Characterization of Human Endothelin B Receptor and Mutant Receptors Expressed in Insect Cells

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Abstract
Endothelin type‐B receptor (ETBR) forms a stable complex with its ligand, endothelin‐1. To facilitate biochemical and biophysical studies of human ETBR, several ETBR 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 ETBR was purified by the use of thiol‐sensitive biotinylated endothelin‐1. While the wild‐type ETBR 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]ETBR, carrying six His residues in the N‐terminal tail, was studied extensively because it was purified most effectively. Ligand‐free [H57–H62, G63–G65]ETBR, 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]ETBR 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]ETBR 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]ETBR showed similar catalytic activity in nucleotide exchange by Gq. 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.