A single amino acid of the cholecystokinin-B/gastrin receptor determines specificity for non-peptide antagonists

Abstract

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.

Molecular Basis for Binding and Subtype Selectivity of 1,4-Benzodiazepine Antagonist Ligands of the Cholecystokinin Receptor

“…Those studies, however, were more indirect, utilizing the orthosteric radioligand based on the natural peptide that is known to bind to extracellular loop and tail regions rather than a radioligand that binds directly to the intramembranous docking site of these small molecules. Two of the residues identified as important in the current work (residues 6.51 and 7.39) have also been identified as important for the binding of the benzodiazepine antagonist L364,718 to the CCK1R (25,46,47). This is helpful in confirming the more general relevance of these residues for small molecule ligand binding to this receptor.…”

“…This, too, provides further evidence for the general relevance of this pocket for small molecule docking while emphasizing the ability of changes in many of these residues to affect indirectly the binding of different types of ligands. It is particularly noteworthy that an earlier study following the effects on CCK radioligand binding reported a critical effect of CCK2R residue 6.51 on benzodiazepine L365,260 binding (46), but the current study in which a benzodiazepine radioligand was utilized observed no such effect.…”

Membrane-tethered ligands are effective probes for exploring class B1 G protein-coupled receptor function

“…A cDNA encoding a membrane-tethered version of pigment-dispersing factor (PDF; a Drosophila class B1 peptide hormone) was chemically synthesized and cloned in pcDNA3.1 (C.C., M.N.N., manuscript submitted). After subcloning into pcDNA1.1, other class B1 GPCR ligand sequences were substituted in place of the PDF coding region by using oligonucleotide-directed, site-specific mutagenesis as previously described (25,26). Point mutations were introduced into the corresponding tethered ligands by using the same mutagenesis approach.…”

Nonpeptide antagonists of neuropeptide receptors: tools for research and therapy

“…However, it has become increasingly clear that this is seldom the case. The use of antagonist-derived radioligands as well as mutational analysis of receptors has suggested the existence of distinct agonist and antagonist binding domains on receptors for tachykinins [7][8][9][10] , CCK 11,12 , angiotensin 13,14 , opioids 15 , neurotensin 16 and vasopressin 17 .…”

“…Although canine and human CCK B receptors share ~90% amino acid identity and have similar agonist binding affinity, they exhibit opposite rank orders of affinity for two nonpeptide CCK antagonists: L365260 shows selectivity for the human receptor, whereas L364718 displays a higher affinity for the dog CCK receptor. Mutational analysis of the CCK B receptor demonstrated that antagonist affinities can be altered dramatically by a single amino acid substitution, which in turn explain species-related differences 11 . The replacement of valine 319 in the sixth transmembrane region of the human receptor with the corresponding amino acid in the canine receptor, leucine, decreases the affinity of L365260 and increases the affinity of L364718 to the values seen in the canine receptor.…”