Thy-1 binds to integrin β3 on astrocytes and triggers formation of focal contact sites

Background: Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Astrocytes, ubiquitous cells of the brain, express a putative Thy-1 ligand that prevents neurite outgrowth. In this paper, a ligand molecule for Thy-1 was identified, and the consequences of Thy-1 binding for astrocyte function were investigated. Results: Thy-1 has been implicated in cell adhesion and, indeed, all known Thy-1 sequences were found to contain an integrin binding, RGD-like sequence. Thy-1 interaction with beta3 integrin on: astrocytes was demonstrated in an adhesion assay using a thymoma line (EL-4) expressing high levels of Thy-1. EL-4 cells bound to astrocytes five times more readily than EL-4(-1), control cells lacking Thy-1. Binding was blocked by either anti-Thy-1 or anti-beta3 antibodies, by RGD-related peptides, or by soluble Thy-1-Fc chimeras. However, neither RGE/RLE peptides nor Thy-1 (RLE)-Fc fusion protein inhibited the interaction. Immobilized Thy-l-Fc, but not Thy-1 (RLE)-Fc fusion protein supported the attachment and spreading of astrocytes in a Mn2+-dependent manner. Binding to Thy-1-Fc was inhibited by RGD peptides. Moreover, vitronectin, fibrinogen, denatured collagen (dcollagen), and a kistrin-derived peptide but not fibronectin, also mediated Mn2+-dependent adhesion, suggesting the involvement of beta3 integrin. The addition of Thy-1 to matrix-bound astrocytes induced recruitment of paxillin, vinculin, and: focal adhesion kinase (FAK) to focal contacts and increased tyrosine phosphorylation of proteins such as p 130(Cas) and FAK. Furthermore, astrocyte binding to immobilized Thy-l-Fc alone was sufficient to promote focal adhesion formation and phosphorylation on tyrosine. Conclusions: Thy-1 binds to beta3 integrin and triggers tyrosine phosphorylation of focal adhesion proteins in: astrocytes, thereby promoting focal adhesion formation, cell; attachment, and spreading.