CLONING AND PHARMACOLOGICAL CHARACTERIZATION OF THE RABBIT BRADYKININB-2 RECEPTOR

Degenerate primers, corresponding to consensus sequences of third and sixth transmembrane domains of G protein-coupled receptor superfamily, were used for the polymerase chain reaction amplification and consecu tive characterization of G protein-coupled receptors present in cultur ed rabbit aortic smooth muscle cells. One of the isolated resulting fr agments was highly homologous to the corresponding region of the brady kinin (BK) B-2 receptor cloned in other species. The polymerase chain reaction fragment was used to screen a rabbit genomic library, which a llowed the identification of an intronless 1101-nucleotide open readin g frame which codes for a 367-amino acid receptor protein. The rabbit B-2 receptor sequence is more than 80% identical to the ones determine d in three other species and retain putative glycosylation, palmitoyla tion and phosphorylation sites. In the rabbit genomic sequence, an acc eptor splice sequence was found 8 base pairs upstream of the start cod on. Northern blot analysis showed a high expression of a major transcr ipt (4.2 kilobases) in the rabbit kidney and duodenum, and a less abun dant expression in other tissues. Southern blot experiments suggest th at a single copy of this gene exists in the rabbit genome. The cloned rabbit B-2, receptor expressed in COS-1 cells binds [H-3]BK in a satur able manner (K-D 2.1 nM) and this ligand competes with a series of kin in agonists and antagonist with a rank order consistent with the B-2 r eceptor identity. The insurmountable character of the antagonism exert ed by Hoe 140 against BK on the rabbit B-2 receptor, previously shown in pharmacological experiments, was confirmed in binding experiments w ith the cloned receptor expressed in a controlled manner. By contrast, Hoe 140 competed with [H-3]BK in a surmountable manner for the human B-2 receptor expressed in COS-I cells. The cloning of the rabbit B-2 r eceptor will be useful notably for the study of the structural basis o f antagonist binding and for studies on receptor regulation in a relat ively large animal.