Identification of a potential effector pathway for the trimeric Go protein associated with secretory granules. Go stimulates a granule-bound phosphatidylinositol 4-kinase by activating RhoA in chromaffin cells.
Besides having a role in signal transduction, heterotrimeric G proteins may be involved in membrane trafficking events. In chromaffin cells, Go is associated with secretory organelles, and its activation inhibits the ATP-dependent priming of exocytosis. By using permeabilized cells, we previously described that the control exerted by the granule-bound Go on exocytosis may be related to effects on the cortical actin network through a sequence possibly involving Rho. To provide further insight into the function of Rho in exocytosis, we focus here on its intracellular localization in chromaffin cells. By immunoreplica analysis, immunoprecipitation, and confocal immunofluorescence, we found that RhoA is specifically associated with the membrane of secretory chromaffin granules. Parallel subcellular fractionation experiments revealed the occurrence of a mastoparan-stimulated phosphatidylinositol 4-kinase activity in purified chromaffin granule membranes. This stimulatory effect of mastoparan was mimicked by GAP-43, an activator of the granule-associated Go, and specifically inhibited by antibodies against Galphao. In addition, Clostridium botulinum C3 exoenzyme completely blocked the activation of phosphatidylinositol 4-kinase by mastoparan. We propose that the control exerted by Go on peripheral actin and exocytosis is related to the activation of a downstream RhoA-dependent phosphatidylinositol 4-kinase associated with the membrane of secretory granules.
Gasman
S
S
INSERM, U-338 Biologie de la Communication Cellulaire, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France.
Chasserot-Golaz
S
S
Hubert
P
P
Aunis
D
D
Bader
M F
MF
D019870:1-Phosphatidylinositol 4-Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D036002:ADP Ribose Transferases / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001905:Botulinum Toxins; D002384:Catalysis; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000201:enzymology / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis; D019204:GTP-Binding Proteins / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D014862:Wasp Venoms / Q000494:pharmacology
D004791:Enzyme Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D014862:Wasp Venoms; D000246:Adenosine Diphosphate Ribose; C037500:mastoparan; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D019870:1-Phosphatidylinositol 4-Kinase; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins