UTILIZATION OF A SOLUBLE INTEGRIN-ALKALINE PHOSPHATASE CHIMERA TO CHARACTERIZE INTEGRIN ALPHA-8-BETA-1 RECEPTOR INTERACTIONS WITH TENASCIN - MURINE ALPHA-8-BETA-1 BINDS TO THE RGD SITE IN TENASCIN-C FRAGMENTS,BUT NOT TO NATIVE TENASCIN-C

The integrin alpha 8 beta 1 has been reported to bind to fibronectin, vitronectin, and tenascin-C in cell adhesion or neurite outgrowth assa ys. Here, we describe cDNA cloning of the murine alpha 8 subunit, puri fication of a recombinant soluble heterodimer consisting of the extrac ellular domains of the murine alpha 8 and beta 1 subunits, and develop ment of a sensitive binding assay using a modified form of this hetero dimer fused to alkaline phosphatase (AP). In binding assays, the purif ied alpha 8 beta 1-AP chimera exhibited the same divalent ion requirem ents for activation and binding specificity as cell surface alpha 8 be ta 1: in the presence of Mn2+ it bound to fibronectin and vitronectin in an RGDS-peptide inhibitable manner. Contrary to previous reports, w e found no evidence that alpha 8 beta 1, expressed on K562 cells or as an AP chimera, interacts strongly with native tenascin-C. In binding, adhesion, and spreading assays, significant interactions were observe d only to short fragments of tenascin-C containing the third fibronect in type III repeat which contains an RGD sequence. Full length tenasci n-C and longer fragments containing this repeat did not appear to serv e as ligands, implying that the RGD site in native tenascin-C is a cry ptic binding site for this integrin, exposed by removal of adjacent do mains. Soluble integrin-AP chimeras should be generally useful for ide ntifying and characterizing integrin interactions with ligands.