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Molecular Pharmacology

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Research ArticleArticle

Sodium Dodecyl Sulfate-Stable Complexes of Echistatin and RGD-Dependent Integrins: A Novel Approach to Study Integrins

Gaétan Thibault
Molecular Pharmacology November 2000, 58 (5) 1137-1145; DOI: https://doi.org/10.1124/mol.58.5.1137
Gaétan Thibault
Laboratoire de Biologie Cellulaire de l'Hypertension, Institut de Recherches Cliniques de Montréal and Universitéde Montréal, Montréal, Quebec, Canada
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Abstract

This study shows that disintegrins, echistatin as a model, can be used as a radiolabeled probe to simultaneously detect the presence of individual RGD-dependent integrins on cardiac fibroblasts. Binding of125I-echistatin to fibroblasts was proportional to cell number, time dependent, reversible, saturable, specific, and membrane bound. SDS-polyacrylamide gel electrophoresis and autoradiograms revealed that 125I-echistatin was associated with three radioactive protein bands of 180, 210, and 220 kDa that were identified by RGD affinity chromatography, immunoblotting, and immunoneutralization as αvβ3, α3β1/α5β1/αvβ1, and α8β1 heterodimeric integrins, respectively. These results suggest that echistatin binds to RGD-dependent integrins, forming SDS-stable complexes in the absence of chemical cross-linkers, reducing conditions and heating. As assessed by radioligand-binding filtration, disintegrins displayed binding characteristics with an IC50 ranging from 0.044 to 1.1 nM, but with slope factors lower than 1, indicating the presence of several binding sites. Resolved by SDS-polyacrylamide gel electrophoresis to reveal echistatin-integrin complexes, disintegrins and RGD peptides displayed different binding affinities to individual RGD-dependent integrins present on cardiac fibroblasts. Elegantin and flavostatin demonstrated the highest affinity toward integrins, whereas flavoridin and acPenRGDC had a greater specificity toward αvβ3-integrin. In summary, echistatin forms SDS-stable complexes with RGD-dependent integrins. This model offers a novel way to visualize RGD-dependent integrins, to investigate their activation state, and to determine the integrin specificity of RGD peptides.

Footnotes

    • Received May 1, 2000.
    • Accepted August 3, 2000.
  • Send reprint requests to: Gaétan Thibault, Ph.D., Laboratoire de Biologie Cellulaire de l'Hypertension, Institut de Recherches Cliniques de Montréal, 110, Avenue des Pins Ouest, Montréal, Québec, Canada H2W 1R7. E-mail:thibaug{at}ircm.qc.ca

  • This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada and from the Medical Research Council of Canada.

  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 58 (5)
Molecular Pharmacology
Vol. 58, Issue 5
1 Nov 2000
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Research ArticleArticle

Sodium Dodecyl Sulfate-Stable Complexes of Echistatin and RGD-Dependent Integrins: A Novel Approach to Study Integrins

Gaétan Thibault
Molecular Pharmacology November 1, 2000, 58 (5) 1137-1145; DOI: https://doi.org/10.1124/mol.58.5.1137

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Research ArticleArticle

Sodium Dodecyl Sulfate-Stable Complexes of Echistatin and RGD-Dependent Integrins: A Novel Approach to Study Integrins

Gaétan Thibault
Molecular Pharmacology November 1, 2000, 58 (5) 1137-1145; DOI: https://doi.org/10.1124/mol.58.5.1137
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