Biochemical characterization of the binding of echistatin to integrin alphavbeta3 receptor

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 alphavbeta3 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 alphavbeta3 receptor and the form expressed on human embryonic kidney 293 cells. We show that both purified and membrane-bound integrin alphavbeta3 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 alphavbeta3 binds to vitronectin in a nondissociable manner, whereas an RGD-containing peptide derived from vitronectin binds in a dissociable manner with a Kd of 9.4 x 10(-7) M. Our studies indicate that radiolabeled echistatin binds to alphavbeta3 in a nondissociable manner, similar to native echistatin. However, echistatin does not support the adhesion of 293 cells expressing alphavbeta3 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 alphavbeta3 is of high affinity and irreversible similar to vitronectin and provides an alternate ligand for high-throughput screening for alphavbeta3 antagonists.