THE ROLE OF THE CHOLECYSTOKININ-B GASTRIN RECEPTOR TRANSMEMBRANE DOMAINS IN DETERMINING AFFINITY FOR SUBTYPE-SELECTIVE LIGANDS

We have examined the role of transmembrane domain amino acids in confe rring subtype-selective ligand affinity to the human cholecystokinin-B (CCK-B)/gastrin receptor. Fifty-eight residues were sequentially repl aced by the corresponding amino acids from the pharmacologically disti nct CCK-A receptor subtype. I-125-CCK-8 competition binding experiment s were performed to compare all mutant CCK-B/gastrin receptor construc ts with the wild type control. Affinities for the nonselective agonist , CCK-8, as well as the subtype-selective peptide (gastrin), peptide d erived (PD135,158), and nonpeptide (L365,260 and L364,718) ligands wer e assessed. Ah of the mutants retained relatively high affinity for CC K-8, suggesting that the tertiary structure of these receptors was wel l maintained. Only eight of the amino acid substitutions had a signifi cant effect on subtype selective binding. When compared with the wild type, single point mutations in the CCK-B/gastrin receptor decreased a ffinity for gastrin, L365,260, and PD135;158 up to 17-, 23-, and 61-fo ld, respectively. In contrast, the affinity for L364,718 increased up to 63-fold. None of the single amino acid substitutions, however, was sufficient to fully account for the subtype selectivity of any tested compound. Rather, CCK-B/gastrin receptor affinity appears to be influe nced by multiple residues acting in concert. The 8 pharmacologically i mportant amino acids cluster in the portion of the transmembrane domai ns adjacent to the cell surface. The spatial orientation of these resi dues was analyzed with a rhodopsin based three dimensional model of G- protein coupled receptor structure (Baldwin, J. M. (1993) EMBO J. 12, 1693-1703). This model predicts that the 8 crucial residues project in to a putative ligand pocket, similar to the one which is well establis hed for biogenic amine receptors (Caron, M. G., and Lefkowitz, R. J. ( 1993) Recent Prog. Horm. Res. 48, 277-290; Strader, C. D., Sigal, I. S ., and Dixon, R. A. (1989) Trends Pharmacol. Sci. 10, Dec. Suppl., 26- 30).