Cholecystokinin activates Gi1-, Gi2-, Gi3- and several Gs-proteins in rat pancreatic acinar cells

Abstract

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.

Characterization of adenosine receptors in brush‐border membranes from pig kidney

“…In fact, the order of potency for A3 receptors described by Ribeiro & Sebastiao (1986) is: R-PIA = cyclohexyladenosine = NECA> chloradenosine, with S-PIA usually being less potent than chloroadenosine. From the similar rank order of potency agonists reported here ( (Gil,Gi2,Gj3) which are linked to cholecystokinin receptors and which may mediate changes in phospholipase C activity (Schnefel et al, 1990). Thus adenosine might mediate changes in phosphatidylinositol metabolism through A3 receptors and cholera-and pertussis-sensitive G proteins.…”

Study of the states and populations of the rat pancreatic cholecystokinin receptor using the full peptide antagonist JMV 179

“…Indeed, numerous previous reports have documented that analogs of GTP increase the dissociation rate of CCK with the pancreatic CCK receptor and that receptor activation involves coupling to GTP-binding proteins [7,[25][26][27][28]. Furthermore, the primary structure of cloned CCK receptor suggests its membership in the family of guanine-nucleotidebinding regulatory protein coupled receptors [6].…”

“…[Thr28, Ahx31]CCK- (25)(26)(27)(28)(29)(30)(31)(32)(33) and human des-S0,H-gastrin were synthesized by Luis Moroder (Max-Planck-Institut fur Biochemie, Munchen, Germany) as described in [15,16] ; partial and full CCK antagonist (JMV 180 and JMV 179) ( Fig. 1) were synthesized as described in [17,14).…”

“…Glu-Glu-Ala-Tyr(S0,H)-Gly-Trp-Met-Asp-Phe-NH, Heptapeptide of gastrin; (2) octapeptide of cholecystokinin; (3) [Thr28, Ahx31]CCK- (25)(26)(27)(28)(29)(30)(31)(32)(33); (4) JMV 179, (5) JMV 180. The peptide (3) is an agonist, (4) is a full antagonist and (5) has both agonist and antagonist activities.…”

A Role for Phosphorylation of Inositol 1,4,5-Trisphosphate Receptors in Defining Calcium Signals Induced by Peptide Agonists in Pancreatic Acinar Cells

“…Furthermore, PKA-mediated phosphorylation of this InsP 3 R can modulate the sensitivity of the receptor to InsP 3 , ultimately resulting in a diminished ability of the channel to release Ca 2ϩ (26). Although CCK is known to couple to G␣ s in pancreatic acinar cells (27), it is not known whether this can account for the phosphorylation of InsP 3 R produced upon CCK stimulation or, in turn, for the specific kinetics of [Ca 2ϩ ] c oscillations observed upon CCK stimulation. The goal of the present study was to determine whether agonist-induced phosphorylation of InsP 3 R contributes to the generation of agonist-specific patterns of [Ca 2ϩ ] c oscillations in pancreatic acinar cells.…”