title: Isolation of alpha 6 beta 1 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, OV-CAR-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 alpha 6 beta 1 integrin by immunoprecipitation with specific antibodies against either the alpha 6 or the beta 1 subunit. The alpha 6 beta 1 integrin did not bind to an affinity column containing fragment P1 originating from a different part of murine laminin which, however, bound the alpha IIb beta 3 integrin from platelets. Furthermore, in immunofluorescence staining, the alpha 6 beta 1 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 alpha 6 beta 1 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 alpha 3 beta 1. When alpha 3 beta 1 was removed from lysates of OVCAR-4 cells by preclearing with an alpha 3-specific monoclonal antibody, alpha 6 beta 1 was able to bind to human laminin as well. Integrin alpha 6 beta 1 on platelets which do not express alpha 3 beta 1 binds directly to human laminin. These results indicate that both alpha 3 beta 1 and alpha 6 beta 1 can act as receptors for human laminin and may interfere by steric hindrance. The alpha 6 beta 4 complex, which is strongly expressed on HBL-100 cells, did not bind to either mouse laminin fragment E8 or human laminin affinity columns.
authors.affiliation: Department of Immunohematology, University of Amsterdam, The Netherlands.
authors.forename: A
authors.initials: A
authors.lastname: Sonnenberg
authors.forename: K R
authors.initials: KR
authors.lastname: Gehlsen
authors.forename: M
authors.initials: M
authors.lastname: Aumailley
authors.forename: R
authors.initials: R
authors.lastname: Timpl
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001792:Blood Platelets / Q000502:physiology*; D002448:Cell Adhesion; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000302:isolation & purification* / Q000378:metabolism; D007797:Laminin / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000138:chemical synthesis; D010434:Pepsin A; D010446:Peptide Fragments
chemical_list: D000911:Antibodies, Monoclonal; D005353:Fibronectins; D016023:Integrins; D007797:Laminin; D009842:Oligopeptides; D010446:Peptide Fragments; D010434:Pepsin A