Gastrin13 and the C-terminal octapeptide of cholecystokinin are differently coupled to G-proteins in guinea-pig brain membranes

Abstract In the course of our study concerning gastrin and cholecystokinin (CCK) receptors, we synthesized and characterized a labelled gastrin ligand, [ 125 I]BH[Leu 15 ]gastrin-(5–17) (3-(3-[ 125 I]iodo-4-hydroxyphenyl)propionyl[Leu 15 ]gastrin-(5–17)). On isolated canine fundic mucosal cells and human Jurkat lymphoblastic cell line, known to express CCK B /gastrin receptors, the binding experiments performed indicate that [ 125 I]BH[Leu 15 ]gastrin-(5–17) provides a convenient biologically active ligand for cholecystokinin/gastrin receptor studies. We showed in this study that, on guinea-pig brain membranes known to possess CCK B and CCK A receptors, [ 125 I]BH[Leu 15 ]gastrin-(5–17) binds to a single class of high-affinity binding sites in a saturable and specific manner. [ 125 I]BH[Leu 15 ]gastrin-(5–17) interacts with guinea-pig brain membranes with a maximal binding capacity that is about three-fold lower than that of [ 125 I]BHCCK 8 (CCK 8 , the C-terminal octapeptide of cholecystokinin). The apparent affinities of CCK analogoues and selective CCK receptor antagonists L-365,260 and MK-329 for the sites labelled by both probes were in accordance with a CCK B -like profile. Association-dissociation kinetics of [ 125 I]BH[Leu 15 ]gastrin-(5–17) and [ 125 I]BHCCK 8 were performed and compared. They showed that [ 125 I]BHCCK 8 equilibrated more slowly than [ 125 I]BH[Leu 15 ]gastrin-(5–17). The effects of pH, monovalent and divalent cations on binding of both probes were investigated. The results obtained did not indicate strong differences between [ 125 I]BH[Leu 15 ]gastrin-(5–17) and [ 125 I]BHCCK 8 binding. Binding experiments in the presence of stable analogues of GTP showed a different behaviour between [ 125 I]BH[Leu 15 ]gastrin-(5–17) and [ 125 I]BHCCK 8 . GTPγS strongly decreased [ 125 I]BH[Leu 15 ]gastrin-(5–17) binding whereas it weakly affected [ 125 I]BHCCK 8 binding. The 5′-adenylylimidodiphosphate was found to exert a similar effect than GTPγS on gastrin and CCK 8 binding. The results of binding studies carried out with [ 125 I]BH[Leu 15 ]gastrin-(5–17) showed that gastrin binds specifically to guinea-pig brain membranes. Furthermore, the different effects of guanyl nucleotides on gastrin and CCK binding strongly suggested that gastrin and CCK trigger a differential G protein coupling through the same binding sites.