The brain cholecystokinin-B/gastrin receptor (CCK-B/gastrin) has been implicated in mediating anxiety, panic attacks, satiety, and the perception of pain. The canine and human CCK-B/gastrin receptors share 90% amino-acid identity and have similar agonist affinities. These receptors can be selectively blocked by the non-peptide benzodiazepine-based antagonists L365260 (ref. 8) and L364718 (ref. 9); however, the binding of these antagonists to the human and canine receptors differs by up to 20-fold, resulting in a reversal of affinity rank order. Here we report the identification of a single amino acid in the sixth transmembrane domain of the CCK-B/gastrin receptor that corresponds to valine 319 in the human homologue and which is critical in determining the binding affinity for these non-peptide antagonists. We show that it is the variability in the aliphatic side chain of the amino acid in position 319 that confers antagonist specificity. Substitution of valine 319 with a leucine residue decreases the affinity for L365260 20-fold while concomitantly increasing the affinity for L364718. An isoleucine in the same position of the human receptor selectively increases affinity for L364718. Interspecies differences in the aliphatic amino acid occupying this single position selectively affect antagonist affinities without altering the agonist binding profile. We therefore conclude that the residues underlying non-peptide antagonist affinity must differ from those that confer agonist specificity. To our knowledge, these findings are the first example in which a critical antagonist binding determinant for a seven-transmembrane-domain peptide hormone receptor has been identified.

We have examined the role of transmembrane domain amino acids in conferring subtype-selective ligand affinity to the human cholecystokinin-B (CCK-B)/gastrin receptor. Fifty-eight residues were sequentially replaced by the corresponding amino acids from the pharmacologically distinct CCK-A receptor subtype. 125I-CCK-8 competition binding experiments were performed to compare all mutant CCK-B/gastrin receptor constructs with the wild type control. Affinities for the nonselective agonist, CCK-8, as well as the subtype-selective peptide (gastrin), peptide-derived (PD135,158), and nonpeptide (L365,260) and L364,718) ligands were assessed. All of the mutants retained relatively high affinity for CCK-8, suggesting that the tertiary structure of these receptors was well maintained. Only eight of the amino acid substitutions had a significant effect on subtype selective binding. When compared with the wild type, single point mutations in the CCK-B/gastrin receptor decreased affinity for gastrin, L365,260, and PD135,158 up to 17-,23-, and 61-fold, 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 influenced by multiple residues acting in concert. The 8 pharmacologically important amino acids cluster in the portion of the transmembrane domains adjacent to the cell surface. The spatial orientation of these residues 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 into a putative ligand pocket, similar to the one which is well established 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).

The development of non-peptide agonists for peptide hormone receptors would markedly expand the treatment options for a large number of diseases. However, difficulty in identifying non-peptide molecules which possess intrinsic activity has been a major obstacle in achieving this goal. At present, most of the known nonpeptide ligands for peptide hormone receptors appear in standard functional assays to be antagonists. Here, we report that a constitutively active mutant of the human cholecystokinin-B/gastrin receptor, Leu 325 3 Glu, offers the potential to detect even trace agonist activity of ligands which, at the wild type receptor isoform, appear to lack efficacy. The enhanced functional sensitivity of the mutant receptor enabled us to detect intrinsic activity of L-365,260, an established non-peptide antagonist for the cholecystokinin-B/gastrin receptor. Extending from this observation, we were able to demonstrate that minor structural modifications could convert L-365,260 into either: (i) an agonist or (ii) an inverse agonist (attenuates ligand-independent signaling). The ability to confer functional activity to small non-peptide ligands suggests that the properties of endogenous peptide hormones can be mimicked, and even extended, by considerably less complex molecules.

The brain cholecystokinin-B͞gastrin receptor (CCK-BR) is a major target for drug development because of its postulated role in modulating anxiety, memory, and the perception of pain. Drug discovery efforts have resulted in the identification of small synthetic molecules that can selectively activate this receptor subtype. These drugs include the peptide-derived compound PD135,158 as well as the nonpeptide benzodiazepine-based ligand, L-740,093 (S enantiomer). We now report that the maximal level of receptor-mediated second messenger signaling that can be achieved by these compounds (drug efficacy) markedly differs among species homologs of the CCK-BR. Further analysis reveals that the observed differences in drug efficacy are in large part explained by single or double aliphatic amino acid substitutions between respective species homologs. This interspecies variability in ligand efficacy introduces the possibility of species differences in receptor-mediated function, an important consideration when selecting animal models for preclinical drug testing. The finding that even single amino acid substitutions can significantly affect drug efficacy prompted us to examine ligandinduced signaling by a known naturally occurring human CCK-BR variant (glutamic acid replaced by lysine in position 288; 288 E 3 K). When examined using the 288 E 3 K receptor, the efficacies of both PD135,158 and L-740,093 (S) were markedly increased compared with values obtained with the wild-type human protein. These observations suggest that functional variability resulting from human receptor polymorphisms may contribute to interindividual differences in drug effects.

The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases D. Vetrie et al.

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