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.