Molecular cloning of the bovine proteolttically-activated thrombin receptor

A significant advance to the understanding of the thrombin's interaction with the vessel wall was the report of the amino acid sequence of the proteolytically-activated thrombin receptor (TR) (human, hamster, mouse and rat). Comparison of the TR from these four species reveals 85-95% homology at the amino acid level. The N-terminal sequence generated after thrombin cleavage is present in all species with significant differences. Although synthetic thrombin receptor activating peptides (TRAP) from 6 to 14-mer can activate TR without cleaving, the sequence of TRAP itserf may be important in determining the maximum thrombin effect. Our laboratory has observed significant increases in PGI? synthesis ( 10 fold) and bamer dysfunction (4 foid) when human TRAP or human thrombin were used in human endothelial cells, with significant less responsiveness when human TRAP or thrombin were used in bovine endothelial cells (3 fold. 2,5 fold respectively). As this suggested that the sequence specificity of the TR may be important, we proposed to done the bovine TR (BTR). In our initial studies, a bovine endothelial cell cDNA library was screened with two polymerase chain reaction (PCR)-generated DNA probes (205 bp & 387 bp) which encoded the most homologous part of TR in all four species. This yielded multiple clones all lacking about 200 bp in the 5′ end when compared with the human TR. PCR cloning using specific internal and vector primers were next used to obtain the very 5' end coding region utilizing a bovine kidney 5′' stretch plus cDNA library as template. The full length BTR cDNA spans a nudeotide sequence stretch of 1609 bp which is 78 % similar, at the nucleotide level, and 89% similar, at the amino acid level, to the human TR. Although the putative thrombin cleavage and binding sites are present, comparison of bovine and human receptors m their newly generated N-terminal sequence (SFFLRNS.NDQYEQJ vs SFLLRNPNDfÇYEPF} reveals differences in 5 amino acids. These differences particularly at amino acid # 13 & # 1, may be important <n understanding species-specific interaction of thrombin with the vascular waif.