Isolation of α6β1 integrins from platelets and adherent cells by affinity chromatography on mouse laminin fragment E8 and human laminin pepsin fragment

Abstract

Ligand affinity chromatography was used to identify receptors on platelets and two adherent cell lines, OVCAR-4 and HBL-100, for the E8 fragment of murine laminin. A complex of two polypeptides (140 and 110 kDa nonreduced) was bound by the E8 affinity columns from all three cell types and was eluted with EDTA. This heterodimeric complex was identified as the α6β1 integrin by immunoprecipitation with specific antibodies against either the α6 or the β1 subunit. The α6β1 integrin did not bind to an affinity column containing fragment P1 originating from a different part of murine laminin which, however, bound the αIIbβ3 integrin from platelets. Furthermore, in immunofluorescence staining, the α6β1 integrin localizes in focal contacts of OVCAR-4 cells attached to laminin and E8 but not to fibronectin substrates. These results, combined with previous antibody inhibition studies, unequivocally identify the α6β1 integrin as a specific receptor for fragment E8. Affinity chromatography of OVCAR-4 and HBL-100 cells on a large pepsin fragment of laminin from human placenta yielded integrin α3β1. When α3β1 was removed from lysates of OVCAR-4 cells by preclearing with an α3-specific monoclonal antibody, α6β1 was able to bind to human laminin as well. Integrin α6β1 on platelets which do not express α3β1 binds directly to human laminin. These results indicate that both α3β1 and α6β1 can act as receptors for human laminin and may interfere by steric hindrance. The α6β4 complex, which is strongly expressed on HBL-100 cells, did not bind to either mouse laminin fragment E8 or human laminin affinity columns.