A segment of five amino acids in the second extracellular loop of the cholecystokinin-B receptor is essential for selectivity of the peptide agonist gastrin.
The two known receptors mediating the actions of cholecystokinin (CCK) and gastrin, CCK type A (CCKAR) and CCK type B (CCKBR) receptors, are G protein-coupled receptors having approximately 50% amino acid homology. Both the CCKAR and CCKBR have high affinity for sulfated CCK peptides, while only the CCKBR has high affinity for gastrin peptides. To determine the structural basis for the selectivity of the CCKBR for gastrin, we first constructed a series of CCKB/AR chimeras in which restriction endonuclease-defined segments of the CCKBR were replaced with the corresponding segments of the CCKAR. Chimeras transiently expressed in COS-1 cells were screened for the selective loss of gastrin affinity according to the displacement of 125I-labeled Bolton-Hunter-CCK-8 binding by gastrin-17-I and CCK-8. The sequence spanning from transmembrane domain III (TM III) to TM V was the only segment that resulted in the selective loss of gastrin affinity. This segment could account for 100 of the expected 300-fold lower affinity of gastrin-17-I observed for the control CCKAR compared to the control CCKBR. Using site-directed mutagenesis in this segment of the CCKBR, we identified a sequence of 5 amino acids in the second extracellular loop responsible for this 100-fold selective loss in gastrin affinity. 125I-labeled Bolton-Hunter-CCK-8 binding displacement by L365,260 (a CCKBR selective antagonist) was unaffected by the changes in these 5 amino acids. These results present for the first time the identification of the amino acid sequence of the CCKBR conferring the majority of the selectivity for gastrin.
Silvente-Poirot
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Digestive Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1804, USA.
Wank
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D000595:Amino Acid Sequence; D000818:Animals; D001570:Benzodiazepinones / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D005755:Gastrins / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010671:Phenylurea Compounds; D017354:Point Mutation; D017433:Protein Structure, Secondary; D043887:Receptor, Cholecystokinin B; D011949:Receptors, Cholecystokinin / Q000819:agonists / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012844:Sincalide / Q000031:analogs & derivatives / Q000378:metabolism; D013388:Succinimides / Q000378:metabolism; D014162:Transfection
D001570:Benzodiazepinones; C044758:Bolton Hunter-cholecystokinin octapeptide; D005755:Gastrins; D007202:Indicators and Reagents; D010671:Phenylurea Compounds; D043887:Receptor, Cholecystokinin B; D011949:Receptors, Cholecystokinin; D011993:Recombinant Fusion Proteins; D013388:Succinimides; C058121:L 365260; D012844:Sincalide