abnormal GPCR/beta -arrestin interacti ons.