title: Integrin alpha 6 beta 4 forms a complex with the cytoskeletal protein HD1 and induces its redistribution in transfected COS-7 cells.
abstract: The integrin alpha 6 beta 4 is a major component of hemidesmosomes, in which it is linked to intermediate filaments. Its presence in these structures is dependent on the beta 4 cytoplasmic domain but it is not known whether beta 4 interacts directly with keratin filaments or by interaction with other proteins. In this study, we have investigated the interaction of GST-cyto beta 4A fusion proteins with cellular proteins and demonstrate that a fragment of beta 4A, consisting of the two pairs of fibronectin type III repeats, separated by the connecting segment, forms a specific complex containing a 500-kDa protein that comigrates with HD1, a hemidesmosomal plaque protein. A similar protein was also bound by a glutathione S-transferase fusion protein containing the cytoplasmic domain of a variant beta 4 subunit (beta 4B), in which a stretch of 53 amino acids is inserted in the connecting segment. Subsequent immunoblot analysis revealed that the 500-kDa protein is in fact HD1. In COS-7 cells, which do not express alpha 6 beta 4 or the hemidesmosomal components BP230 and BP180, HD1 is associated with the cytoskeleton, but after transfecting the cells with cDNAs for human alpha 6 and beta 4, it was, instead, colocalized with alpha 6 beta 4 at the basal side of the cells. The organization of the vimentin, keratin, actin, and tubulin cytoskeletal networks was not affected by the expression of alpha 6 beta 4 in COS-7 cells. The localization of HD1 at the basal side of the cells depends on the same region of beta 4 that forms a complex containing HD1 in vitro, since the expression of alpha 6 with a mutant beta 4 subunit that lacks the four fibronectin type III repeats and the connecting segment did not alter the distribution of HD1. The results indicate that for association of alpha 6 beta 4 with HD1, the cytoplasmic domain of beta 4 is essential. We suggest that this association may be crucial for hemidesmosome assembly.
authors.affiliation: Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
authors.forename: C M
authors.initials: CM
authors.lastname: Niessen
authors.forename: E H
authors.initials: EH
authors.lastname: Hulsman
authors.forename: E S
authors.initials: ES
authors.lastname: Rots
authors.forename: P
authors.initials: P
authors.lastname: Sánchez-Aparicio
authors.forename: A
authors.initials: A
authors.lastname: Sonnenberg
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D039161:Integrin alpha6beta4; D016023:Integrins / Q000235:genetics / Q000378:metabolism*; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D051190:Plectin; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D000954:Antigens, Surface; D003598:Cytoskeletal Proteins; D039161:Integrin alpha6beta4; D016023:Integrins; D007381:Intermediate Filament Proteins; C495332:PLEC protein, human; C495333:Plec protein, mouse; D051190:Plectin; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase