Mutations within the Cholecystokinin-B/Gastrin Receptor Ligand ‘Pocket’ Interconvert the Functions of Nonpeptide Agonists and Antagonists

Abstract

We have reported previously that the transmembrane domains of the cholecystokinin-B/gastrin receptor (CCK-BR) comprise a putative ligand binding pocket. In the present study, we examined whether amino acid substitutions within the CCK-BR pocket altered the affinities and/or functional activities of L-365,260 (the prototypical nonpeptide CCK-BR antagonist) and two structural derivatives, YM022 (a higher affinity antagonist) and L-740,093S (a partial agonist). Eight amino acids that project into the CCK-BR pocket were individually replaced by alanine, using site-directed mutagenesis. Affinities for the nonpeptide molecules, as well as ligand-induced inositol phosphate production, were assessed with the wild-type and mutant receptors. For each of the nonpeptide ligands examined, a distinct series of mutations altered the affinity, suggesting that each ligand possessed a characteristic pattern of interactions within the CCK-BR pocket. Basal signaling levels and inositol phosphate formation induced by the full agonist CCK octapeptide were comparable for the wild-type receptor and all of the mutant CCK-BR forms. In contrast to the peptide agonist CCK octapeptide, the functional activities of the nonpeptide molecules were selectively altered by single point mutations within the CCK-BR pocket, resulting in interconversion of agonists and antagonists. These findings suggest that interactions between nonpeptide molecules and transmembrane domain amino acids of the CCK-BR can determine the functional activity and affinity of the ligands.

Identification of a series of CCK-2 receptor nonpeptide agonists: Sensitivity to stereochemistry and a receptor point mutation

“…The binding of each compound was also determined at a mutant CCK-2R, N353L, that is known to have reduced affinity (21-fold) for L-365,260 (14). As observed with the prototype CCK-2R antagonist, the affinity of each R compound is highly sensitive to the N353L mutation.…”

“…We have previously shown that small-molecule ligands recognize a putative binding pocket within the CCK-2R that is comprised of the side chains of transmembrane domain amino acids (24). Mutation of individual residues within this pocket, including N353, may differentially alter the affinity and͞or efficacy of small-molecule ligands (14). Extending this theme, we now demonstrate that steric configuration of a given ligand may markedly influence the magnitude and even the direction of affinity͞efficacy changes resulting from a point mutation.…”

“…The cDNA expression constructs encoding the wild-type human CCK-2R and the L325E (third intracellular loop) and N353L (sixth transmembrane domain) mutants have been described (2,14). COS-7 cells (10 6 ) were transfected with 5 g of either wild-type or mutant human CCK-2R cDNA subcloned into the expression vector pcDNA1.1 (Invitrogen).…”

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.…”

A substance P-opioid chimeric peptide as a unique nontolerance-forming analgesic

“…For this reason, the potency of ESP7 at the SPR was assessed by quantifying its ability to stimulate IP production. The protocol of Blaker et al (28) was followed. Then 10 6 COS-7 cells͞10-cm plate were transfected with 5 g of rat NK-1 receptor cDNA (29) or pcDNA1.1 (Invitrogen).…”