IDENTIFICATION OF A POTENTIAL EFFECTOR PATHWAY FOR THE TRIMERIC G(O) PROTEIN ASSOCIATED WITH SECRETORY GRANULES - G(O) STIMULATES A GRANULE-BOUND PHOSPHATIDYLINOSITOL 4-KINASE BY ACTIVATING RHOA IN CHROMAFFIN CELLS

Besides having a role in signal transduction, heterotrimeric G protein s may be involved in membrane trafficking events. In chromaffin cells, G, is associated with secretory organelles, and its activation inhibi ts the ATP-dependent priming of exocytosis, By using permeabilized cel ls, we previously described that the control exerted by the granule-bo und G, on exocytosis may be related to effects on the cortical actin n etwork through a sequence possibly involving Rho, To provide further i nsight into the function of Rho in exocytosis, we focus here on its in tracellular localization in chromaffin cells. By immunoreplica analysi s, immunoprecipitation, and confocal immunofluorescence, we found that RhoA is specifically associated with the membrane of secretory chroma ffin granules, Parallel subcellular fractionation experiments revealed the occurrence of a mastoparan-stimulated phosphatidylinositol 4-kina se activity in purified chromaffin granule membranes. This stimulatory effect of mastoparan was mimicked by GAP-43, an activator of the gran ule-associated G,, and specifically inhibited by antibodies against G alpha(0). In addition, Clostridium botulinum C3 exoenzyme completely b locked the activation of phosphatidylinositol 4-kinase by mastoparan. We propose that the control exerted by G, on peripheral actin and exoc ytosis is related to the activation of a downstream RhoA-dependent pho sphatidylinositol 4-kinase associated with the membrane of secretory g ranules.