Characterization of human endothelin B receptor and mutant receptors expressed in insect cells.
Endothelin type-B receptor (ET(B)R) forms a stable complex with its ligand, endothelin-1. To facilitate biochemical and biophysical studies of human ET(B)R, several ET(B)R mutants carrying a hexahistidine tag sequence at the N or C terminus were expressed in Sf9 cells and were purified by a combination of biotinylated endothelin-1-ligand-affinity and nickel-affinity chromatographies. The ligand-free receptor was purified by dissociating the ligand x receptor complex with 2 M NaSCN, whereas the ligand-bound ET(B)R was purified by the use of thiol-sensitive biotinylated endothelin-1. While the wild-type ET(B)R was expressed at about 100 pmol 125I-endothelin-1-binding activity/mg membrane protein, the deletion of 36 residues from the N-terminus reduced the expressed activity to about 30%. On the other hand, the lack of glycosylation and the replacement of 2-9 residues in the N-terminal tail resulted in a 20-40% reduction in the expressed activity. Among the mutant proteins, [H57-H62, G63-G65]ET(B)R, carrying six His residues in the N-terminal tail, was studied extensively because it was purified most effectively. Ligand-free [H57-H62, G63-G65]ET(B)R, purified in digitonin, retained full ligand-binding activity, while other detergents led to partial denaturation of the receptor after solubilization or after elution with NaSCN. On the other hand, ligand-bound [H57-H62, G63-G65]ET(B)R could be purified in various detergents, such as n-octyl-beta-D-glucopyranoside or n-decyl-beta-D-maltopyranoside. Ligand-free [H57-H62, G63-G65]ET(B)R reconstituted in phospholipid vesicles stimulated the binding of guanosine 5'-3-O-(thio)triphosphate by Gq in the presence of endothelin-1. Ligand-bound [H57-H62, G63-G65]ET(B)R showed similar catalytic activity in nucleotide exchange by Gq. These results indicate that the ligand x receptor complex in a detergent-micellar solution retained the biologically active structure, and that the presence of ligand, endothelin-1, in the receptor molecule reinforces the stable assembly of a helical bundle and therefore the active structure.
Doi
T
T
International Institute for Advanced Research, Matsushita Electric Industrial Co., Ltd, Seika, Kyoto, Japan. tomoko@crl.mei.co.jp
Hiroaki
Y
Y
Arimoto
I
I
Fujiyoshi
Y
Y
Okamoto
T
T
Satoh
M
M
Furuichi
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003902:Detergents; D019332:Endothelin-1 / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008024:Ligands; D008081:Liposomes; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D018411:Spodoptera
D003902:Detergents; D019332:Endothelin-1; D008024:Ligands; D008081:Liposomes; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin