The Ras-dependent activation of mitogen-activated protein (MAP) kinase pathways by many receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) requires the activation of Src family tyrosine kinases. Stimulation of beta2 adrenergic receptors resulted in the assembly of a protein complex containing activated c-Src and the receptor. Src recruitment was mediated by beta-arrestin, which functions as an adapter protein, binding both c-Src and the agonist-occupied receptor. beta-Arrestin 1 mutants, impaired either in c-Src binding or in the ability to target receptors to clathrin-coated pits, acted as dominant negative inhibitors of beta2 adrenergic receptor-mediated activation of the MAP kinases Erk1 and Erk2. These data suggest that beta-arrestin binding, which terminates receptor-G protein coupling, also initiates a second wave of signal transduction in which the "desensitized" receptor functions as a critical structural component of a mitogenic signaling complex.

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 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.

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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.

G protein-coupled receptors (GPCR) represent the single most important drug targets for medical therapy, and information from genome sequencing and genomic data bases has substantially accelerated their discovery. The lack of a systematic approach either to identify the function of a new GPCR or to associate it with a cognate ligand has added to the growing number of orphan receptors. In this work we provide a novel approach to this problem using a ␤-arrestin2/green fluorescent protein conjugate (␤arr2-GFP). It provides a real-time and single cell based assay to monitor GPCR activation and GPCR-G protein-coupled receptor kinase or GPCR-arrestin interactions. Confocal microscopy demonstrates the translocation of ␤arr2-GFP to more than 15 different ligand-activated GPCRs. These data clearly support the common hypothesis that the ␤-arrestin binding of an activated receptor is a convergent step of GPCR signaling, increase by 5-fold the number of GPCRs known to interact with ␤-arrestins, demonstrate that the cytosol is the predominant reservoir of biologically active ␤-arrestins, and provide the first direct demonstration of the critical importance of G protein-coupled receptor kinase phosphorylation to the biological regulation of ␤-arrestin activity and GPCR signal transduction in living cells. The use of ␤arr2-GFP as a biosensor to recognize the activation of pharmacologically distinct GPCRs should accelerate the identification of orphan receptors and permit the optical study of their signal transduction biology intractable to ordinary biochemical methods.The G protein-coupled receptor (GPCR) 1 superfamily is growing rapidly (1-3), creating many new orphan receptors whose properties remain undefined (4 -6). 2 Currently characterized GPCRs display many distinct pharmacologies. For example, they interact with a vast array of ligands and generate intracellular signals by multiple second messenger pathways (4,8,9). Based on work with rhodopsin and the ␤ 2 -adrenergic receptor (␤ 2 AR), it has been postulated that members of the GPCR superfamily desensitize via a common mechanism involving the arresting proteins visual arrestin, ␤-arrestin1 and ␤-arrestin2 (10 -13). However, mainly due to the inherent difficulties of examining the interaction of the components mediating desensitization in their native environment or the need for purified reconstituted systems, this has not been clearly established for many GPCRs. Biochemical studies indicate that arrestins regulate GPCR signal transduction (desensitization) by binding agonist-activated receptors that have been phosphorylated by G protein-coupled receptor kinases (GRKs) (12). While the functional source of arrestin molecules targeted to receptors remains unknown, it is apparent that arrestin binding terminates signaling by interdicting receptor interaction with G proteins (12).To characterize the interaction between ␤-arrestin and different GPCRs and to assess the contribution of GRKs to this process, we examined using confocal microscopy how a green fluorescent protein...

High-throughput in vitro toxicity screening can provide an efficient way to identify potential biological targets for chemicals. However, relying on nominal assay concentrations may misrepresent potential in vivo effects of these chemicals due to differences in bioavailability, clearance, and exposure. Hepatic metabolic clearance and plasma protein binding were experimentally measured for 239 ToxCast Phase I chemicals. The experimental data were used in a population-based in vitro-to-in vivo extrapolation model to estimate the daily human oral dose, called the oral equivalent dose, necessary to produce steady-state in vivo blood concentrations equivalent to in vitro AC(50) (concentration at 50% of maximum activity) or lowest effective concentration values across more than 500 in vitro assays. The estimated steady-state oral equivalent doses associated with the in vitro assays were compared with chronic aggregate human oral exposure estimates to assess whether in vitro bioactivity would be expected at the dose-equivalent level of human exposure. A total of 18 (9.9%) chemicals for which human oral exposure estimates were available had oral equivalent doses at levels equal to or less than the highest estimated U.S. population exposures. Ranking the chemicals by nominal assay concentrations would have resulted in different chemicals being prioritized. The in vitro assay endpoints with oral equivalent doses lower than the human exposure estimates included cell growth kinetics, cytokine and cytochrome P450 expression, and cytochrome P450 inhibition. The incorporation of dosimetry and exposure provide necessary context for interpretation of in vitro toxicity screening data and are important considerations in determining chemical testing priorities.

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). ...