RESUMO
A rabbit B1 bradykinin receptor cDNA was isolated from a rabbit aorta smooth muscle cell library. The 1223 bp cDNA clone encodes a protein of 352 amino acids which is 78% identical to the human bradykinin B1(3) receptor protein. Heterologous expression of the rabbit B1 receptor cDNA in COS-7 cells imparts a high affinity specific binding for 3H-labeled [des-Arg10,Leu9]kallidin. Scatchard analysis indicates that the receptor binds the radiolabeled ligand with a Kd of 0.5 nM. The ability of kallidin (Lys-bradykinin) and bradykinin analogues to compete with binding of 3H-labeled [des-Arg10,Leu9]kallidin was determined and defined a rank order of potency: [des-Arg10,Leu9]kallidin = [des-Arg10]kallidin > [des- Arg9]bradykinin = kallidin >> bradykinin. This receptor exhibits the classical B1 pharmacological property of preferentially binding to kinin analogues which lack the C-terminal arginine. In addition, the affinities for [des-Arg10]kallidin and [des-Arg10,Leu9]kallidin are 100-fold higher than those for the corresponding bradykinin analogues [des-Arg9]bradykinin and [des-Arg9,Leu8]bradykinin which lack the N-terminal lysine. This pharmacological profile is characteristic of the B1 receptor subtype.
Assuntos
Aorta/metabolismo , Músculo Liso Vascular/metabolismo , Receptores da Bradicinina/biossíntese , Receptores da Bradicinina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Ligação Competitiva , Bradicinina/análogos & derivados , Bradicinina/metabolismo , Bradicinina/farmacologia , Clonagem Molecular/métodos , Biblioteca Gênica , Humanos , Cinética , Dados de Sequência Molecular , Coelhos , Receptor B1 da Bradicinina , Receptores da Bradicinina/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
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.
