Hemidesmosome formation is initiated by the β4 integrin subunit, requires complex formation of β4 and HD1/plectin, and involves a direct interaction between β4 and the bullous pemphigoid antigen 180

Hemidesmosomes (HDs) are stable anchoring structures that mediate the link between the intermediate filament cytoskeleton and the cell substratum. We investigated the contribution of various segments of the beta 4 integrin cytoplasmic domain in the formation of HDs in transient transfection studies using immortalized keratinocytes derived from an epidermolysis bullosa patient deficient in beta 4 expression, We found that the expression of wild-type beta 4 restored the ability of the beta 4-deficient cells to form HDs and that distinct domains in the NH2- and COOH-terminal regions of the beta 4 cytoplasmic domain are required for the localization of HD1/plectin and the bullous pemphigoid antigens 180 (BP180) and 230 (BP230) in these HDs. The tyrosine activation motif located in the connecting segment (CS) of the beta 4 cytoplasmic domain was dispensable for HD formation, although it may be involved in the efficient localization of BP180. Using the yeast two-hybrid system, we could demonstrate a direct interaction between beta 4 and BP180 which involves sequences within the COOH-terminal part of the CS and the third fibronectin type III (FNIII) repeat. Immunoprecipitation studies using COS-7 cells transfected with cDNAs for alpha 6 and beta 4 and a mutant BP180 which lacks the collagenous extracellular domain confirmed the interaction of beta 4 with BP180. Nevertheless, beta 4 mutants which contained the BP180-binding region, but lacked sequences required 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 pair of FNIII repeats and the CS, suggesting that the cytoplasmic domain of beta 4 is folded back upon itself. Unfolding of the cytoplasmic domain may be part of a mechanism by which the interaction of beta 4 with other hemidesmosomal components, e.g., BP180, is regulated.