title: Phospholipase D in rat myometrium: occurrence of a membrane-bound ARF6 (ADP-ribosylation factor 6)-regulated activity controlled by betagamma 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.
authors.affiliation: Signalisation et Régulations Cellulaires, CNRS UMR 8619, Bâtiment 432, Université Paris-Sud, 91405 Orsay Cedex, France.
authors.forename: H
authors.initials: H
authors.lastname: Le Stunff
authors.forename: L
authors.initials: L
authors.lastname: Dokhac
authors.forename: S
authors.initials: S
authors.lastname: Bourgoin
authors.forename: M F
authors.initials: MF
authors.lastname: Bader
authors.forename: S
authors.initials: S
authors.lastname: Harbon
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D017607:Aluminum Compounds / Q000494:pharmacology; D000645:Ammonium Sulfate; D000818:Animals; D004789:Enzyme Activation; D005260:Female; D005459:Fluorides / Q000494:pharmacology; D020962:Heterotrimeric GTP-Binding Proteins / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism*; D009215:Myometrium / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D019301:Oleic Acid / Q000494:pharmacology; D010739:Phospholipase D / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D000089982:ADP-Ribosylation Factor 6; D017607:Aluminum Compounds; D008565:Membrane Proteins; D019301:Oleic Acid; D010739:Phospholipase D; D020962:Heterotrimeric GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; C000715349:Arf6 protein, rat; D005459:Fluorides; D000645:Ammonium Sulfate; C032311:aluminum fluoride