G(alpha)(i3) in pancreatic zymogen granules participates in vesicular fusion

Earlier studies indicated that a G(i)-like protein localized in pancreatic zymogen granule (ZG) membrane mediates vesicle swelling, and is a potentially important prerequisite for vesicle fusion at the cell plasma membrane (PM) [Jena et al. (1997) Proc. Natl. Acad. Sci. USA 94, 13317-13322]. In the present study, we demonstrate the presence of G(alpha)(i3) immunoreactivity in ZGs of rat exocrine pancreas using immunoblot assays, light and electron immunomicroscopy. Since GTP has been implicated in the fusion of isolated ZG with PM fractions [Nadin et al. (1989) J. Cell Biol. 109, 2801-2808], the potential role of ZG-associated G(alpha)(i3) was investigated. Immunoblot assays demonstrate an increase in G(alpha)(i3) protein in ZGs isolated from carbamylcholine stimulated pancreas. Thin layer chromatography shows an increase in GTP hydrolysis by GTPase in ZGs isolated from stimulated compared to resting pancreas. In vitro fusion assays demonstrate that ZGs isolated from carbamylcholine-stimulated pancreatic lobules fuse with the PM at a greater potency in the presence of GTP, mastoparan (G protein agonist) and its analogue mas7. Furthermore, G(alpha)(i3)-specific a recombinant GAIP (G alpha interacting protein), potentiates ZG-PM fusion in the presence of GTP but not in presence of the non-hydrolyzable GTP analogue Gpp(NH)p. Our immunoblot analysis demonstrates the recruitment of Galpha(i3) immunoreactivity to ZG from stimulated acinar cells, and these isolated ZGs are more potent and efficient in fusing with plasma membrane fractions, suggesting the possible involvement of G(alpha)(i3) in ZG-PM fusion. The participation of ZG-associated G(alpha)(i3) in ZG-PM fusion is further confirmed by the influence of the G(alpha)(i3)-specific GAIP, which is known to interact specifically with G(alpha)(i3), and not with G(alpha)(i2) or G(alpha)(q) [DeVries et al. (1995) Proc. Natl. Acad. Sci. USA 92, 11916-11920]. Additionally, our data suggest that GTP hydrolysis is a requirement for ZG-PM fusion since GAIP in the presence of Gpp(NH)p shows little or no effect on fusion, whereas GAIP in the presence of GTP significantly potentiates ZG-PM fusion. Our studies suggest a possible role for ZG-associated G(alpha)(i3) in ZG-PM fusion.