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.
- SourceAvailable from: Vladan Rankovic[Show abstract] [Hide abstract]
ABSTRACT: We demonstrated recently that opioid-induced activation of phospholipase D2 (PLD2) enhances mu- (MOPr) and delta-opioid receptor endocytosis/recycling and thus reduces the development of opioid receptor desensitization and tolerance. However, the mechanistic basis for the PLD2-mediated induction of opioid receptor endocytosis is currently unknown. Here we show that PLD2-generated phosphatidic acid (PA) might play a key role in facilitating the endocytosis of opioid receptors. However, PLD2-derived PA is known to be further converted to diacylglycerol (DAG) by PA phosphohydrolase (PPAP2). In fact, blocking of PA phosphohydrolase activity by propranolol or PPAP2-short interfering RNA (siRNA) transfection significantly attenuated agonist-induced opioid receptor endocytosis. The primary importance of PA-derived DAG in the induction of opioid receptor endocytosis was further supported by the finding that increasing the DAG level by inhibiting the reconversion of DAG into PA with the DAG kinase inhibitor 3-[2-(4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl)ethyl]-2,3-dihydro-2-thioxo-4(1H)quinazolinone (R59949) or the addition of the synthetic cell-permeable DAG analog 1,2-dioctanoyl-sn-glycerol (DOG), further increased the agonist-induced opioid receptor endocytosis. Moreover, the addition of DOG bypasses the PLD2-siRNA- or PPAP2-siRNA-mediated impairment of DAG synthesis and resulted in a restoration of agonist-induced opioid receptor internalization. Further studies established a functional link between PA-derived DAG and the activation of p38 mitogen-activated protein kinase (MAPK) and the subsequent phosphorylation of the Rab5 effector early endosome antigen 1, which has been demonstrated recently to be required for the induction of MOPr endocytosis. Taken together, our results revealed that the regulation of opioid receptor endocytosis by PLD2 involves the conversion of its product PA to DAG resulting in an activation of the p38 MAPK pathway.Molecular pharmacology 03/2010; 78(1):105-13. · 4.53 Impact Factor
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ABSTRACT: Physiological effects of opioids are mediated through binding to specific G protein-coupled opioid receptors. The mu-opioid receptor (MOPr) is of particular importance for the mediation of both the analgesic and the adaptive effects of clinically relevant opioid drugs. After opioid binding, the ligand-receptor complex is endocytosed via clathrin coated vesicles. Internalized receptors are then either recycled back to the plasma membrane or degraded in the lysosome. Previous studies have shown that endocytosis of MOPr plays a protective role in the development of tolerance to opioid drugs by facilitating receptor reactivation and recycling. It has been further demonstrated, that the opioid-mediated activation of phospholipase D2 (PLD2) is a prerequisite for MOPr endocytosis and is dependent on small GTPases of ADP-ribosylation factor (ARF) family. However, precise identity of ARF protein (ARF1 or ARF6) as well as the mechanisms involved in opioid-mediated PLD2 activation by ARF proteins are still not clear. By coexpressing the MOPr and different ARF mutants in human embryonic kidney (HEK) 293 cells and cultured primary cortical neurons, we have identified the ARF6 protein to be involved in the regulation of MOPr endocytosis. This conclusion was based on the two facts: 1) overexpression of dominant negative ARF6 mutant blocked receptor internalization after treatment with potent endocytotic drug DAMGO and 2) receptor endocytosis was increased in the presence of an active, “fast cycling” ARF6 mutant after treatment with morphine, an agonist that is unable to induce MOPr endocytosis by itself. Moreover, siRNA-mediated knock down of endogenous ARF6 protein expression significantly decreased receptor internalization. Presented study also documents that expression of an effector domain mutant of ARF6 which is incapable of activating PLD2 (“PLD-defective” mutant) blocked agonist-induced receptor endocytosis showing that ARF6 function in MOPr trafficking is PLD2-mediated. Analogously, opioid-mediated activation of PLD2 is blocked in the presence of dominant negative ARF6 mutants. Furthermore, we have also shown that ARF6 protein influences the recycling/reactivation of internalized MOPr and thus modulates agonist-induced MOPr desensitization. And finally, we demonstrated the importance of GTP hydrolysis of activated ARF6 protein and full GDP/GTP cycle for the trafficking of internalized MOPr back to the plasma membrane since locking ARF6 in its GTP-bound, active state blocked the recycling of the receptor. Taken together, these results provide evidence that ARF6 protein regulates MOPr trafficking and signaling via PLD2 activation and hence affects the development of opioid receptor desensitization and tolerance to opioid drugs.
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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