Somatostatin receptors signal through EFA6A-ARF6 to activate phospholipase D in clonal beta-cells.
ABSTRACT Somatostatin (SS) is a peptide hormone that inhibits insulin secretion in beta-cells by activating its G(i/o)-coupled receptors. Our previous work indicated that a betagamma-dimer of G(i/o) coupled to SS receptors can activate phospholipase D1 (PLD1) (Cheng, H., Grodnitzky, J. A., Yibchok-anun, S., Ding, J., and Hsu, W. H. (2005) Mol. Pharmacol. 67, 2162-2172). The aim of the present study was to elucidate the mechanisms underlying SS-induced PLD activation. We demonstrated the presence of ADP-ribosylation factor Arf1 and Arf6 in clonal beta-cells, HIT-T15. We also determined that the activation of PLD1 was mediated through Arf6. Overexpression of dominant-negative (dn) Arf6 mutant, Arf6(T27N), and suppression of mRNA levels using siRNA, both abolished SS-induced PLD activation, while overexpression of wild type Arf6 further enhanced this PLD activation. In contrast, overexpression of dn-Arf1 mutant Arf1(T31N) or dn-Arf5 mutant Arf5(T31N) failed to reduce SS-induced PLD activation. These findings suggested that Arf6, but not Arf1 or Arf5, mediates the effect of SS. We further determined the involvement of the Arf6 guanine nucleotide exchange factor (GEF) EFA6A, a GEF previously thought to be found predominantly in the brain, in the activation of PLD1 in HIT-T15 cells. Using Northern and Western blot analyses, both mRNA and protein of EFA6A were found in these cells. Overexpression of dn-EFA6A mutant, EFA6A(E242K), and suppression of mRNA levels using siRNA, both abolished SS-induced PLD activation, whereas overexpression of dn-EFA6B mutant, EFA6B(E651K), failed to reduce SS-induced PLD activation. In addition, overexpression of dn-ARNO mutant, ARNO(E156K), another GEF of Arf6, had no effect on SS-induced activation of PLD. Taken together, these results suggest that SS signals through EFA6A to activate Arf6-PLD cascade.
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ABSTRACT: Glucose-stimulated insulin secretion [GSIS] involves interplay between small G-proteins and their regulatory factors. Herein, we tested the hypothesis that Arf nucleotide binding site opener [ARNO], a guanine nucleotide-exchange factor [GEF] for the small G-protein Arf6, mediates the functional activation of Arf6, and that ARNO/Arf6 signaling axis, in turn, controls the activation of Cdc42 and Rac1, which have been implicated in GSIS. Molecular biological [i.e., expression of inactive mutants or siRNA] and pharmacological approaches were employed to assess the roles for ARNO/Arf6 signaling pathway in insulin secretion in normal rat islets and INS 832/13 cells. Degrees of activation of Arf6 and Cdc42/Rac1 were quantitated by GST-GGA3 and PAK-1 kinase pull-down assays, respectively. ARNO is expressed in INS 832/13 cells, rat islets and human islets. Expression of inactive mutants of Arf6 [Arf6-T27N] or ARNO [ARNO-E156K] or siRNA-ARNO markedly reduced GSIS in isolated β-cells. SecinH3, a selective inhibitor of ARNO/Arf6 signaling axis, also inhibited GSIS in INS 832/13 cells and rat islets. Stimulatory concentrations of glucose promoted Arf6 activation, which was inhibited by secinH3 or siRNA-ARNO, suggesting that ARNO/Arf6 signaling cascade is necessary for GSIS. SecinH3 or siRNA-ARNO also inhibited glucose-induced activation of Cdc42 and Rac1 suggesting that ARNO/Arf6 might be upstream to Cdc42 and Rac1 activation steps, which are necessary for GSIS. Lastly, co-immunoprecipitation and confocal microscopic studies suggested increased association between Arf6 and ARNO in glucose-stimulated β-cells. These findings provide the first evidence to implicate ARNO in the sequential activation of Arf6, Cdc42 and Rac1 culminating in GSIS.Biochemical pharmacology 01/2011; 81(8):1016-27. · 4.25 Impact Factor
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ABSTRACT: Glucose-stimulated insulin secretion from the islet beta-cell involves a sequence of metabolic events and an interplay between a wide range of signaling pathways leading to the generation of second messengers (e.g., cyclic nucleotides, adenine and guanine nucleotides, soluble lipid messengers) and mobilization of calcium ions. Consequent to the generation of necessary signals, the insulin-laden secretory granules are transported from distal sites to the plasma membrane for fusion and release of their cargo into the circulation. The secretory granule transport underlies precise changes in cytoskeletal architecture involving a well-coordinated cross-talk between various signaling proteins, including small molecular mass GTP-binding proteins (G proteins) and their respective effector proteins. The purpose of this article is to provide an overview of current understanding of the identity of small G proteins (e.g., Cdc42, Rac1, and ARF-6) and their corresponding regulatory factors (e.g., GDP/GTP-exchange factors, GDP-dissociation inhibitors) in the pancreatic beta-cell. Plausible mechanisms underlying regulation of these signaling proteins by insulin secretagogues are also discussed. In addition to their positive modulatory roles, certain small G proteins also contribute to the metabolic dysfunction and demise of the islet beta-cell seen in in vitro and in vivo models of impaired insulin secretion and diabetes. Emerging evidence also suggests significant insulin secretory abnormalities in small G protein knockout animals, further emphasizing vital roles for these proteins in normal health and function of the islet beta-cell. Potential significance of these experimental observations from multiple laboratories and possible avenues for future research in this area of islet research are highlighted.Endocrine reviews 11/2009; 31(1):52-78. · 19.76 Impact Factor
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ABSTRACT: Cancer cells are characterized by their intrinsic ability to rapidly divide and migrate and to invade other tissues. How these processes are regulated at a molecular level is largely unknown. Here, we identify the oncogenic TBC (Tre-2/Bub2/Cdc16) domain protein USP6 (also termed TRE17) as a regulator of both cell migration and division. We show that manipulating USP6 expression levels alters the ability of cells to migrate and to divide. Furthermore, we observe that cell proliferation and progression through cytokinesis depend on USP6 expression via a pathway that involves the small GTPase Arf6 and its GTPase-activating protein ACAP1. Our data suggest a model whereby the oncogenic potential of USP6 is linked to its ability to integrate cell migration and cytokinesis by regulating Arf6/ACAP1.Biology of the Cell 11/2011; 104(1):22-33. · 3.49 Impact Factor