␤-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...

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...

Differentiation of CNS glia is regulated by Notch signaling through neuron-glia interaction. Here, we identified Delta/Notch-like EGF-related receptor (DNER), a neuron-specific transmembrane protein, as a previously unknown ligand of Notch during cellular morphogenesis of Bergmann glia in the mouse cerebellum. DNER binds to Notch1 at cell-cell contacts and activates Notch signaling in vitro. In the developing cerebellum, DNER is highly expressed in Purkinje cell dendrites, which are tightly associated with radial fibers of Bergmann glia expressing Notch. DNER specifically binds to Bergmann glia in culture and induces process extension by activating gamma-secretase- and Deltex-dependent Notch signaling. Inhibition of Deltex-dependent, but not RBP-J-dependent, Notch signaling in Bergmann glia suppresses formation and maturation of radial fibers in organotypic slice cultures. Additionally, deficiency of DNER retards the formation of radial fibers and results in abnormal arrangement of Bergmann glia. Thus, DNER mediates neuron-glia interaction and promotes morphological differentiation of Bergmann glia through Deltex-dependent Notch signaling.

Cyclic ADP-ribose (cADPR) is a second messenger for Ca 2؉ mobilization via the ryanodine receptor (RyR) from islet microsomes for insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370 -373). In the present study, FK506, an immunosuppressant that prolongs allograft survival, as well as cADPR were found to induce the release of Ca 2؉ from islet microsomes. After islet microsomes were treated with FK506, the Ca 2؉ release by cADPR from microsomes was reduced. cADPR as well as FK506 bound to FK506-binding protein 12.6 (FKBP12.6), which we also found occurs naturally in islet microsomes. When islet microsomes were treated with cADPR, FKBP12.6 dissociated from the microsomes and moved to the supernatant, releasing Ca 2؉ from the intracellular stores. The microsomes that were then devoid of FKBP12.6 did not show Ca 2؉ release by cADPR. These results strongly suggest that cADPR may be the ligand for FKBP12.6 in islet RyR and that the binding of cADPR to FKBP12.6 frees the RyR from FKBP12.6, causing it to release Ca 2؉ .

Intracellular Ca2؉ mobilization occurs in a variety of cellular processes and is mediated by two major systems, the inositol 1,4,5-trisphosphate (IP 3 ) and cyclic ADP-ribose (cADPR) systems. cADPR has been proposed to be a second messenger for insulin secretion induced by glucose in pancreatic ␤-cells (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370 -373). Here we show that the cADPR signal system for insulin secretion is replaced by the IP 3 system in diabetic ␤-cells such as ob/ob mouse islets and RINm5F cells. We measured the cADPR content in these ␤-cells by radioimmunoassay and found that the increase of the cADPR content by glucose did not occur in ob/ob mouse islets and RINm5F cells, whereas the increased cADPR level by glucose was observed in normal rat and mouse islets. Microsomes of these diabetic ␤-cells released Ca 2؉ in response to IP 3 but not to cADPR. In the diabetic ␤-cells, CD38 (ADP-ribosyl cyclase/cADPR hydrolase) and type 2 ryanodine receptor mRNAs were scarcely detected and, in contrast, an increased expression of IP 3 receptor mRNAs was observed. The diabetic ␤-cells secreted insulin rather by carbamylcholine than by glucose.