Human Endothelin Receptors ETA and ETB Expressed in Baculovirus‐Infected Insect Cells

We expressed human endothelin receptors, ETA and ETB, in insect Sf9 cells infected by recombinant baculoviruses that contained the respective cDNAs. Ligand-binding experiments showed that the two expressed receptors have the same affinities as observed for the receptors in mammalian cells, i.e. the ETA receptor showed an affinity order of ET-1≥ET-21≫ET-3, and the ETa receptor remained nonselective for three isopeptide ligands. The ETB receptor was purified by affinity chromatography with K9-biotinyl-ET-1 without losing the ligand-binding activity from the membrane of infected Sf9 cells. Protein chemical analysis of the purified ETB receptor showed that it is glycosylated, and that the N-terminal 38-amino-acid peptide is susceptible to proteolytic digestion, resulting in a small 35-kDa receptor like that found in the human placenta. Surprisingly, the infected and unlysed cells showed a strong intracellular Ca2+ concentration increase ([Ca2+]i), which was generated by a unique signal-transduction pathway consisting of the insect GTP-binding protein and human endothelin receptors expressed in the late phase of virus infection. Due mainly to an efficient expression (over 200000 receptors/cell), to a low background owing to no endogenous homolog receptor in insect Sf9 cells, and to a sensitive fluorescent reagent Fura-2, this insect Sf9 cell system can detect the [Ca2+]i induced by picomolar levels of endothelin-receptor. We propose that this highly sensitive system be used to screen for potential antagonists/agonists of endothelin receptors.