Visual arrestin, ␤arrestin1, and ␤arrestin2 comprise a family of intracellular proteins that desensitize G protein-coupled receptors (GPCRs). In addition, ␤arrestin1 and ␤arrestin2 target desensitized receptors to clathrincoated pits for endocytosis. Whether arrestins differ in their ability to interact with GPCRs in cells is not known. In this study, we visualize the interaction of arrestin family members with GPCRs in real time and in live cells using green fluorescent protein-tagged arrestins. In the absence of agonist, visual arrestin and ␤arrestin1 were found in both the cytoplasm and nucleus of HEK-293 cells, whereas ␤arrestin2 was found only in the cytoplasm. Analysis of agonist-mediated arrestin translocation to multiple GPCRs identified two major classes of receptors. Class A receptors (␤2 adrenergic receptor, mu opioid receptor, endothelin type A receptor, dopamine D1A receptor, and ␣1b adrenergic receptor) bound ␤arrestin2 with higher affinity than ␤arrestin1 and did not interact with visual arrestin. In contrast, class B receptors (angiotensin II type 1A receptor, neurotensin receptor 1, vasopressin V2 receptor, thyrotropin-releasing hormone receptor, and substance P receptor) bound both ␤arrestin isoforms with similar high affinities and also interacted with visual arrestin. Switching the carboxyl-terminal tails of class A and class B receptors completely reversed the affinity of each receptor for the visual and non-visual arrestins. In addition, exchanging the ␤arrestin1 and ␤arrestin2 carboxyl termini reversed their extent of binding to class A receptors as well as their subcellular distribution. These results reveal for the first time marked differences in the ability of arrestin family members to bind GPCRs at the plasma membrane. Moreover, they show that visual arrestin can interact in cells with GPCRs other than rhodopsin. These findings suggest that GPCR signaling may be differentially regulated depending on the cellular complement of arrestin isoforms and the ability of arrestins to interact with other cellular proteins.

beta-Arrestins, originally discovered in the context of heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) desensitization, also function in internalization and signaling of these receptors. We identified c-Jun amino-terminal kinase 3 (JNK3) as a binding partner of beta-arrestin 2 using a yeast two-hybrid screen and by coimmunoprecipitation from mouse brain extracts or cotransfected COS-7 cells. The upstream JNK activators apoptosis signal-regulating kinase 1 (ASK1) and mitogen-activated protein kinase (MAPK) kinase 4 were also found in complex with beta-arrestin 2. Cellular transfection of beta-arrestin 2 caused cytosolic retention of JNK3 and enhanced JNK3 phosphorylation stimulated by ASK1. Moreover, stimulation of the angiotensin II type 1A receptor activated JNK3 and triggered the colocalization of beta-arrestin 2 and active JNK3 to intracellular vesicles. Thus, beta-arrestin 2 acts as a scaffold protein, which brings the spatial distribution and activity of this MAPK module under the control of a GPCR.

Resensitization of G protein-coupled receptors (GPCRs)following agonist-mediated desensitization is a necessary step for maintaining physiological responsiveness. However, the molecular mechanisms governing the nature of GPCR resensitization are poorly understood. Here, we examine the role of ␤-arrestin in the resensitization of the ␤ 2 adrenergic receptor (␤ 2 AR), known to recycle and resensitize rapidly, and the vasopressin V2 receptor (V2R), known to recycle and resensitize slowly. Upon agonist activation, both receptors recruit ␤-arrestin to the plasma membrane and internalize in a ␤-arrestin-and clathrin-dependent manner. However, whereas ␤-arrestin dissociates from the ␤ 2 AR at the plasma membrane, it internalizes with the V2R into endosomes. The differential trafficking of ␤-arrestin and the ability of these two receptors to dephosphorylate, recycle, and resensitize is completely reversed when the carboxyl-terminal tails of these two receptors are switched. Moreover, the ability of ␤-arrestin to remain associated with desensitized GPCRs during clathrin-mediated endocytosis is mediated by a specific cluster of phosphorylated serine residues in the receptor carboxyl-terminal tail. These results demonstrate that the interaction of ␤-arrestin with a specific motif in the GPCR carboxyl-terminal tail dictates the rate of receptor dephosphorylation, recycling, and resensitization, and thus provide direct evidence for a novel mechanism by which ␤-arrestins regulate the reestablishment of GPCR responsiveness.

