Identification of receptor binding and activation sites in endothelin-1 by use of site-directed mutagenesis

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.