Abstract
Integrins are cell-surface glycoproteins that mediate cell activities, including tissue morphogenesis, development, immune response, and cancer, through interaction with extracellular proteins. Here we report a novel means by which integrin signaling and functions are regulated. In pull-down assays and immunoprecipitation, beta(1)-integrin bound to the C-terminal domain of PG-M/versican, an extracellular chondroitin sulfate proteoglycan. This was confirmed by cell-surface binding assays. Binding was calcium- and manganese-dependent. Upon native gel electrophoresis, beta(1)-integrin comigrated with the C-terminal domain of PG-M/versican. The interaction of beta(1)-integrin with the C-terminal domain of PG-M/versican activated focal adhesion kinase, enhanced integrin expression, and promoted cell adhesion. As a result, cells expressing the C-terminal domain of PG-M/versican were resistant to free radical-induced apoptosis. As the PG-M/versican peptide used in this study does not contain the RGD consensus-binding motif for integrins, the mechanism of the observed binding represents an entirely new function.
Highlights
Integrins are the major cell-surface receptors expressed by all cell types
The G3 Domain of PG-M/Versican Enhances 1-Integrin Expression and Protects Cells from Free Radical-induced Apoptosis—As integrin expression is always associated with cell adhesion, we examined whether G3⌬epidermal growth factor (EGF)-induced cell adhesion affects integrin expression
As integrin-mediated cell adhesion is essential for cell survival [1,2,3,4,5,6,7], we investigated whether G3 fragment lacking the two EGFlike motifs (G3⌬EGF)-induced cell adhesion and integrin expression can promote cell viability
Summary
Materials—Lipofectin, Geneticin (G418), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Hanks’ balanced salt solution, and trypsin/EDTA were from Invitrogen (Burlington, Ontario, Canada). The cell lysate was prepared in lysis buffer for phosphorylation assay (50 mM HEPES (pH 7.5), 0.5% Triton X-100, 100 mM NaF, 10 mM sodium phosphate, 4 mM EDTA, 2 mM sodium vanadate, 2 mM sodium molybdate, 2 g/ml aprotinin, 2 g/ml leupeptin, and 2 g/ml phenylmethylsulfonyl fluoride), followed by incubation with protein G that had been presaturated overnight with anti-FAK monoclonal antibody (Transduction Laboratories) at 4 °C. The beads were boiled in 1ϫ protein loading dye for 5 min, followed by Western blot analysis and probing with anti-FAK or anti-phosphotyrosine (clone PY-20, Transduction Laboratories) antibody to detect FAK phosphorylation. U87 cells (5 ϫ 104 cells) were incubated with the culture medium containing purified His6-G3 or eluate from the vector control in the presence of 2 mM each CaCl2 and MnCl2 at room temperature for 30 min. Cells were subjected to annexin V-FITC staining (15 min in the dark) using an annexin V apoptosis detection kit (Santa Cruz Biotechnology, Inc.) to monitor apoptosis as described [37, 38]
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