Fiche publication
Date publication
mai 2000
Journal
The Journal of biological chemistry
Auteurs
Membres identifiés du Cancéropôle Est :
Dr BADER Marie-France
,
Dr VITALE Nicolas
Tous les auteurs :
Caumont AS, Vitale N, Gensse M, Galas MC, Casanova JE, Bader MF
Lien Pubmed
Résumé
ADP-ribosylation factors (ARFs) constitute a family of structurally related proteins that forms a subset of the Ras superfamily of regulatory GTP-binding proteins. Like other GTPases, activation of ARFs is facilitated by specific guanine nucleotide exchange factors (GEFs). In chromaffin cells, ARF6 is associated with the membrane of secretory granules. Stimulation of intact cells or direct elevation of cytosolic calcium in permeabilized cells triggers the rapid translocation of ARF6 to the plasma membrane and the concomitant activation of phospholipase D (PLD) in the plasma membrane. Both calcium-evoked PLD activation and catecholamine secretion in permeabilized cells are strongly inhibited by a synthetic peptide corresponding to the N-terminal domain of ARF6, suggesting that the ARF6-dependent PLD activation near the exocytotic sites represents a key event in the exocytotic reaction in chromaffin cells. In the present study, we demonstrate the occurrence of a brefeldin A-insensitive ARF6-GEF activity in the plasma membrane and in the cytosol of chromaffin cells. Furthermore, reverse transcriptase-polymerase chain reaction and immunoreplica analysis indicate that ARNO, a member of the brefeldin A-insensitive ARF-GEF family, is expressed and predominantly localized in the cytosol and in the plasma membrane of chromaffin cells. Using permeabilized chromaffin cells, we found that the introduction of anti-ARNO antibodies into the cytosol inhibits, in a dose-dependent manner, both PLD activation and catecholamine secretion in calcium-stimulated cells. Furthermore, co-expression in PC12 cells of a catalytically inactive ARNO mutant with human growth hormone as a marker of secretory granules in transfected cells resulted in a 50% inhibition of growth hormone secretion evoked by depolarization with high K(+). The possibility that the plasma membrane-associated ARNO participates in the exocytotic pathway by activating ARF6 and downstream PLD is discussed.
Mots clés
ADP-Ribosylation Factors, metabolism, Adrenal Glands, drug effects, Animals, Antibodies, pharmacology, Brefeldin A, pharmacology, Calcium, pharmacology, Catecholamines, metabolism, Cattle, Cell Membrane, metabolism, Chromaffin Cells, metabolism, Cytoplasmic Granules, drug effects, Cytosol, metabolism, Enzyme Activation, drug effects, Exocytosis, drug effects, GTPase-Activating Proteins, genetics, Guanine Nucleotide Exchange Factors, metabolism, PC12 Cells, Peptide Fragments, pharmacology, Phospholipase D, metabolism, RNA, Messenger, metabolism, Rats
Référence
J. Biol. Chem.. 2000 May;275(21):15637-44
