Open AccessCharacterization of Human Endothelin B Receptor and Mutant Receptors Expressed in Insect Cells
Author(s) -
Doi Tomoko,
Hiroaki Yoko,
Arimoto Ikuyo,
Fujiyoshi Yoshinori,
Okamoto Tomoyuki,
Satoh Misako,
Furuichi Yasuhiro
Publication year - 1997
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1997.00139.x
Subject(s) - ligand (biochemistry) , biotinylation , digitonin , receptor , affinity chromatography , endothelin receptor , biochemistry , mutant , biology , chemistry , microbiology and biotechnology , enzyme , gene
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 · 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 125 I‐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‐β‐ d ‐glucopyranoside or n ‐decyl‐β‐ 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 G q in the presence of endothelin‐1. Ligand‐bound [H57–H62, G63–G65]ET B R showed similar catalytic activity in nucleotide exchange by G q . These results indicate that the ligand 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.