Identification of a ligand-binding site of the human endothelin-A receptor and specific regions required for ligand selectivity.
To investigate the ligand-binding site of the human endothelin-A-receptor subtype (ETA), we have produced various chimeric and mutated receptors in chinese hamster ovary cells. The substitution of Lys140 with Ile located in the C-terminus of the second transmembrane region caused a 13-fold reduction in affinity for endothelin-1 (ET-1) and 3.6-fold lower Bmax than those values for the original receptor. Correspondingly, the mutated ETA receptor with the Lys140-->Ile substitution failed to induce an increase in the intracellular calcium concentration in the presence of 1 nM ET-1. Thus, the Lys140 in the ETA receptor is important in ligand binding. ETA and ETB receptors possess the ET isopeptides selective and non-selective binding activities, respectively. Displacement experiments and the binding of 125I-ET-3 to various chimera receptors demonstrated that both the third and fourth extracellular regions, including the flanking transmembrane regions, are responsible for the ligand-binding selectivity of the ETA receptor.
Adachi
M
M
Department of Molecular Genetics, Nippon Roche Research Center, Kamakura, Japan.
Furuichi
Y
Y
Miyamoto
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D008024:Ligands; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin; D011993:Recombinant Fusion Proteins