Differential distribution of a subunits and βγ subunits of heterotrimeric G proteins on golgi membranes of the exocrine pancreas

Abstract

Heterotrimeric G proteins are well known to be involved in signaling via plasma membrane (PM) receptors. Recent data indicate that heterotrimeric G proteins are also present on intracellular membranes and may regulate vesicular transport along the exocytic pathway. We have used subcellular fractionation and immunocytochemical localization to investigate the distribution of Gα and Gβγ subunits in the rat exocrine pancreas which is highly specialized for protein secretion. We show that Gαs, Gαi3 and Gαq/11 are present in Golgi fractions which are >95% devoid of PM. Removal of residual PM by absorption on wheat germ agglutinin (WGA) did not deplete Gα subunits. Gαs was largely restricted to TGN-enriched fractions by immunoblotting, whereas Gαi3 and Gαq/11 were broadly distributed across Golgi fractions. Gαs did not colocalize with TGN38 or caveolin, suggesting that Gαs is associated with a distinct population of membranes. Gβ subunits were barely detectable in purified Golgi fractions. By immunofluorescence and immunogold labeling, Gβ subunits were detected on PM but not on Golgi membranes, whereas Gαs and Gαi3 were readily detected on both Golgi and PM. Gα and Gβ subunits were not found on membranes of zymogen granules. These data indicate that Gαs, Gαq/11, and Gαi3 associate with Golgi membranes independent of Gβ subunits and have distinctive distributions within the Golgi stack. Gβ subunits are thought to lock Gα in the GDP-bound form, prevent it from activating its effector, and assist in anchoring it to the PM. Therefore the presence of free Gα subunits on Golgi membranes has several important functional implications: it suggests that Gα subunits associated with Golgi membranes are in the active, GTP-bound form or are bound to some other unidentified protein(s) which can substitute for Gβγ subunits. It further implies that Gα subunits are tethered to Golgi membranes by posttranslational modifications (e.g., palmitoylation) or by binding to another protein(s).