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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 alpha i/alpha o G-protein subunits including apparently alpha i3, but also alpha s. Pertussis toxin treatment of cells, which resulted in the stoichiometric ADP-ribosylation of alpha i/alpha o, 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 alpha i/alpha o on the TGN. Mastoparan-7, a peptide known to mimic an activated receptor and to stimulate nucleotide exchange on alpha i/alpha o, inhibited cell-free vesicle formation, an effect abolished by pertussis toxin. In contrast, activation of alpha s 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. alpha i/alpha o, were activated by GTP gamma S and [AIF4]-. Our results show that both inhibitory and stimulatory trimeric G-proteins on the TGN participate in the regulation of secretory vesicle formation.

Type

Journal article

Journal

EMBO J

Publication Date

12/1992

Volume

11

Pages

4795 - 4804

Keywords

Adenosine Diphosphate Ribose, Animals, Binding Sites, Blotting, Western, Cell-Free System, Cholera Toxin, Cytoplasmic Granules, Fluorescent Antibody Technique, GTP-Binding Proteins, Golgi Apparatus, PC12 Cells, Pertussis Toxin, Virulence Factors, Bordetella