Hechler U, Becker A, Haendler B, Schleuning W D
Research Laboratories of Schering AG, Berlin, Germany.
Biochem Biophys Res Commun. 1993 Aug 16;194(3):1305-10. doi: 10.1006/bbrc.1993.1966.
The cDNAs for human endothelin receptors ETA and ETB were subcloned into the eukaryotic expression vector pMPSV/CMV and transfected, in parallel with plasmids carrying resistances for hygromycin B and puromycin, into baby hamster kidney (BHK) cells. Cell clones constitutively expressing high levels of either receptor were obtained through the combined selective pressure of both antibiotics. This was further confirmed by Northern blot analysis using ETA- or ETB-specific oligonucleotide probes. The calculated KD for endothelin (ET)-1 binding to ETA and ETB were 2.2 x 10(-10) M and 5.3 x 10(-10) M, respectively. Competitive binding experiments using the different ET forms showed the expected isopeptide-selective and non-isopeptide-selective profiles for ETA and ETB, respectively. BQ 123, a specific ETA antagonist, competed with ET-1 for binding to ETA but not to ETB.
将人内皮素受体ETA和ETB的cDNA亚克隆到真核表达载体pMPSV/CMV中，并与携带潮霉素B和嘌呤霉素抗性的质粒一起转染到幼仓鼠肾（BHK）细胞中。通过两种抗生素的联合选择压力获得了组成性表达高水平任一受体的细胞克隆。使用ETA或ETB特异性寡核苷酸探针进行的Northern印迹分析进一步证实了这一点。计算得出内皮素（ET）-1与ETA和ETB结合的KD分别为2.2×10（-10）M和5.3×10（-10）M。使用不同ET形式的竞争性结合实验分别显示了ETA和ETB预期的异肽选择性和非异肽选择性谱。特异性ETA拮抗剂BQ 123与ET-1竞争结合ETA，但不与ETB结合。
