Biochemical Characterization of the Binding of Echistatin to Integrin avb3 Receptor

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 avb3 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 avb3 receptor and the form expressed on human embryonic kidney 293 cells. We show that both purified and membrane-bound integrin avb3 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 avb3 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 3 10 27 M. Our studies indicate that radiolabeled echistatin binds to avb3 in a nondissociable manner, similar to native echistatin. However, echistatin does not support the adhesion of 293 cells expressing avb3 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 avb3 is of high affinity and irreversible similar to vitronectin and provides an alternate ligand for high-throughput screening for avb3 antagonists. Adhesion receptors of the integrin family are responsible for a wide range of cell-extracellular matrix and cell-cell interactions (Hynes, 1992; Clark and Brugge, 1995). Each integrin consists of noncovalently associated alpha and beta subunits which pair to create heterodimers (ab) with distinct adhesive capabilities. As receptors of extracellular matrix proteins, integrins provide anchorage and convey signals that regulate cell growth, differentiation and migration (Juliano and Haskill, 1993; Sastry and Horowitz, 1993). The integrin avb3 is expressed on endothelial cells, osteoclasts, melanoma and other cell types (Cheresh and Spiro, 1987; Cheresh,1987; Miyuchi et al., 1991; Horton, 1990), where it plays a role in physiological processes that include angiogenesis and tissue repair as well as pathological conditions such as osteoporosis, tumor cell metastasis, adenoviral infections and tumor-induced angiogenesis (Schwartz, 1993; Brunhilde,1992; Davis et al., 1993; Ross et al., 1993; Brooks et al., 1994a,b; Wickham et al., 1993). Recent results which demonstrate a critical role for integrin avb3 in angiogenesis have sparked an interest in this receptor (Brooks et al., 1994a,b). Brooks et al. (1994a,b) have shown that antibody and peptide antagonists of integrin avb3 inhibit angiogenesis on the chick chorioallantoic membrane when introduced intravenously into the chick embryo. Evidence has been presented indicating that antagonists of integrin avb3 inhibit this process by selectively promoting apoptosis of vascular endothelial cells (Brooks et al., 1994a). These findings indicate a key role for integrin avb3 in a signaling event critical for the survival and ultimately differentiation of vascular cells undergoing angiogenesis in vivo. These results also provide evidence that antagonists of integrin avb3 may provide a novel therapeutic approach for the treatment of neoplasia or other diseases characterized by angiogenesis. Although originally isolated as a receptor for vitronectin, avb3 actually recognizes a broad range of extracellular matrix protein ligands such as vitronectin, fibronectin, fibrinogen, von Willebrand factor, thrombospondin and osteopontin, all of which contain the classical integrin recognition motif, Arg-Gly-Asp (RGD) (Leavesly et al., 1992; Charo et al., 1990). The relaxed specificity of avb3 contrasts sharply with the selectivity of a5b1 integrin which binds to only fibronectin and fibrinogen (D’Souza et al., 1991; Suehiro et al., 1997). Thus, even though the RGD motif has been firmly established as a key determinant in the recognition of extracellular matrix protein ligands by avb3, avb1 and other integrins, Received for publication February 13, 1997. 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-2hydroxyethylpiperazine-N9-2-ethanesulfonic acid. 0022-3565/97/2832-0843$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 283, No. 2 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 283:843–853, 1997 843 at A PE T Jornals on N ovem er 9, 2017 jpet.asjournals.org D ow nladed from the molecular basis for differences in receptor-ligand specificity remains poorly understood. Investigation of the role of RGD-interactive adhesion molecules has been facilitated by the identification of small RGD-containing proteins derived from snake venoms termed disintegrins (Gould et al., 1990). Disintegrins are a family of naturally occurring, cysteine-rich, small (5–9 kDa) polypeptides that potently inhibit platelet aggregation and cell adhesion (Gould et al., 1990; Niewiaroski et al., 1994). The biological activity of disintegrins depends on the structure of an RGD-containing loop maintained in an appropriate conformation by disulfide bridges. Because they are relatively small (each is 50–80 amino acids), they provide a unique opportunity to gain insight into the three-dimensional structure of RGD-active proteins and the factors that are important in controlling specificity. Echistatin from the venom of Echis carinatus is the smallest (49 amino acids) member of the family and has been the focus of intense research (Gan et al., 1988). Echistatin is believed to bind to the avb3 integrin expressed on osteoclasts (Sato et al., 1990, 1994; Fisher, et al., 1993). Sato et al. (1990) have