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*
- From INSERM, U-338 Biologie de la Communication Cellulaire, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France
Abstract
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 Gαo. 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.
Footnotes
-
↵* This work was supported by Association de la Recherche sur le Cancer ARC Grant 9101 and the Ligue Nationale Contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵‡ To whom correspondence should be addressed. Tel.: 33 3 88 45 67 13; Fax: 33 3 88 60 08 06; E-mail: bader{at}neurochem.u-strasbg.fr.
-
↵1 The abbreviations used are: PtdIns, phosphatidylinositol; gPtdIns-4-P, glycerophosphatidylinositol 4-phosphate; HPLC, high pressure liquid chromatography; PBS, phosphate-buffered saline; DTT, dithiothreitol; CHO, Chinese hamster ovary; DβH, dopamine-β-hydroxylase; PtdInsP, phosphatidylinositol monophosphate; PtdInsP2, phosphatidylinositol 4,5-bisphosphate; DTAF, dichlorotriazinyl aminofluorescein; TRITC, tetramethylrhodamine isothiocyanate; CGA, chromogranin A.
-
- Received January 14, 1998.
- Revision received April 13, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
