The Role of the Cholecystokinin-B/Gastrin Receptor Transmembrane Domains in Determining Affinity for Subtype-selective Ligands(∗)

151

183

An important question in taste research is how 25 receptors of the human TAS2R family detect thousands of structurally diverse compounds. An answer to this question may arise from the observation that TAS2Rs in general are broadly tuned to interact with numerous substances. Ultimately, interaction with chemically diverse agonists requires architectures of binding pockets tailored to combine flexibility with selectivity. The present study determines the structure of hTAS2R binding pockets. We focused on a subfamily of closely related hTAS2Rs exhibiting pronounced amino acid sequence identities but unique agonist activation spectra. The generation of chimeric and mutant receptors followed by calcium imaging analyses identified receptor regions and amino acid residues critical for activation of hTAS2R46, -R43, and -R31. We found that the carboxyl-terminal regions of the investigated receptors are crucial for agonist selectivity. Intriguingly, exchanging two residues located in transmembrane domain seven between hTAS2R46, activated by strychnine, and hTAS2R31, activated by aristolochic acid, was sufficient to invert agonist selectivity. Further mutagenesis revealed additional positions involved in agonist interaction. The transfer of functionally relevant amino acids identified in hTAS2R46 to the corresponding positions of hTAS2R43 and -R31 resulted in pharmacological properties indistinguishable from the parental hTAS2R46. In silico modeling of hTAS2R46 allowed us to visualize the putative mode of interaction between agonists and hTAS2Rs. Detailed structure-function analyses of hTAS2Rs may ultimately pave the way for the development of specific antagonists urgently needed for more sophisticated analyses of human bitter taste perception.calcium imaging | G protein-coupled receptors | hTAS2R

Section: Discussionsupporting

confidence: 77%

Section: Discussionsupporting

confidence: 77%

Structural requirements of bitter taste receptor activation

Brockhoff

Behrens²,

Niv³

et al. 2010

151

183

“…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).…”

Section: Discussionmentioning

confidence: 99%

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

Kopin

McBride

Chen

et al. 2003

Self Cite

“…AF019371), and canine (14) receptor cDNAs were subcloned into the expression vector pcDNA I (Invitrogen). To generate human͞murine chimeras, MluI and HindIII sites (corresponding to human cDNA nucleotides 987-992 and 1116-1121, respectively) were introduced into the receptor cDNAs using oligonucleotide-directed mutagenesis (20). Naturally occurring PstI sites, found in the same relative position in both the mouse and human receptors (corresponding to human cDNA nucleotides 607-612), were utilized to generate the PstI chimera.…”

Section: Methodsmentioning

confidence: 99%

Inter- and intraspecies polymorphisms in the cholecystokinin-B/gastrin receptor alter drug efficacy

Kopin

McBride

Gordon

et al. 1997

Self Cite

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.