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

Adviye Ergul, Randall L. Tackett, David Puett

Research output: Contribution to journalArticlepeer-review

10 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)1087-1094
Number of pages8
JournalCirculation Research
Issue number6
StatePublished - Jan 1 1995
Externally publishedYes


  • endothelin-1
  • receptor activation
  • receptor binding
  • site-directed mutagenesis

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine


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