MULTIPLE TRIMERIC G-PROTEINS ON THE TRANS-GOLGI NETWORK EXERT STIMULATORY AND INHIBITORY EFFECTS ON SECRETORY VESICLE FORMATION

The role of heterotrimeric G-proteins on the formation of constitutive secretory vesicles (CSVs) and immature secretory granules (ISGs) from the trans-Golgi network (TGN) of PC12 cells was investigated. Using immunofluorescence and subcellular fractionation in conjunction with immunoblotting or ADP-ribosylation by either pertussis toxin or cholera toxin, TGN membranes were found to contain not only several alphai/alphao G-protein subunits including apparently alphai3, but also alphas. Pertussis toxin treatment of cells, which resulted in the stoichiometric ADP-ribosylation of alphai/alphao, a modification known to prevent their coupling to receptors, led to the stimulation of cell-free CSV and ISG formation, suggesting the presence of a guanine nucleotide exchange factor for alphai/alphao on the TGN. Mastoparan-7, a peptide known to mimic an activated receptor and to stimulate nucleotide exchange on alphai/alphao, inhibited cell-free vesicle formation, an effect abolished by pertussis toxin. In contrast, activation of alphas by cholera toxin treatment of cells resulted in a stimulation of cell-free CSV and ISG formation. This stimulation could be reversed when the alpha subunits not activated by cholera toxin, i.e. alphai/alphao, were activated by GTP(gamma)S and [AlF4]-. Our results show that both inhibitory and stimulatory trimeric G-proteins on the TGN participate in the regulation of secretory vesicle formation.