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

摘要: 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 hexahistidine tag sequence at the N or C terminus were expressed in Sf9 cells purified by combination biotinylated endothelin-1-ligand-affinity nickel-affinity chromatographies. The ligand-free was dissociating ligand · 2 M NaSCN, whereas ligand-bound use thiol-sensitive While wild-type about 100 pmol 125I-endothelin-1-binding activity/mg membrane protein, deletion 36 residues from N-terminus reduced activity to 30%. On other hand, lack glycosylation replacement 2–9 N-terminal tail resulted 20–40% reduction activity. Among mutant proteins, [H57–H62, G63–G65]ETBR, six His tail, studied extensively because it most effectively. Ligand-free digitonin, retained full ligand-binding activity, while detergents led partial denaturation after solubilization elution NaSCN. G63–G65]ETBR could be various detergents, such as n-octyl-β-d-glucopyranoside n-decyl-β-d-maltopyranoside. reconstituted phospholipid vesicles stimulated binding guanosine 5'-3-O-(thio)triphosphate Gq presence Ligand-bound showed similar catalytic nucleotide exchange Gq. These results indicate that detergent-micellar solution biologically active structure, endothelin-1, molecule reinforces assembly helical bundle therefore structure.