Thy-1 binds to integrin beta(3) 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 putat ive Thy-1 ligand that prevents neurite outgrowth. In this paper, a ligand m olecule 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 sequenc e. Thy-1 interaction with beta3 integrin on: astrocytes was demonstrated in an adhesion assay using a thymoma line (EL-4) expressing high levels of Th y-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 an ti-beta3 antibodies, by RGD-related peptides, or by soluble Thy-1-Fc chimer as. However, neither RGE/RLE peptides nor Thy-1 (RLE)-Fc fusion protein inh ibited the interaction. Immobilized Thy-l-Fc, but not Thy-1 (RLE)-Fc fusion protein supported the attachment and spreading of astrocytes in a Mn2+-dep endent manner. Binding to Thy-1-Fc was inhibited by RGD peptides. Moreover, vitronectin, fibrinogen, denatured collagen (dcollagen), and a kistrin-der ived peptide but not fibronectin, also mediated Mn2+-dependent adhesion, su ggesting the involvement of beta3 integrin. The addition of Thy-1 to matrix -bound astrocytes induced recruitment of paxillin, vinculin, and: focal adh esion 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 formati on and phosphorylation on tyrosine. Conclusions: Thy-1 binds to beta3 integrin and triggers tyrosine phosphoryl ation of focal adhesion proteins in: astrocytes, thereby promoting focal ad hesion formation, cell; attachment, and spreading.