Utilization of a Soluble Integrin-Alkaline Phosphatase Chimera To Characterize Integrin R8â1 Receptor Interactions with Tenascin: Murine R8â1 Binds to the

ABSTRACT: The integrin R8â1 has been reported to bind to fibronectin, vitronectin, and tenascin-C in cell adhesion or neurite outgrowth assays. Here, we describe cDNA cloning of the murine R8 subunit, purification of a recombinant soluble heterodimer consisting of the extracellular domains of the murine R8 and â1 subunits, and development of a sensitive binding assay using a modified form of this heterodimer fused to alkaline phosphatase (AP). In binding assays, the purified R8â1-AP chimera exhibited the same divalent ion requirements for activation and binding specificity as cell surface R8â1: in the presence of Mn2+ it bound to fibronectin and vitronectin in an RGDS-peptide inhibitable manner. Contrary to previous reports, we found no evidence that R8â1, expressed on K562 cells or as an AP chimera, interacts strongly with native tenascin-C. In binding, adhesion, and spreading assays, significant interactions were observed only to short fragments of tenascin-C containing the third fibronectin type III repeat which contains an RGD sequence. Full length tenascin-C and longer fragments containing this repeat did not appear to serve as ligands, implying that the RGD site in native tenascin-C is a cryptic binding site for this integrin, exposed by removal of adjacent domains. Soluble integrin-AP chimeras should be generally useful for identifying and characterizing integrin interactions with ligands. Integrins are a family of noncovalently linked Râ het