Ligand binding domain of the human endothelin-B subtype receptor.

We have employed both protein chemical and molecular biological approaches to determine the ligand binding domain of the endothelin-B subtype (ETB) receptor. The human ETB receptor purified from human placenta by using affinity chromatography was cross-linked with 125I-labeled endothelin-1 (ET-1) and then incubated in the presence of trypsin or thermolysin under nondenaturing conditions. The N-terminal amino acid sequence of the radiolabeled polypeptide encompassed approximately 115 amino acid residues starting from Ile85 of the human ETB receptor. This was confirmed by experiments in which the binding activity of endothelin-1 to various chimeric endothelin receptors was monitored in the presence and absence of competitive endothelin receptor antagonists such as BQ-123 and bosentan. The region from Ile138 to Ile197 (60 amino acid residues) of the ETB receptor was found to interact with both antagonists. Therefore, this sequence was determined to be the ligand binding domain. In addition, we found that part of the N-terminal domain in close proximity to the first transmembrane region was required for the ligand binding activity of the ETB receptor, and the 12 amino acid residues from Ser390 to Leu401 at the proximal cytoplasmic tail are perhaps necessary to maintain the ligand binding site in active form. The cysteine rich region from residue 400 to residue 403 in the C-terminus of the ETB receptor is involved in coupling of the guanine nucleotide-binding regulatory protein for ET-1-induced signal transduction.