HEMIDESMOSOME FORMATION IS INITIATED BY THE BETA-4 INTEGRIN SUBUNIT, REQUIRES COMPLEX-FORMATION OF BETA-4 AND HD1 PLECTIN, AND INVOLVES A DIRECT INTERACTION BETWEEN BETA-4 AND THE BULLOUS PEMPHIGOID ANTIGEN-180/

Hemidesmosomes (HDs) are stable anchoring structures that mediate the link between the intermediate filament cytoskeleton and the cell subst ratum. We investigated the contribution of various segments of the bet a 4 integrin cytoplasmic domain in the formation of HDs in transient t ransfection studies using immortalized keratinocytes derived from an e pidermolysis bullosa patient deficient in beta 4 expression, We found that the expression of wild-type beta 4 restored the ability of the be ta 4-deficient cells to form HDs and that distinct domains in the NH2- and COOH-terminal regions of the beta 4 cytoplasmic domain are requir ed for the localization of HD1/plectin and the bullous pemphigoid anti gens 180 (BP180) and 230 (BP230) in these HDs. The tyrosine activation motif located in the connecting segment (CS) of the beta 4 cytoplasmi c domain was dispensable for HD formation, although it may be involved in the efficient localization of BP180. Using the yeast two-hybrid sy stem, we could demonstrate a direct interaction between beta 4 and BP1 80 which involves sequences within the COOH-terminal part of the CS an d the third fibronectin type III (FNIII) repeat. Immunoprecipitation s tudies using COS-7 cells transfected with cDNAs for alpha 6 and beta 4 and a mutant BP180 which lacks the collagenous extracellular domain c onfirmed the interaction of beta 4 with BP180. Nevertheless, beta 4 mu tants which contained the BP180-binding region, but lacked sequences r equired for the localization of HD1/plectin, failed to localize BP180 in HDs. Additional yeast two-hybrid assays indicated that the 85 COOH- terminal residues of beta 4 can interact with the first NH2-terminal p air of FNIII repeats and the CS, suggesting that the cytoplasmic domai n of beta 4 is folded back upon itself. Unfolding of the cytoplasmic d omain may be part of a mechanism by which the interaction of beta 4 wi th other hemidesmosomal components, e.g., BP180, is regulated.