Phospholipase D in rat myometrium: occurrence of a membrane-bound ARF6 (ADP-ribosylation factor 6)-regulated activity controlled by beta gamma subunits of heterotrimeric G-proteins

Both protein kinase C and protein tyrosine kinases have been shown to be in volved in phospholipase D (PLD) activation in intact rat myometrium [Le Stu nff, Dokhac and Harbon (2000) J. Pharmacol. Exp, Ther, 292, 629-637]. In th is study we assessed the involvement of monomeric G-proteins in PLD activat ion in a cell-free system derived from myometrial tissue. Both the PLD1 and PLD2 isoforms were detected. Two forms of PLD activity, essentially membra ne-bound, were found in myometrial preparations. One form was stimulated by oleate and insensitive to guanosine 5'-[gamma -thio] triphosphate (GTP[S]) . The second required ammonium sulphate to be detected and was stimulated b y GTP[S]. ADP-ribosylation factors (ARF1 and ARF6) and RhoA were immunodete cted in myometrial preparations. ARF1 and RhoA were present in the membrane and cytosolic fractions whereas ARF6 was detected exclusively in the membr ane fraction. A synthetic myristoylated peptide corresponding to the N-term inal domain of ARF6 [myrARF6((2-13))] totally abolished PLD activation in t he presence of ammonium sulphate and GTP[S], whereas myrARF1((2-17)) and th e inhibitory GDP/GTP-exchange factor, Rho GDI, did not. These data are cons istent with a membrane-bound ARF6-regulated PLD activity. Finally, the stim ulation of PLD by ARF6 was inhibited by AlF4- and this inhibition was count eracted by the fusion protein glutathione S-transferase-beta -adrenergic re ceptor kinase 1 (495-689) and by the QEHA peptide (from adenylate cyclase A CII), which act as G-protein beta gamma -subunit scavengers. It is conclude d that G-protein subunits beta gamma are involved in a pathway modulating P LD activation by ARF6, illustrating cross-talk between heterotrimeric and m onomeric G-proteins.