INTEGRIN ALPHA-6-BETA-4 FORMS A COMPLEX WITH THE CYTOSKELETAL PROTEINHD1 AND INDUCES ITS REDISTRIBUTION IN TRANSFECTED COS-7 CELLS

The integrin alpha 6 beta 4 is a major component of hemidesmosomes, in which it is linked to intermediate filaments. Its presence in these s tructures is dependent on the beta 4 cytoplasmic domain but it is not known whether beta 4 interacts directly with keratin filaments or by i nteraction with other proteins. In this study, we have investigated th e interaction of GST-cyto beta 4A fusion proteins with cellular protei ns and demonstrate that a fragment of beta 4A, consisting of the two p airs of fibronectin type III repeats, separated by the connecting segm ent, forms a specific complex containing a 500-kDa protein that comigr ates with HD1, a hemidesmosomal plaque protein. A similar protein was also bound by a glutathione S-transferase fusion protein containing th e cytoplasmic domain of a variant beta 4 subunit (beta 4B), in which a stretch of 53 amino acids is inserted in the connecting segment. Subs equent immunoblot analysis revealed that the 500-kDa protein is in fac t HD1. In COS-7 cells, which do not express alpha 6 beta 4 or the hemi desmosomal components BP230 and BP180, HD1 is associated with the cyto skeleton, 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, ac tin, and tubulin cytoskeletal networks was not affected by the express ion of alpha 6 beta 4 in COS-7 cells. The localization of HD1 at the b asal 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 wi th a mutant beta 4 subunit that lacks the four fibronectin type III re peats 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.