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 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.