beta-Arrestins are proteins that bind phosphorylated heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) and contribute to the desensitization of GPCRs by uncoupling the signal transduction process. Resensitization of GPCR responsiveness involves agonist-mediated receptor sequestration. Overexpression of beta-arrestins in human embryonic kidney cells rescued the sequestration of beta 2-adrenergic receptor (beta 2AR) mutants defective in their ability to sequester, an effect enhanced by simultaneous overexpression of beta-adrenergic receptor kinase 1. Wild-type beta 2AR sequestration was inhibited by the overexpression of two beta-arrestin mutants. These findings suggest that beta-arrestins play an integral role in GPCR internalization and thus serve a dual role in the regulation of GPCR function.

The process of agonist-promoted internalization (sequestration) of G protein-coupled receptors (GPCRs) is intimately linked to the regulation of GPCR responsiveness. Following agonist-mediated desensitization, sequestration of GPCR is presumably associated with the dephosphorylation and recycling of functional receptors. However, the exact mechanisms responsible for GPCR sequestration, even for the prototypic ␤ 2 -adrenergic receptor (␤ 2 AR), have remained controversial. We demonstrate here that dynamin, a GTPase that regulates the formation and internalization of clathrincoated vesicles, is essential for the agonist-promoted sequestration of the ␤ 2 AR, suggesting that the ␤ 2 AR internalizes via the clathrin-coated vesicle-mediated endocytic pathway. In contrast, internalization of the angiotensin II type 1A receptor (AT 1A R), another typical GPCR, does not require dynamin. In addition, the AT 1A R internalizes independent of the function of ␤-arrestin, a critical component for ␤ 2 AR cellular trafficking, but additional AT 1A Rs are mobilized to the dynamin-dependent pathway upon overexpression of ␤-arrestin. These findings demonstrate that GPCRs can utilize distinct endocytic pathways, distinguishable by dynamin and ␤-arrestin, and that ␤-arrestins function as adaptor proteins specifically targeting GPCRs for dynamin-dependent endocytosis via clathrin-coated vesicles. G protein-coupled receptor (GPCR)1 internalization is a phenomenon triggered by agonist stimulation. This rapid internalization, referred to as sequestration (1), follows the agonistmediated phosphorylation and desensitization of the signaling function of these receptors and is thought to contribute to the resensitization of GPCR responsiveness (2-5). Although the process of sequestration has been documented for many GPCRs, the cellular nature and molecular determinants of sequestration have remained elusive. Recently, however, phosphorylation of GPCRs by specific G protein-coupled receptor kinases has been demonstrated to facilitate agonist-mediated receptor sequestration (6, 7). This receptor phosphorylation serves to increase the affinity of GPCRs for ␤-arrestin proteins, which, in addition to uncoupling receptor-G protein interactions, can act as adaptor-like molecules for receptor trafficking (8).Considerable evidence exists that GPCRs internalize via the clathrin-coated vesicle-mediated endocytic pathway (9 -12). However, even for the prototypic GPCR, ␤ 2 -adrenergic receptor (␤ 2 AR), this issue has remained controversial (9, 10, 13). Dynamin, a 100-kDa GTPase, originally isolated as a nucleotidedependent microtubule binding protein, has been identified as a major component and marker of the clathrin-mediated endocytic pathway (14 -18). Dynamin colocalizes with clathrin (19,20) and binds to the appendage domain of ␣-adaptin, a component of the clathrin-coated pits (21). Functionally, dynamin contributes to the early stages of endocytosis by catalyzing a GTP-dependent pinching off of endocytic vesicles from the plasma membrane (22,23). ...