Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of ␤-arrestins in the activation and targeting of extracellular signal-regulated kinase 2 (ERK2) following stimulation of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinintagged AT1aR, angiotensin stimulation triggered ␤-arrestin-2 binding to the receptor and internalization of AT1aR-␤-arrestin complexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redistribution of activated ERK2 into endosomal vesicles that also contained AT1aR-␤-arrestin complexes. This targeting of ERK2 reflects the formation of multiprotein complexes containing AT1aR, ␤-arrestin-2, and the component kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-tagged ␤-arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with ␤-arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with ␤-arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to ␤-arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in ␤-arrestin complexes was markedly enhanced by coexpression of cRaf-1, and this effect is blocked by expression of a catalytically inactive dominant inhibitory mutant of MEK1. Stimulation with angiotensin increased the binding of both cRaf-1 and ERK2 to ␤-arrestin-2, and the association of ␤-arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that ␤-arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.

By binding to agonist-activated G protein-coupled receptors (GPCRs), ␤-arrestins mediate homologous receptor desensitization and endocytosis via clathrincoated pits. Recent data suggest that ␤-arrestins also contribute to GPCR signaling by acting as scaffolds for components of the ERK mitogen-activated protein kinase cascade. Because of these dual functions, we hypothesized that the stability of the receptor-␤-arrestin interaction might affect the mechanism and functional consequences of GPCR-stimulated ERK activation. In transfected COS-7 cells, we found that angiotensin AT1a and vasopressin V2 receptors, which form stable receptor-␤-arrestin complexes, activated a ␤-arrestin-bound pool of ERK2 more efficiently than ␣1b and ␤2 adrenergic receptors, which form transient receptor-␤-arrestin complexes. We next studied chimeric receptors in which the pattern of ␤-arrestin binding was reversed by exchanging the C-terminal tails of the ␤2 and V2 receptors. The ability of the V2␤2 and ␤2V2 chimeras to activate ␤-arrestin-bound ERK2 corresponded to the pattern of ␤-arrestin binding, suggesting that the stability of the receptor-␤-arrestin complex determined the mechanism of ERK2 activation. Analysis of covalently cross-linked detergent lysates and cellular fractionation revealed that wild type V2 receptors generated a larger pool of cytosolic phospho-ERK1/2 and less nuclear phospho-ERK1/2 than the chimeric V2␤2 receptor, consistent with the cytosolic retention of ␤-arrestin-bound ERK. In stably transfected HEK-293 cells, the V2␤2 receptor increased ERK1/2-mediated, Elk-1-driven transcription of a luciferase reporter to a greater extent than the wild type V2 receptor. Furthermore, the V2␤2, but not the V2 receptor, was capable of eliciting a mitogenic response. These data suggest that the C-terminal tail of a GPCR, by determining the stability of the receptor-␤-arrestin complex, controls the extent of ␤-arrestin-bound ERK activation, and influences both the subcellular localization of activated ERK and the physiologic consequences of ERK activation.Homologous desensitization of most heptahelical, or G protein-coupled, receptors (GPCRs) 1 results from the physical uncoupling of receptor and G protein as a consequence of arrestin binding. Agonist-occupied GPCRs are rapidly phosphorylated by specialized G protein-coupled receptor kinases (GRKs). Subsequent high affinity binding of arrestin to the GRK-phosphorylated receptor results in steric inhibition of receptor-G protein coupling. In addition, the two nonvisual arrestins, ␤-arrestin 1 and ␤-arrestin 2, function as adapter proteins, binding to clathrin and the ␤2 adaptin subunit of the AP-2 complex, and leading to targeting of GPCRs to clathrin-coated pits where they are internalized (1, 2).Data obtained using green fluorescent protein (GFP)-tagged ␤-arrestins and epitope-tagged GPCRs to visualize ␤-arrestin and receptor trafficking in live cells have demonstrated that most GPCRs exhibit one of two characteristic patterns of agonist-induced ␤-arrestin interaction that al...

