Distinct heterotrimeric GTP-binding-proteins act in series to control the exocytotic machinery in chromaffin cells.
Regulated exocytosis requires both calcium and MgATP. Although the biochemical events responsible for ATP-dependent calcium-activated secretion have not been elucidated yet, some progress has been made in determining the relative order of the ATP- and calcium-dependent steps. Studies on permeabilized secretory cells have shown that MgATP acts before calcium and maintains the secretory apparatus in a "primed" state. In this paper, we examine the possible role of heterotrimeric G-proteins in these two steps of exocytosis in permeabilized chromaffin cells. We show that mastoparan and other activators of heterotrimeric G-proteins inhibit the MgATP-dependent reaction, but stimulate the late calcium-dependent step of exocytosis. Non-hydrolyzable GTP analogues (GTP-gamma-S and GMP-PNP) mimic the dual effects of mastoparan on secretion, but with different potencies, suggesting the involvement of two distinct heterotrimeric G-proteins in regulated exocytosis. GPAnt-2, a substance P related peptide known to inhibit the stimulation of Gi and Go by mastoparan, reverses, in a dose-dependent manner, both the inhibitory and stimulatory effects of mastoparan on secretion. These results indicate that two distinct heterotrimeric G-proteins from the Gi/o family may act in series in the exocytotic pathway in chromaffin cells: one controls the ATP-dependent priming step, whereas the second is involved in the late calcium-dependent fusion step which does not require ATP.
Vitale
N
N
Institut National de la Santé et de la Recherche Médicale, U388 Biologie de la Communication Cellulaire, Strasbourg, France.
Aunis
D
D
Bader
M F
MF
D000255:Adenosine Triphosphate / Q000494:pharmacology; D017607:Aluminum Compounds / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002838:Chromaffin System / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005459:Fluorides / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009638:Norepinephrine / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology; D010455:Peptides; D011487:Protein Conformation; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D013373:Substance P / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D014862:Wasp Venoms / Q000494:pharmacology
D017607:Aluminum Compounds; C090711:GPAnt-2; D036341:Intercellular Signaling Peptides and Proteins; D009842:Oligopeptides; D010455:Peptides; D014862:Wasp Venoms; D013373:Substance P; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C037500:mastoparan; D000255:Adenosine Triphosphate; D019204:GTP-Binding Proteins; D005459:Fluorides; D002118:Calcium; D011761:Pyrrolidonecarboxylic Acid; D009638:Norepinephrine; C032311:aluminum fluoride