On separation of rat pancreatic plasma membrane proteins by two-dimensional gel electrophoresis, 15 GTP-binding protein (G-protein) alpha-subunits could be detected immunochemically using an alpha common antibody. These consisted of five 48 kDa proteins (pI 5.70, 5.80, 5.90, 6.10 and 6.25) and five 45 kDa proteins (pI 5.90, 6.05, 6.25, 6.30 and 6.70), presumably corresponding to low- and high-molecular mass forms of the Gs-protein, as well as three 40/41 kDa proteins (pI 5.50, 5.70 and 6.00) and two 39 kDa proteins (pI 5.50 and 6.00). All of these proteins except for the more acidic 39 kDa protein were ADP-ribosylated by cholera toxin (CT). In addition, the three 40/41 kDa proteins and the more alkaline 39 kDa protein were also ADP-ribosylated by pertussis toxin (PT). CT- and PT-induced ADP-ribosylation changed the pI values of G-protein alpha-subunits by 0.2 pI units to more acidic values. Preincubation of isolated pancreatic membranes with cholecystokinin octapeptide (CCK-OP), which stimulates phospholipase C in acinar cells, decreased CT-induced as well as PT-induced ADP-ribosylation of the three 40/41 kDa proteins, whereas CT-induced ADP-ribosylation of one 45 kDa (pI 5.80) and all 48 kDa proteins was enhanced in the presence of CCK. Carbachol, another stimulant of phospholipase C, had no effect. The three 40/41 kDa proteins and one 48 kDa protein could be labelled with the GTP analogue [alpha-32P]GTP-gamma-azidoanilide. CCK, but not carbachol, stimulated incorporation of the GTP analogue into all of these four proteins. Using different anti-peptide antisera specific for alpha-subunits of G-proteins we identified the three 40/41 kDa Gi-proteins as Gi1 (pI 6.00), Gi2 (pI 5.50) and Gi3 (pI 5.70). The Gi3-protein was found to be the major Gi-protein of pancreatic plasma membranes. One of the 39 kDa proteins (pI 6.0) was identified as Go. These results indicate that CCK receptors functionally interact with six Gs-proteins and with Gi1, Gi2 and Gi3-proteins. Since evidence suggests that a 40/41 kDa CT substrate is involved in the stimulation of phospholipase C in pancreatic acinar cells, it is likely that one, two or all three 40/41 kDa Gi-proteins are involved in the coupling of CCK receptors with phospholipase C.

We have studied the involvement of GTP-binding proteins in the stimulation of phospholipase C from rat pancreatic acinar cells. Pretreatment of permeabilized cells with activated cholera toxin inhibited both cholecystokinin-octapeptide (CCK-OP) and GTPyS but not carbachol (CCh)-induced production of inositol trisphosphate. Pertussis toxin had no effect. Neither vasoactive intestinal polypeptide, a stimulator, of adenylyl cyclase, nor the CAMP-analogue, 8-bromo CAMP, mimicked the inhibitory effect of cholera toxin on agonist-induced phospholipase C activation. This indicates that inhibition by cholera toxin could not be attributed to a direct interaction of cholera toxin activated G, with phospholipase C or to an elevation of CAMP. In isolated rat pancreatic plasma membranes cholera toxin ADP-ribosylated a 40 kDa protein, which was inhibited by CCK-OP but not by CCh. We conclude from these data that both CCK-and muscarinic acetylcholine receptors functionally couple to phospholipase C by two different GTP-binding proteins.

We have detected high- and small-molecular-weight GTP-binding proteins (G- and smg-proteins, respectively) in zymogen granule membranes (ZGM) of rat pancreatic acinar cells. G_i-proteins with molecular weights of 40/41 kDa were detected by pertussis-toxin induced ADP ribosylation and by [α^-32P] GTP photoaffinity labelling in ZGM. Smg-proteins were analysed by [α^-32P] GTP binding, ADP ribosylation with Clostridium botulinum ADP ribosyltransferase C3 and immunoblotting following one- and two-dimensional gel electrophoresis. Two-dimensional separation of ZGM proteins revealed the presence of up to 30 differently charged smg-proteins with molecular masses between 18 and 27 kDa in ZGM. A monoclonal antibody, which recognizes both rab3A and rab3B (clone 42.1), identified 2 proteins with isoelectric points (pI) of 4.89 and 4.96 as proteins closely related to rab3. These proteins were mainly present in ZGM and to a slight extent also in plasma membranes (PM), microsomal membranes (MM) and cytosol. An antibody, which recognizes rab3A only (clone 42.2), did not react with pancreatic proteins. An anti-rap IB antibody recognized a 23 kDa protein localized in the ZGM, PM and to a lesser extent in MM. A monoclonal antibody against all 3 known ras proteins (p21^Ha-ras, p21^ĸi-ras, p21^N-ras) identified ras proteins only in PM and not in ZGM or other membranes. C3-induced ADP ribosylation as indication for the presence of rho and/or rac proteins was detected in PM, MM and cytosol but not in ZGM. The presence of multiple GTP-binding proteins in ZGM suggests a possible role of these proteins in the regulation of exocytosis in pancreatic acinar cells.

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