Identification of Receptor Binding and Activation Sites in Endothelin-1 by Use of Site-Directed Mutagenesis

• Published in: Circulation research Vol. 77; no. 6; pp. 1087 - 1094
• Main Authors: Ergul, Adviye, Tackett, Randall L, Puett, David
• Format: Journal Article
• Language: English
• Published: Hagerstown, MD American Heart Association, Inc 01.12.1995; Lippincott; Lippincott Williams & Wilkins Ovid Technologies
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Biological and medical sciences; Blood vessels and receptors; Cell Line; Chromatography, High Pressure Liquid; DNA, Complementary - genetics; Dogs; Endothelins - analysis; Endothelins - chemistry; Endothelins - genetics; Epithelial Cells; Epithelium - metabolism; Female; Fundamental and applied biological sciences. Psychology; Haplorhini; Humans; Kidney; Logistic Models; Molecular Sequence Data; Muscle, Smooth, Vascular - cytology; Muscle, Smooth, Vascular - metabolism; Mutagenesis; Peptides - genetics; Radioimmunoassay; Rats; Receptors, Endothelin - genetics; Receptors, Endothelin - metabolism; Transfection; Vertebrates: cardiovascular system; Rat; Rodentia; Site directed mutagenesis; Monkey; Vasoconstriction; Smooth muscle; Activation; Endothelin 1; Binding site; Kidney; Vertebrata; Membrane receptor; Cell line; Mammalia; Binding capacity; Structure activity relation; Blood vessel; Primates; Circulatory system
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/01.res.77.6.1087

This study addresses the structural requirements for the intracellular processing and receptor binding properties of endothelin-1 (ET-1). Point mutants of preproendothelin-1 cDNA, with replacement of the codons for Lys (Ref. 9) of ET-1 by ones for Ala and Glu and of Ile (Ref. 20) and Trp (Ref. 21) by ones encoding Ala, were expressed in COS-7 cells. Competitive binding experiments on rat vascular smooth muscle cells (A-10), which were shown to be an ETA receptor-rich cell line, between [sup 125 I]ET-1 and synthetic ET-1, wild-type recombinant ET-1, and recombinant [Ala (Ref. 9)]ET-1, [Glu (Ref. 9)]ET-1, [Ala (Ref. 20)]ET-1, and [Ala (Ref. 21)]ET-1 yielded Ki values of 0.2 plus minus 0.02, 0.2 plus minus 0.02, 0.04 plus minus 0.01, 1.4 plus minus 0.2, 1.6 plus minus 0.2, and more than 50 nmol/L, respectively. In similar experiments with ETB receptor-rich human Girardi heart cells, the corresponding values were 0.2 plus minus 0.03, 0.2 plus minus 0.03, 0.2 plus minus 0.04, 0.2 plus minus 0.06, 1.4 plus minus 0.4, and more than 50 nmol/L. The ETA receptor-mediated contractile responses to [Glu (Ref. 9)]ET-1 and [Ala (Ref. 20)]ET-1, measured by using canine coronary artery rings, were decreased approximately fourfold to fivefold compared with the response produced by synthetic or wild-type recombinant ET-1, whereas [Ala (Ref. 9)]ET-1 was found to be more potent, and [Ala (Ref. 21)]ET-1 did not produce any contraction. These results demonstrate that Ile (Ref. 20) and Trp (Ref. 21) are involved in binding to both receptor subtypes. Of considerable interest was the observation that [Glu (Ref. 9)]ET-1 also blunts the ETA receptor subtype-mediated contractile response to ET-1 stimulus.(Circ Res. 1995;77:1087-1094.)