title: Conserved cholecystokinin receptor transmembrane domain IV amino acids confer peptide affinity.
abstract: The Cholecystokinin type 1 and type 2 receptors (CCK-1R and CCK-2R) share >50% amino acid identity, as well as subnanomolar affinity for the endogenous peptide cholecystokinin octapeptide (CCK-8). Although it is likely that these two receptor subtypes share amino acids that confer CCK-8 affinity, it has been difficult to identify such residues. We have examined the role of several transmembrane domain (TMD) IV residues that are common to both CCK receptor subtypes. In both the CCK-1R and CCK-2R, we demonstrate that alanine substitution of two TMD IV residues, which are highly conserved among all known CCK receptor subtypes and species homologs, significantly decrease CCK-8 affinity. Despite the observed decrease in peptide binding, the mutant receptors maintain close to wild-type affinity for the respective subtype selective nonpeptide ligands, 3H-labeled L-364,714 (CCK-1R) and 3H-labeled L-365,260 (CCK-2R), suggesting conserved tertiary structure of these mutants. Assessment of CCK-8-induced inositol phosphate production at each of the mutant CCK receptors revealed normal peptide efficacy. In contrast, peptide potencies are reduced in parallel with the observed decreases in affinity. Taken together, these findings suggest that important peptide affinity determinants are localized on TMD IV, a region that has not previously been considered a major contributor to ligand affinity in either CCK receptors or other G protein-coupled peptide receptors.
authors.affiliation: Department of Medicine and Molecular Pharmacology Research Center, Tufts-New England Medical Center, Boston, MA 02111, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Ren
authors.forename: Michael
authors.initials: M
authors.lastname: Bläker
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Seshadri
authors.forename: Edward W
authors.initials: EW
authors.lastname: McBride
authors.forename: Martin
authors.initials: M
authors.lastname: Beinborn
authors.forename: Alan S
authors.initials: AS
authors.lastname: Kopin
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects / Q000235:genetics; D019556:COS Cells; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000378:metabolism*; D019143:Evolution, Molecular; D006801:Humans; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011869:Radioligand Assay; D043886:Receptor, Cholecystokinin A; D043887:Receptor, Cholecystokinin B; D011949:Receptors, Cholecystokinin / Q000737:chemistry* / Q000235:genetics; D012844:Sincalide / Q000378:metabolism / Q000494:pharmacology; D014316:Tritium
chemical_list: D043886:Receptor, Cholecystokinin A; D043887:Receptor, Cholecystokinin B; D011949:Receptors, Cholecystokinin; D014316:Tritium; D012844:Sincalide; D000409:Alanine