␤-Arrestins are cytosolic proteins that mediate homologous desensitization of G protein-coupled receptors (GPCRs) by binding to agonist-occupied receptors and by uncoupling them from heterotrimeric G proteins. The recent finding that ␤-arrestins bind to some mitogen-activated protein (MAP) kinases has suggested that they might also function as scaffolds for GPCR-stimulated MAP kinase activation. To define the role of ␤-arrestins in the regulation of ERK MAP kinases, we examined the effect of ␤-arrestin overexpression on ERK1/2 activation and nuclear signaling in COS-7 cells expressing angiotensin II type 1a receptors (AT1aRs). Expression of either ␤-arrestin1 or ␤-arrestin2 reduced angiotensin-stimulated phosphatidylinositol hydrolysis but paradoxically increased angiotensin-stimulated ERK1/2 phosphorylation. The increase in ERK1/2 phosphorylation in ␤-arrestin-expressing cells correlated with activation of a ␤-arrestin-bound pool of ERK2. The ␤-arrestindependent increase in ERK1/2 phosphorylation was accompanied by a significant reduction in ERK1/2-mediated, Elk1-driven transcription of a luciferase reporter. Analysis of the cellular distribution of phospho-ERK1/2 by confocal immunofluorescence microscopy and cellular fractionation revealed that overexpression of ␤-arrestin resulted in a significant increase in the cytosolic pool of phospho-ERK1/2 and a corresponding decrease in the nuclear pool of phospho-ERK1/2 following angiotensin stimulation. ␤-Arrestin overexpression resulted in formation of a cytoplasmic pool of ␤-arrestinbound phospho-ERK, decreased nuclear translocation of phospho-ERK1/2, and inhibition of Elk1-driven luciferase transcription even when ERK1/2 was activated by overexpression of cRaf-1 in the absence of AT1aR stimulation. These data demonstrate that ␤-arrestins facilitate GPCR-mediated ERK activation but inhibit ERKdependent transcription by binding to phospho-ERK1/2, leading to its retention in the cytosol. The G protein-coupled receptor (GPCR)1 superfamily is composed of a diverse array of membrane receptors that share a conserved seven-transmembrane domain architecture. In response to receptor occupancy, GPCRs promote the activation of heterotrimeric G proteins by catalyzing the exchange of GDP for GTP on the G␣ subunit and dissociation of the G␣ subunit from the G␤␥ subunit heterodimer. Once dissociated, free G␣-GTP and G␤␥ subunits regulate the activity of enzymatic effectors, such as adenylyl cyclases and phospholipase C isoforms.For the majority of GPCRs, productive G protein coupling in the continued presence of agonist is terminated by receptor phosphorylation followed by the binding of arrestins (1, 2). Specialized G protein-coupled receptor kinases phosphorylate agonist-occupied GPCRs, increasing their affinity for arrestins. Upon binding the receptor, arrestins sterically block further coupling between GPCR and G protein. In addition, the two non-visual arrestins, ␤-arrestin1 and 2, target GPCRs for endocytosis by linking the GPCR to components of the cellular endocytic machi...

The dermatonecrotic toxin produced by Pasteurella multocida is one of the most potent mitogenic substances known for fibroblasts in vitro. Exposure to recombinant P. multocida toxin (rPMT) causes phospholipase C-mediated hydrolysis of inositol phospholipids, calcium mobilization, and activation of protein kinase C via a poorly characterized mechanism involving G q/11 family heterotrimeric G proteins. To determine whether the regulation of G protein pathways contributes to the mitogenic effects of rPMT, we have examined the mechanism whereby rPMT stimulates the Erk mitogen-activated protein kinase cascade in cultured HEK-293 cells. Treatment with rPMT resulted in a dose and time-dependent increase in Erk 1/2 phosphorylation that paralleled its stimulation of inositol phospholipid hydrolysis. Both rPMT-and ␣-thrombin receptor-stimulated Erk phosphorylation were selectively blocked by cellular expression of two peptide inhibitors of G q/11 signaling, the dominant negative mutant G protein-coupled receptor kinase, GRK2(K220R), and the G␣ q carboxyl-terminal peptide, G␣ q -(305-359). Like ␣-thrombin receptor-mediated Erk activation, the effect of rPMT was insensitive to the protein kinase C inhibitor GF109203X, but was blocked by the epidermal growth factor receptor-specific tyrphostin, AG1478 and by dominant negative mutants of mSos1 and Ha-Ras. These data indicate that rPMT employs G q/11 family heterotrimeric G proteins to induce Ras-dependent Erk activation via protein kinase C-independent "transactivation" of the epidermal growth factor receptor.Pasteurella multocida is a wide ranging bacterium found in the respiratory tracts of over 60 avian and 40 mammalian species (1). It is a significant veterinary, and occasional human, pathogen and the cause of swine atrophic rhinitis, a condition characterized by osteoclastic bone resorption and severe progressive turbinate damage. The pathogenicity of P. multocida is related to the production of a 146-kDa toxin (2, 3) that exhibits little functional homology to other known toxins or proteins. Both native and recombinant PMT 1 are potently mitogenic for several cell types in vitro (4). In Rat1 fibroblasts, rPMT produces anchorage-independent DNA synthesis and growth in soft agar (5). In primary osteoblastic cells in culture, rPMT induces cell proliferation and down-regulation of markers of osteoblast differentiation (6). The mechanisms whereby rPMT exerts its mitogenic effects are poorly understood. The toxin interacts with a gangliosidetype cell surface receptor and is internalized via both coated and noncoated endocytic structures (7,8). The mitogenic effects of rPMT require its internalization, since exposure of cells to rPMT at 4°C or incubation with the weak base methylamine prevents the response (4). Exposure to rPMT stimulates the hydrolysis of inositol phospholipids, calcium mobilization, and phosphorylation of protein kinase C (PKC) substrates, including the myristoylated alanine-rich C kinase substrate protein (4, 5, 9). In addition, rPMT has been shown to stimu...