A novel endothelin receptor type-B-like gene enriched in the brain

We report here our effort of cloning and characterization of a novel human gene, which encodes a putative human endothelin receptor type B like protein (hET(B)R-LP), from a human hippocampus tissue cDNA library. hET(B)R-LP consists of 614 amino acids with seven putative transmembrane domains. The deduced amino acid sequence of hET(B)R-LP is 52% similar and 26.7% identical to human endothelin type B receptor. A 4.0 kb mRNA of hET(B)R-LP is abundantly expressed in the human brain. The results of in situ hybridization and reverse transcriptase in situ gene amplification reveal tissue distribution and cellular localization of signals of hET(B)R-LP mRNA in the neuronal cells, particularly concentrated in Purkinje cells of the cerebellum, and neuronal cells of the hippocampus of human brain, including pyramidal cells of Ammon's horn and granule cells of the dentate gyrus. A 4.0 kb mRNA of hET(B)R-LP is also less abundantly expressed in the liver and the placenta. Expression of recombinant protein, hET(B)R-LP/HA, in cells of COS7 and HEK293 transfected with plasmid DNA, hET(B)R-LP/HA/pcDNA1/Amp, was confirmed by Northern blot analysis and by immunofluorescence staining of cells with anti-HA antibody. Specific binding of radiolabeled ET-1 and ET-3 to membrane preparations and to intact cells expressing recombinant protein of hET(B)R-LP/HA did not show any significant difference of binding properties between cells transfected with plasmid DNA, hET(B)R-LP/HA/pcDNA1/Amp, and cells untransfected, including both COS7 cells and HEK293 cells. The results of assays of measuring Ca++ mobilization and cAMP production in HEK293 cells indicate that ET-1, ET-3, bombesin and neuropeptide Y are unable to produce any kind of significant difference of Ca++ mobilization and cAMP production between HEK293 cells expressing recombinant protein and HEK293 cells untransfected or HEK293 cells transfected with vector DNA only (pcDNA1/Amp) in functional assays performed. Therefore, its ligand and physiological significance of hET(B)R-LP remains to be discovered.