shown that echistatin inhibits both excavation of bone by rat osteoclasts and the release of H-proline from prelabeled bone particles by chicken osteoclasts (Sato et al., 1994). Because avb3 is the predominant receptor expressed on osteoclasts, these studies suggested that echistatin can bind to integrin avb3. These activities depend on the RGD domain, because substitutions of the arginine of the RGD sequence of the echistatin with alanine resulted in loss of activity (Fisher et al., 1993). However, detailed biochemical studies on the binding of echistatin to avb3 receptor are lacking. This study provides a quantitative biochemical characterization of the binding of echistatin to integrin avb3. In this study, we show that integrin avb3 receptor binds to echistatin with a high affinity both in its purified form and the form expressed on the cell surface. Echistatin can also inhibit the adhesion of human embryonic kidney cells expressing avb3 receptor to vitronectin, which suggests that it can function as an antagonist of the receptor. We also demonstrate that echistatin binds to avb3 in a nondissociable manner similar to vitronectin. Materials and Methods Materials. Human embryonic kidney (HEK 293) cells were obtained from American Type Culture Collection (CRL 1573). DMEM, L-glutamine, nonessential amino acids, gentamycin and synthetic RGD-containing peptides were purchased from Gibco-BRL (Gaithersburg, MD). Fetal bovine serum was from Hazleton Biologicals (Lenox, KS). Octyl-b-D-glucopyranoside and Nonidet P-40 were purchased from Sigma Chemical Company (St. Louis, MO). Microlite-2 plates were obtained from Dynatech Corporation (Chantilly, VA). Multiscreen-FB opaque plates (1.0 mm Glass Fiber Type B filter) were from Millipore (Billerica, MA). Falcon Microtest III microtiter plates are from Falcon (Franklin Lakes, NJ). avb3 specific (LM609) monoclonal antibodies, anti-avb5 specific antibodies (mAb 1961) and LM609-coupled to Affi-Gel matrix were purchased from Chemicon International Inc. (Temecula, CA). Anti av specific monoclonals (12084–018) were from Gibco-BRL, and anti-b3 (550036) and anti-b1 (55034) specific monoclonal antibodies were purchased from Becton Dickinson (Franklin Lakes, NJ). I-Echistatin labeled by the lactoperoxidase method to a specific activity of 2000 Ci/mmol was from Amersham International (Chicago, IL). Echistatin was purchased from Bachem (Torrence, CA). Protein purification. avb3 was purified as described by Orlando and Cheresh (1991). Human placenta was cut into 2-cm pieces and washed with 0.05% Digitonin, 2 mM PMSF, 2 mM CaCl2 in water for 60 min on ice. The placenta was then extracted by incubation with 100 mM octylglucoside, 2 mM CaCl2, 1 mM PMSF in PBS for 60 min on ice. The resulting extract was filtered through sterile gauze and centrifuged at 50,000 3 g for 30 min. The supernatant was then recirculated over LM609-Affi-Gel column overnight at 4°C. The column was washed with 50 column volumes of 0.1% Nonidet P-40, 2 mM CaCl2 in PBS, followed by 50 column volumes of 0.01 M NaHOAc, pH 4.5, 0.1% Nonidet P-40, 2 mM CaCl2, in PBS. avb3 was then eluted with 0.01 M NaHOAc, pH 3.0, 0.1% Nonidet P-40 and 2 mM CaCl2. The column fractions were rapidly neutralized by collecting the fractions directly into 3.0 M Tris, pH 8.8. Fractions containing the receptor, as judged by SDS-PAGE, were pooled and concentrated against high molecular weight Polyethylene glycol. The receptor preparation was then dialyzed against 0.1% Nonidet P-40, 2 mM CaCl2 in PBS and stored at 280°C. The identity and purity of the protein was confirmed by Western blot analysis with monoclonal antibodies specific for av and b3 subunits. Solid-phase receptor binding assay. The receptor binding assay was performed as described previously (Orlando and Cheresh, 1991). avb3 was diluted at 500 ng/ml in coating buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2) and an aliquot of 100 ml/well was added to a 96-well microtiter plate (Microlite-2 from Dynatech) and incubated overnight at 4°C. The plate was washed once with blocking/binding buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 1% bovine serum albumin), and incubated an additional 2 h at room temperature. The plate was rinsed twice with the same buffer and incubated with radiolabeled ligand at the indicated concentrations for 3 h at room temperature. For coincubations, unlabeled competitor was included at the concentrations described. For preincubations, after the 3-h incubation with radiolabeled ligand, the plate was washed three times with blocking/binding buffer and further incubated for indicated times in the presence of either competitor or buffer alone. After an additional three washes, the plates were counted by liquid scintillation method with Top count (Packard, Meriden, CT). When I-ligand incubations were performed without receptor, no interaction was detected because of nonspecific adsorption with the microtiter well. Nonspecific binding of ligand to the receptor was determined with molar excess (200-fold) of the unlabeled ligand. Each data point is a result of the average of triplicate