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(Circulation Research. 1995;77:1087-1094.)
© 1995 American Heart Association, Inc.

Articles

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

Adviye Ergul, Randall L. Tackett, David Puett

From the Departments of Biochemistry and Molecular Biology (A.E., D.P.) and Pharmacology and Toxicology, College of Pharmacy (R.L.T.), University of Georgia, Athens, and the Departments of Biochemistry and Molecular Biology and the Reproductive Sciences and Endocrinology Laboratories (A.E., D.P.), University of Miami (Fla) School of Medicine.

Correspondence to Dr David Puett, Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602.E-mail puett@bchiris.biochem.uga.edu.

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 ET_A 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 K_i 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 ET_B 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 ET_A 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 ET_A receptor subtype–mediated contractile response to ET-1 stimulus.

Key Words: endothelin-1 • site-directed mutagenesis • receptor binding • receptor activation

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