CHOLECYSTOKININ ACTIVATES GI1-PROTEINS, GI2-PROTEINS, GI3-PROTEINS AND SEVERAL GS-PROTEINS IN RAT PANCREATIC ACINAR-CELLS

On separation of rat pancreatic plasma membrane proteins by two-dimensional gel electrophoresis, 15 GTP-binding protein (G-protein) α-subunits could be detected immunochemically using an α(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 G(s)-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 α-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 [α-32P]GTP-γ-azidoanilide. CCK, but not carbachol, stimulated incorporation of the GTP analogue into all of these four proteins. Using different anti-peptide antisera specific for α-subunits of G-proteins we identified the three 40/41 kDa G(i)-proteins as G(i)1 (pI 6.00), G(i)2 (pI 5.50) and G(i)3 (pI 5.70). The G(i)3-protein was found to be the major G(i)-protein of pancreatic plasma membranes. One of the 39 kDa proteins (pI 6.0) was identified as G(o). These results indicate that CCK receptors functionally interact with six G(s)-proteins and with G(i)1, G(i)2 and G(i)3-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 G(i)-proteins are involved in the coupling of CCK receptors with phospholipase C.