ABSTRACT
A genomic clone encoding the mouse B1 receptor was isolated by homology to the human B1 receptor cDNA. The deduced amino acid sequence of the mouse B1 receptor is 72% identical to the human B1 receptor and 73% identical to the rabbit B1 receptor. Ligand binding studies of the mouse B1 receptor expressed in COS cells indicate that it has the pharmacological properties associated with the B1 receptor subtype. However the pharmacology of the mouse receptor is unique in that it possesses a 2-3-fold selectivity for the 'classical' B1 agonist des-Arg9BK over the agonist des-Arg10 kallidin. In contrast, the human and rabbit B1 receptors exhibit an approx. 2000- and 150-fold selectivity, respectively, for des-Arg10kallidin over des-Arg9BK. Thus relative to the human and rabbit B1 receptors the mouse B1 receptor has the opposite selectivity for kinin agonists. The DNA sequence of the region encoding bradykinin was determined for two different mouse kininogen cDNA clones, both encode the sequence Arg-BK. Antipeptide antibodies directed against a C-terminal peptide of the human B1 receptor were produced. Initial characterization of this antibody indicates that it detects specific bands by Western blot analyses that are present in membranes prepared from COS cells transfected with the human B1 receptor cDNA but not from mock transfected COS cells.
Subject(s)
Receptors, Bradykinin/agonists , Receptors, Bradykinin/genetics , Amino Acid Sequence , Animals , Base Sequence , Bradykinin/analogs & derivatives , Bradykinin/metabolism , COS Cells , Cattle , Cloning, Molecular , DNA Primers/genetics , DNA, Complementary/genetics , Humans , Kallidin/analogs & derivatives , Kallidin/metabolism , Mice , Molecular Sequence Data , Rabbits , Receptor, Bradykinin B1 , Receptors, Bradykinin/metabolism , Sequence Homology, Amino Acid , Species Specificity , TransfectionABSTRACT
A cDNA clone encoding a human B1 bradykinin receptor was isolated from a human embryonic lung fibroblast cDNA library by expression cloning. The photoprotein aequorin was utilized as an indicator of the ability of the B1 receptor agonist [des-Arg10]kallidin to mediate Ca2+ mobilization in Xenopus laevis oocytes injected with RNA. A clone was isolated with a 1307-nucleotide insert which contains an open reading frame encoding a 353-amino acid protein with the characteristics of a G-protein-coupled receptor. The amino acid sequence of the B1 bradykinin receptor is 36% identical to the amino acid sequence of the B2 bradykinin receptor. The cloned B1 bradykinin receptor expressed in mammalian cells exhibits high affinity binding for 3H-labeled [des-Arg10]kallidin and low affinity for bradykinin. The B1 receptor antagonist [des-Arg10,Leu9]kallidin effectively displaces 3H-labeled [des-Arg10]kallidin from the cloned receptor, whereas the B2 receptor antagonist Hoe-140 (D-Arg0-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin, where Thi is L-[3-(2-thienyl)alanyl], Tic is D-(1,2,3,4-tetrahydroisoquinolin-3-yl-carbonyl), and Oic is L-[(3aS, 7aS)-octahydroindol-2-yl-carbonyl]) does not. Therefore, the expressed receptor has the pharmacological characteristics of the B1 receptor subtype. The availability of both the cloned human B1 and B2 bradykinin receptors should allow the elucidation of the relative contributions of these two receptor subtypes in acute and chronic inflammatory processes.