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

Abstract

Both protein kinase C and protein tyrosine kinases have been shown to be involved in phospholipase D (PLD) activation in intact rat myometrium [Le Stunff, Dokhac and Harbon (2000) J. Pharmacol. Exp. Ther. 292, 629-637]. In this study we assessed the involvement of monomeric G-proteins in PLD activation in a cell-free system derived from myometrial tissue. Both the PLD1 and PLD2 isoforms were detected. Two forms of PLD activity, essentially membrane-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 by GTP[S]. ADP-ribosylation factors (ARF1 and ARF6) and RhoA were immunodetected in myometrial preparations. ARF1 and RhoA were present in the membrane and cytosolic fractions whereas ARF6 was detected exclusively in the membrane fraction. A synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 [myrARF6((2-13))] totally abolished PLD activation in the presence of ammonium sulphate and GTP[S], whereas myrARF1((2-17)) and the inhibitory GDP/GTP-exchange factor, Rho GDI, did not. These data are consistent with a membrane-bound ARF6-regulated PLD activity. Finally, the stimulation of PLD by ARF6 was inhibited by AlF(-)(4) and this inhibition was counteracted by the fusion protein glutathione S-transferase-beta-adrenergic receptor kinase 1 (495-689) and by the QEHA peptide (from adenylate cyclase ACII), which act as G-protein betagamma-subunit scavengers. It is concluded that G-protein subunits betagamma are involved in a pathway modulating PLD activation by ARF6, illustrating cross-talk between heterotrimeric and monomeric G-proteins.

Activation of the Luteinizing Hormone/Choriogonadotropin Hormone Receptor Promotes ADP Ribosylation Factor 6 Activation in Porcine Ovarian Follicular Membranes

“…Synthetic peptides corresponding to the N terminus of the ARFs inhibit such ARF activities as intra-Golgi transport, the accumulation of coated vesicles and buds from Golgi preparations, phospholipase D (PLD) activation, and catecholamine secretion (30,(43)(44)(45)(46). We have shown that a pool of ␤arrestin1 is docked at the plasma membrane at a site distinct from the LH/CG receptor and that, upon LH/CG receptor activation, ␤arrestin1 binds to the LH/CG receptor resulting in an uncoupling of receptor and G s (1-3).…”

Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins

“…Possible pathways connecting the Ras superfamily GTPases and phospholipases have been reported. The GTPases RhoA and Arf 6 ( Table 1D ) are direct activators of PLD 2 ( 39, 40 ) from rat or human origin ( 41 ).…”

Ceramide inhibition of phospholipase D and its relationship to RhoA and ARF1 translocation in GTPγS-stimulated polymorphonuclear leukocytes

“…Both ARF1 and ARF6 have been implicated in PLD activation in phagocytic cells. 13,14 Sphingolipid signaling is involved in cell growth, differentiation, and apoptosis. 15 During PMN phagocytosis, plasma membraneassociated neutral sphingomyelinase is activated, resulting in accumulation of ceramide within the cell.…”