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 (ET(A)), 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 B-max than those values for the original receptor. Correspondingly, the mutated ET(A) 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 ET(A) receptor is important in ligand binding. ET(A) and ET(B) receptors possess the ET isopeptides selective and non-selective binding activities, respectively Displacement experiments and the binding of I-125-ET-3 to various chimera receptors demonstrated that both the third and fourth extracellular regions, including the flanking transmenbrane regions, are responsible for the ligand-binding selectivity of the ET(A) receptor.