Human endothelin receptors ET(A) and ET(B) expressed in baculovirus-infected insect cells--direct application for signal transduction analysis.
We expressed human endothelin receptors, ET(A) and ET(B), 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 ET(A) receptor showed an affinity order of ET-1 > or = ET-2 >> ET-3, and the ET(B) receptor remained nonselective for three isopeptide ligands. The ET(B) 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 ET(B) 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 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 endothelin-receptor. We propose that this highly sensitive system be used to screen for potential antagonists/agonists of endothelin receptors.
Satoh
M
M
AGENE Research Institute, Kajiwara, Kamakura, Japan.
Miyamoto
C
C
Terashima
H
H
Tachibana
Y
Y
Wada
K
K
Watanabe
T
T
Hayes
A E
AE
Gentz
R
R
Furuichi
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D019332:Endothelin-1 / Q000378:metabolism / Q000494:pharmacology; D005470:Fluorometry; D016257:Fura-2 / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011233:Precipitin Tests; D017466:Receptors, Endothelin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D015398:Signal Transduction; D018411:Spodoptera
D019332:Endothelin-1; D017466:Receptors, Endothelin; D011994:Recombinant Proteins; D002118:Calcium; D016257:Fura-2