Somatostatin receptors signal through EFA6A-ARF6 to activate phospholipase D in clonal β-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: 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: The peptide somatostatin (SRIF) has important physiological effects, mostly inhibitory, which have formed the basis for the clinical use of SRIF compounds. SRIF binding to its 5 guanine nucleotide-binding proteins-coupled receptors leads to the modulation of multiple transduction pathways. However, our current understanding of signaling exerted by receptors endogenously expressed in different cells/tissues reflects a rather complicated picture. On the other hand, the complexity of SRIF receptor signaling in pathologies, including pituitary and nervous system diseases, may be studied not only as alternative intervention points for the modulation of SRIF function but also to exploit new chemical space for drug-like molecules.Pharmacology [?] Therapeutics 12/2007; 116(2):322-41. DOI:10.1016/j.pharmthera.2007.06.010 · 7.75 Impact Factor
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ABSTRACT: Endocytosis of the mu-opioid receptor (MOPr) has been shown to play a protective role against the development of tolerance to opioid drugs by facilitating receptor reactivation and recycling. It has been further demonstrated, that the opioid-mediated and ADP-ribosylation factor (ARF)-dependent activation of phospholipase D2 (PLD2) is a prerequisite for MOPr endocytosis. In this study, we investigated which particular ARF protein is involved in opioid-mediated PLD2 activation and what are the mechanisms of ARF function in MOPr trafficking and signaling. By coexpressing the MOPr and dominant negative or constitutively active ARF mutants in human embryonic kidney (HEK) 293 cells and primary cultured cortical neurons as well as by using siRNA technology, we identified the ARF6 protein to be involved in the regulation of MOPr endocytosis. We also found that expression of an effector domain mutant of ARF6, which is incapable of activating PLD, blocked agonist-induced endocytosis suggesting 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. Finally, we also showed that ARF6 protein influences the recycling/reactivation of internalized MOPr and thus modulates agonist-induced MOPr desensitization. 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.Cellular Signalling 09/2009; 21(12):1784-93. DOI:10.1016/j.cellsig.2009.07.014 · 4.47 Impact Factor