HUMAN ENDOTHELIN RECEPTORS ETA AND ETB EXPRESSED IN BACULOVIRUS-INFECTED INSECT CELLS DIRECT APPLICATION FOR SIGNAL-TRANSDUCTION ANALYSIS

We expressed human endothelin receptors, ETA and ETB, in insect Sf9 ce lls infected by recombinant baculoviruses that contained the respectiv e cDNAs. Ligand-binding experiments showed that the two expressed rece ptors have the same affinities as observed for the receptors in mammal ian cells, i.e. the ETA receptor showed an affinity order of ET-1 grea ter than or equal to ET-2 much greater than ET-3, and the ETB receptor remained nonselective for three isopeptide ligands. The ETB receptor was purified by affinity chromatography with K-9-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 t hat it is glycosylated, and that the N-terminal 38-amino-acid peptide is susceptible to proteolytic digestion, resulting in a small 35-kDa r eceptor like that found in the human placenta. Surprisingly, the infec ted and unlysed cells showed a strong intracellular Ca2+ concentration increase ([Ca2+](i)), which was generated by a unique signal-transduc tion pathway consisting of the insect GTP-binding protein and human en dothelin receptors expressed in the late phase of virus infection. Due mainly to an efficient expression (over 200 000 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 en dothelin-receptor. We propose that this highly sensitive system be use d to screen for potential antagonists/agonists of endothelin receptors .