title: Identification of receptor binding and activation sites in endothelin-1 by use of site-directed mutagenesis.
abstract: 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 Lys9 of ET-1 by ones for Ala and Glu and of Ile20 and Trp21 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 [125I]ET-1 and synthetic ET-1, wild-type recombinant ET-1, and recombinant [Ala9]ET-1, [Glu9]ET-1, [Ala20]ET-1, and [Ala21]ET-1 yielded Ki values of 0.2 +/- 0.02, 0.2 +/- 0.02, 0.04 +/- 0.01, 1.4 +/- 0.2, 1.6 +/- 0.2, and > 50 nmol/L, respectively. In similar experiments with ETB receptor-rich human Girardi heart cells, the corresponding values were 0.2 +/- 0.03, 0.2 +/- 0.03, 0.2 +/- 0.04, 0.2 +/- 0.06, 1.4 +/- 0.4, and > 50 nmol/L. The ETA receptor-mediated contractile responses to [Glu9]ET-1 and [Ala20]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 [Ala9]ET-1 was found to be more potent, and [Ala21]ET-1 did not produce any contraction. These results demonstrate that Ile20 and Trp21 are involved in binding to both receptor subtypes. Of considerable interest was the observation that [Glu9]ET-1 also blunts the ETA receptor subtype-mediated contractile response to ET-1 stimulus.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Pharmacy, University of Georgia, Athens 30602, USA.
authors.forename: A
authors.initials: A
authors.lastname: Ergul
authors.forename: R L
authors.initials: RL
authors.lastname: Tackett
authors.forename: D
authors.initials: D
authors.lastname: Puett
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000235:genetics; D004285:Dogs; D016232:Endothelins / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D005260:Female; D000882:Haplorhini; D006801:Humans; D007668:Kidney; D016015:Logistic Models; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D016296:Mutagenesis; D010455:Peptides / Q000235:genetics; D011863:Radioimmunoassay; D051381:Rats; D017466:Receptors, Endothelin / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D016232:Endothelins; D010455:Peptides; D017466:Receptors, Endothelin