GASTRIN(13) AND THE C-TERMINAL OCTAPEPTIDE OF CHOLECYSTOKININ ARE DIFFERENTLY COUPLED TO G-PROTEINS IN GUINEA-PIG BRAIN MEMBRANES

In the course of our study concerning gastrin and cholecystokinin (CCK) receptors, we synthesized and characterized a labelled gastrin ligand, [I-125]BH[Leu(15)]gastrin-(5-17) (3-(3-[I-125]iodo-4-hydroxyphenyl)propionyl[Leu(15)]gastrin-(5-17)). On isolated canine fundic mucosal cells and human Jurkat lymphoblastic cell line, known to express CCKB/gastrin receptors, the binding experiments performed indicate that [I-125]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 CCKB and CCKA receptors, [I-125]BH[Leu(15)]gastrin-(5-17) binds to a single class of high-affinity binding sites in a saturable and specific manner. [I-125]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 [I-125]BHCCK8 (CCK8, the C-terminal octapeptide of cholecystokinin). The apparent affinities of CCK analogues and selective CCK receptor antagonists L-365,260 and MK-329 for the sites labelled by both probes were in accordance with a CCKB-like profile. Association-dissociation kinetics of [I-125]BH[Leu(15)]gastrin-(5-17) and [I-125]BHCCK8 were performed and compared. They showed that [I-125]BHCCK8 equilibrated more slowly than [I-125]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 [I-125]BH[Leu(15)]gastrin-(5-17) and [I-125]BHCCK8 binding. Binding experiments in the presence of stable analogues of GTP showed a different behaviour between [I-125]BH[Leu(15)]gastrin-(5-17) and [I-125]BHCCK8. GTP gamma S strongly decreased [I-125]BH[Leu(15)]gastrin-(5-17) binding whereas it weakly affected [I-125]BHCCK8 binding. The 5'-adenylylimidodiphosphate was found to exert a similar effect than GTP gamma S on gastrin and CCK8 binding. The results of binding studies carried out with [I-125]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.