The G protein-coupled -opioid receptor (OR) mediates the physiological effects of endogenous opioid peptides as well as the structurally distinct opioid alkaloids morphine and etorphine. An intriguing feature of OR signaling is the differential receptor trafficking and desensitization properties following activation by distinct agonists, which have been proposed as possible mechanisms related to opioid tolerance. Here we report that the ability of distinct opioid agonists to differentially regulate OR internalization and desensitization is related to their ability to promote G protein-coupled receptor kinase (GRK)-dependent phosphorylation of the OR. Although both etorphine and morphine effectively activate the OR, only etorphine elicits robust OR phosphorylation followed by plasma membrane translocation of ␤-arrestin and dynamin-dependent receptor internalization. In contrast, corresponding to its inability to cause OR internalization, morphine is unable to either elicit OR phosphorylation or stimulate ␤-arrestin translocation. However, upon the overexpression of GRK2, morphine gains the capacity to induce OR phosphorylation, accompanied by the rescue of ␤-arrestin translocation and receptor sequestration. Moreover, overexpression of GRK2 also leads to an attenuation of morphine-mediated inhibition of adenylyl cyclase. These findings point to the existence of marked differences in the ability of different opioid agonists to promote OR phosphorylation by GRK. These differences may provide the molecular basis underlying the different analgesic properties of opioid agonists and contribute to the distinct ability of various opioids to induce drug tolerance.

␤-Arrestins bind agonist-activated G protein-coupled receptors (GPCRs) and mediate their desensitization and internalization. Although ␤-arrestins dissociate from some receptors at the plasma membrane, such as the ␤2 adrenergic receptor, they remain associated with other GPCRs and internalize with them into endocytic vesicles. Formation of stable receptor-␤-arrestin complexes that persist inside the cell impedes receptor resensitization, and the aberrant formation of these complexes may play a role in GPCR-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 investigate the molecular determinants responsible for sustained receptor/␤-arrestin interactions. We show in real time and in live human embryonic kidney (HEK-293) cells that a ␤-arrestin-2-green fluorescent protein conjugate internalizes into endocytic vesicles with agonistactivated neurotensin-1 receptor, oxytocin receptor, angiotensin II type 1A receptor, and substance P receptor. Using receptor mutagenesis, we demonstrate that the ability of ␤-arrestin to remain associated with these receptors is mediated by specific clusters of serine and threonine residues located in the receptor carboxyl-terminal tail. These clusters are remarkably conserved in their position within the carboxyl-terminal domain and serve as primary sites of agonist-dependent receptor phosphorylation. In addition, we identify a ␤-arrestin mutant with enhanced affinity for the agonist-activated ␤2-adrenergic receptor that traffics into endocytic vesicles with receptors that lack serine/threonine clusters and normally dissociate from wild-type ␤-arrestin at the plasma membrane. By identifying receptor and ␤-arrestin residues critical for the formation of stable receptor-␤-arrestin complexes, these studies provide novel targets for regulating GPCR responsiveness and treating diseases resulting from abnormal GPCR/␤-arrestin interactions.

Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq.

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ABSTRACT␤arrestins mediate the desensitization of the ␤ 2 -adrenergic receptor (␤ 2 AR) and many other G proteincoupled receptors (GPCRs). Additionally, ␤arrestins initiate the endocytosis of these receptors via clathrin coated-pits and interact directly with clathrin. Consequently, it has been proposed that ␤arrestins serve as clathrin adaptors for the GPCR family by linking these receptors to clathrin lattices. AP-2, the heterotetrameric clathrin adaptor protein, has been demonstrated to mediate the internalization of many types of plasma membrane proteins other than GPCRs. AP-2 interacts with the clathrin heavy chain and cytoplasmic domains of receptors such as those for epidermal growth factor and transferrin. In the present study we demonstrate the formation of an agonist-induced multimeric complex containing a GPCR, ␤arrestin 2, and the ␤2-adaptin subunit of AP-2. ␤2-Adaptin binds ␤arrestin 2 in a yeast two-hybrid assay and coimmunoprecipitates with ␤arrestins and ␤ 2 AR in an agonist-dependent manner in HEK-293 cells. Moreover, ␤2-adaptin translocates from the cytosol to the plasma membrane in response to the ␤ 2 AR agonist isoproterenol and colocalizes with ␤ 2 AR in clathrin-coated pits. Finally, expression of ␤arrestin 2 minigene constructs containing the ␤2-adaptin interacting region inhibits ␤ 2 AR endocytosis. These findings point to a role for AP-2 in GPCR endocytosis, and they suggest that AP-2 functions as a clathrin adaptor for the endocytosis of diverse classes of membrane receptors.