EVALUATION OF INTEGRIN MOLECULES INVOLVED IN SUBSTRATE ADHESION

Integrins were cross-linked to their extracellular matrix ligands usin g non-penetrating chemical cross-linkers. This procedure did not distu rb the distribution of integrin in the adhesion structure and adhesion plaque integrin staining remained even when the cultures were extract ed with ionic detergents. 80-90% of the beta1 integrin in the cross-li nked culture was extracted with RIPA buffer and the remaining 10-20% w as recovered following reversal of the cross-linking. This separated t wo distinct integrin pools, one which can be cross-linked to substrate bound extracellular matrix and one which is not. The specificity of t his procedure for cross-linking of integrins involved in substrate adh esion was demonstrated using NIH 3T3 cells which express both alpha5be ta1 and alpha6beta1 integrins. Alpha6 was cross-linked only in cells p lated on laminin whereas alpha5 was cross-linked when fibronectin was present. Using antisera directed to the cytoplasmic domains of either alpha5 or beta1 integrin, it was demonstrated that these domains can b e blocked in the intact cell but the blocking can be removed using ion ic detergent extraction after chemical cross-linking. The extracellula r matrix associated with the substrate surface but not that associated with the media exposed surface is both cross-linked and retained on t he plastic dish following cross-linking.