Title

Molecular aspects of endothelin-1

Date of Award

1995

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

David Puett, Committee Chair

Abstract

Endothelin-1 (ET-1) is a 21-amino acid residue peptide with potent vasoconstricting and mitogenic properties. Three members of the ET family, ET-1, ET-2, and ET-3, have been cloned, and these peptides were found to have sequence homology and similar bioactivity to a peptide toxin family, sarafotoxins (STX). A comparison of the amino acid sequences of the ETs and STXs revealed that certain residues are highly conserved, suggesting that they might be important in post-translational modification, cellular transport, receptor binding, or receptor activation.To test these possibilities, the codons for Lys-9, Ile-20, and Trp-21 in the PPET-1 cDNA were replaced by ones that encode for Ala. Lys-9 was also replaced by Glu, and the mutant and wild-type PPET-1 cDNAs were transiently expressed in COS-7 cells. Levels of the expression of recombinant mutant ET-1s were measured by radioimmunoassay. Interestingly, immunoreactive ET-1 levels in the transfection medium obtained from cells transfected with (Ala-21-ET-1)PPET-1 cDNA were 10-fold lower that those of cells transfected with wild-type and other mutant cDNAs along with a corresponding increase in the big ET-1 levels in the extracts of (Ala-21-ET-1)PPET-1 cDNA-transfected cells.The receptor binding properties of the mutant peptides were studied on two cell lines, rat vascular smooth muscle cells A-10 and human Girardi heart cells that possess ET$\sb{\rm A}$- and ET$\sb{\rm B}$-class receptors, respectively. The contractile effects of these peptides were tested on canine coronary artery rings that are enriched in the ET$\sb{\rm A}$ receptor subtype. The results of the binding and contractility experiments demonstrated that Lys-9, Ile-20, and Trp-21 are important for binding and activation of ET$\sb{\rm A}$ receptors, whereas replacement of Lys-9 by either Ala or Glu does not have any appreciable effect on binding to the ET$\sb{\rm B}$ receptor subtype. In addition, the (Glu-9)ET-1 mutant peptide blocked the contractile response mediated by 50 nM ET-1, indicating that it has inhibitory properties as well.

Keywords

Biology, Molecular; Chemistry, Biochemistry

Link to Full Text

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