Somatostatin receptors signal through EFA6A-ARF6 to activate phospholipase D in clonal beta-cells.
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.
Grodnitzky
Justin A
JA
Department of Biomedical Sciences, Iowa State University, Ames, Iowa 50011, USA.
Syed
Nasser
N
Kimber
Michael J
MJ
Day
Tim A
TA
Donaldson
Julie G
JG
Hsu
Walter H
WH
D020823:ADP-Ribosylation Factor 1 / Q000235:genetics / Q000378:metabolism; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000201:enzymology* / Q000378:metabolism; D020125:Mutation, Missense; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017481:Receptors, Somatostatin / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013004:Somatostatin / Q000378:metabolism / Q000494:pharmacology
D000089982:ADP-Ribosylation Factor 6; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin; C504475:Psd protein, rat; D012333:RNA, Messenger; D017481:Receptors, Somatostatin; D013004:Somatostatin; D010739:Phospholipase D; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; C486478:Arf5 protein, rat; C000715349:Arf6 protein, rat