Abstract
Echistatin is a 49-amino-acid peptide belonging to the family of disintegrins that are derived from snake venoms and are potent inhibitors of platelet aggregation and cell adhesion. Integrin αvβ3 receptor plays a critical role in several physiological processes such as tumor-induced angiogenesis, tumor cell metastasis, osteoporosis and wound repair. In this study, we have characterized the binding of echistatin to purified integrin αvβ3 receptor and the form expressed on human embryonic kidney 293 cells. We show that both purified and membrane-bound integrin αvβ3 binds to echistatin with a high affinity, which can be competed efficiently by linear and cyclic peptides containing the RGD sequence. Previous studies have shown that αvβ3 binds to vitronectin in a nondissociable manner, whereas an RGD-containing peptide derived from vitronectin binds in a dissociable manner with aKd of 9.4 × 10−7 M. Our studies indicate that radiolabeled echistatin binds to αvβ3 in a nondissociable manner, similar to native echistatin. However, echistatin does not support the adhesion of 293 cells expressing αvβ3 receptor because of poor binding to plastic dishes and is a potent antagonist of the adhesion of these cells to vitronectin. These studies demonstrate that echistatin binding to αvβ3 is of high affinity and irreversible similar to vitronectin and provides an alternate ligand for high-throughput screening for αvβ3 antagonists.
Footnotes
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Send reprint requests to: Dr. C. Chandra Kumar, Dept. of Tumor Biology, Schering Research Institute, 2015, Galloping Hill Road, Kenilworth, NJ 07033.
- Abbreviations:
- DMEM
- Dulbecco’s modified Eagle’s medium
- PMSF
- phenylmethylsulfonyl fluoride
- SDS
- sodium dodecyl sulfate
- PBS
- phosphate-buffered saline
- FACS
- fluorescence-activated cell sorter
- VN
- vitronectin
- PAGE
- polyacrylamide gel electrophoresis
- HEPES
- N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
- Received February 13, 1997.
- Accepted July 16, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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