BRADYKININ-B-2 RECEPTORS IN HUMANS AND RATS - CDNA STRUCTURES, GENE STRUCTURES, POSSIBLE ALTERNATIVE SPLICING, AND HOMOLOGY SEARCHING FOR SUBTYPES

1. To identify and isolate cDNAs encoding rat and human bradykinin-B-2 receptor subtypes we isolated a human bradykinin receptor cDNA homolo gous to a rat B-2 receptor cDNA. 2. The cDNA was expressed in the brad ykinin receptor negative cell line, CHO; membranes prepared from these cells bound bradykinin and had specificity similar to that of the kno wn rat B-2 receptor. In addition, the expressed receptor has a low aff inity for des-Arg(9)-bradykinin. Thus, the cDNA encodes a human B-2-br adykinin receptor. 3. Comparison of the human and rat cDNAs suggested that the human and rat genes are composed of three exons. Cloning, seq uencing and characterization of parts of the human and rat B-2-bradyki nin receptor genes demonstrated the postulated three-exon structure. T his structure includes two 5' exons upstream of the most favorable tra nslation initiation methionine in exon-3. 4. The two 5' exons each con tain methionines, which if independently spliced to the third exon, wo uld yield an open reading frame that includes all of exon-3. This arra ngement could thus vary the amino-terminal region of the protein. Do t hese potential arrangements occur in human RNAs, and will they lead to proteins with differing amino-termini? 5. Reverse transcriptase-polym erase chain reactions (RT-PCR) using human mRNA, nested primers from e xon-1 and exon-3, and detection of the products by hybridization using an independent exon-1 oligonucleotide showed thai the arrangement of exon-1 with exon-2 and exon-3 could not be detected in eight human RNA s. Furthermore, exon-1 spliced with exon-3 was a common arrangement. 6 . Low stringency examination of human and rat Southern blots revealed only bands attributable to the known human or rat B-2-bradykinin recep tor. 7. Reduced stringency hybridization searches of seven different g enomic and cDNA libraries - including two different human genomic libr aries, a rat genomic library, two different rat uterus cDNA libraries, a rat brain library and a human lung library - yielded only rat or hu man B-2-bradykinin receptors. The results of our low stringency hybrid ization experiments suggest that other bradykinin receptors are less t han 60% identical, on the nucleotide level, to the known B-2 receptor. 8. Degenerate polymerase chain reactions using rat genomic DNA as a t emplate and degenerate primers, designed based on the homology of a B- 2-bradykinin receptor with angiotensin-II type-1 receptor, identified B-2-bradykinin receptors, angiotensin-II-type-1 receptors and three no vel orphan receptors.