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Receptor phosphorylation in response to agonist stimulation is a key regulatory principle in signal transduction. Previous work has suggested the concerted action of protein kinase C (PKC) and a staurosporine-insensitive receptor kinase in homologous phosphorylation of the cholecystokinin (CCK) receptor in freshly isolated rat pancreatic acinar cells [Gates, Ulrich, Miller (1993) Am J Physiol 264:G840-G847]. The present study shows that down-regulation of PKC by prolonged (2 h) treatment with 0.1 muM 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly reduced basal CCK receptor phosphorylation as well as that induced by TPA (0.1 muM) and cholecystokinin-(26-33)-peptide amide (CCK8, 0.1 muM). The phosphorylation level reached was the same with both stimulants and equalled basal phosphorylation in untreated control cells. The absence of any CCK8-stimulated phosphorylation reflecting the activity of a putative staurosporine-insensitive receptor kinase raises the intriguing possibility that a basal level of PKC-mediated receptor phosphorylation is required for the action of such a receptor kinase. Immunoblot analysis revealed that the decrease in receptor phosphorylation coincided with a marked reduction of PKC-alpha and, to a lesser extent, PKC-epsilon. In addition, TPA-induced inhibition of the increase in cytosolic free Ca2+ concentration ([Ca2+]i) evoked by the high-affinity CCK receptor agonist JMV-180 was completely reversed. The time-course of recovery closely matched that of the reduction of PKC-alpha. Finally, digital imaging microscopy of individual PKC down-regulated cells revealed a marked increase in the duration of JMV-180-evoked oscillatory changes in [Ca2+]i. Taken together, the present findings are in agreement with the idea that PKC-alpha-mediated receptor phosphorylation leads to a shortening of the duration of the [Ca2+]i oscillations and eventually to inhibition of high-affinity Ca2+ signalling through the native CCK receptor in pancreatic acinar cells.

Recently a molecular model was proposed for the binding site of the antagonist 3 S (−)- N -(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1 H -1,4-benzodiazepine-3-yl)-1 H -indole-2-carboxamide (devazepide) on the cholecystokinin-A (CCK A ) receptor ( Van der Bent et al., 1994 . Drug Design Discov. 12, 129–148). Fifteen amino acids were identified, including hydrophilic ones such as Ser 139 , Asn 349 and Ser 379 , that might interact with the carboxamide moiety in devazepide. To provide mutational evidence for this model, wild-type and mutant receptors (S139A, N349A and S379A) were transiently expressed and compared with respect to the ability of devazepide to inhibit binding of radiolabelled cholecystokinin-(26–33)-peptide amide (CCK-8) and CCK-8-evoked Ca 2+ mobilization. The data presented suggest the involvement of the three residues in antagonist binding, although to a different extent. However, it does not seem likely that hydrogen bonds are the driving force in view of the relatively minor changes in receptor affinity and activity. © 1997 Elsevier Science B.V.

Digital-imaging microscopy of Fura-2-loaded Chinese hamster ovary cells, stably expressing the cholecystokinin-A receptor, revealed that both the C-terminal octapeptide of cholecystokinin (CCKB) and its analogue JMV-180, which acts as an agonist at the high-affinity CCK-A receptor, recruited CHO-CCK-A cells dose-dependently in terms of receptor-mediated Ca2+ mobilization. Agonist-evoked cell recruitment was inhibited by short-term (10 min) pretreatment with 0.1 microM 12-O-tetradecanoylphorbol 13-acetate (TPA). In the case of CCKB, inhibition was overcome with increasing of the hormone concentration. In contrast, increasing of the JMV-180 concentration did not reverse the inhibitory action of TPA. CHO-CCK-A cells gradually regained their responsiveness to JMV-180 during prolonged TPA pretreatment. Complete recovery was observed within 1 h following addition of TPA. Western blot analysis using antibodies directed against the various PKC isotypes revealed that recovery was paralleled by the disappearance of PKC-alpha. Surprisingly, short-term (10 min) TPA pretreatment virtually completely inhibited the formation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in response to CCKB concentrations at which the effect on cell recruitment was not affected by short term phorbol ester pretreatment. Together with the finding that JMV-180 does not detectably increase the cellular Ins(1,4,5)P3 content, this suggests a large overproduction of this second messenger by CCKB concentrations supramaximal in terms of cell recruitment. Again, full responsiveness was observed after long term TPA pretreatment. The present observations are in agreement with the idea that in CHO-CCK-A cells activation of PKC-alpha leads to inhibition of agonist-evoked Ca2+ mobilization through inhibition of receptor-stimulated Ins(1,4,5)P3 formation.