Stable Expression of Human Endothelin Receptors ET A and ET B by Transfected Baby Hamster Kidney Cells

Abstract

The cDNAs for human endothelin receptors ET A and ET B 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 ET A- or ET B-specific oligonucleotide probes. The calculated K D for endothelin (ET)- 1 binding to ET A and ET B were 2.2 × 10 −10 M and 5.3 × 10 −10 M, respectively. Competitive binding experiments using the different ET forms showed the expected isopeptide-selective and non-isopeptide-sclective profiles for ET A and ET B, respectively. BQ 123, a specific ET A antagonist, competed with ET-1 for binding to ET A but not to ET B.