Characterization of human endothelin B receptor and mutant receptors expressed in insect cells

Endothelin type-B receptor (ETBR) forms a stable complex with its ligand, endothelin-l. 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-l-ligand-affinity and nickel-affinity chromatographies. The ligand-free receptor was purified by dissociating the ligand receptor complex with 2M NaSCN, whereas the ligand-bound ETBR was purified by the use of thiol-sensitive biotinylated endothelin-l. While the wild-type ETBR was expressed at about 100 pmol I-125-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-K62, 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-beta-D-glucopyranoside or n-decyl-beta-D-maltopyranoside. Ligand-free [H57-H62, G63-G65]ETBR 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]ETBR 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.