The CCK‐B/Gastrin Receptor

Gastrin and cholecystokinin (CCK) share an identical carboxy-terminal pentapeptide amide, a domain critical for receptor binding. Based on the binding of agonists as well as antagonists, receptors within the gastrin/CCK family have traditionally been divided into two major subtypes: CCK-A and CCK-B/gastrin.l The CCK-A receptor is found in pancreas, gallbladder, and isolated brain nuclei. CCK-B/gastrin receptors are present on smooth muscle cells and parietal cells (“gastrin” receptors) and are the predominant brain CCK receptor (“CCK-B”). Although CCK-A and CCK-B/gastrin receptors can easily be distinguished on the basis of both agonist and antagonist binding, small differences in agonist binding have created controversy regarding the existence of distinct “CCK-B” and “gastrin” receptor subtypes. Our data suggest that the CCK-B and gastrin receptors represent a single subtype2 and that species differences underlie part of the heterogeneity in ligand binding reported within the CCK-B/gastrin receptor ~ubclass.~ As a first step towards understanding receptor function at a molecular level, we used a COS-7 cell expression cloning system to isolate a cDNA encoding the parietal cell gastrin receptor (FIG. 1). The first requirement in this cloning strategy was to isolate mRNA from cells with abundant gastrin receptors. On the basis of A. H. Soll’s earlier observations,4 we used an enriched (95%) preparation of canine parietal cells as starting material. From these cells, size selected (> 1.5 kb) mRNA was isolated and used to make a cDNA expression library in the vector pcDNAl (Invitrogen). Pools of library DNA, representing 3,000-10,000 primary recombinants, were transfected into COS-7 cells. Forty-eight hours after transfection, cells were incubated with IzI CCK-8 as well as with lSI D-Tyr-Gly-[(Nle28*31)-CCK-26-33], a CCK analog that includes a free amino group available for chemical cross-linking? Inasmuch as COS-7 cells do not normally have gastrin or CCK receptors, only cells expressing the recombinant gastrin receptor bound the radioligand. Following the binding assay, excess radioligand was washed away, and the cells were fixed in glutaraldehyde. Slides were then dipped in photoemulsion, exposed in the dark for 72 hours, developed, and examined under the microscope. After two million primary recombinants were screened, a pool expressing the