Gene correction of integrin β4-dependent pyloric atresia-functional epidermolysis bullosa keratinocytes establishes a role for β4 tyrosines 1422 and 1440 in hemidesmosome assembly.

The cytoplasmic domain of beta (4) integrin contains two pairs of fibronectin-like repeats separated by a connecting segment. The connecting segment harbors a putative tyrosine activation motif in which tyrosines 1422 and 1440 are phosphorylated in response to alpha (6)beta (4) binding to laminin-5. Primary beta (4)-null keratinocytes, obtained from a newborn suffering from lethal junctional epidermolysis bullosa, were stably transduced with retroviruses carrying a full-length beta (4) cDNA or a beta (4) cDNA with phenylalanine substitutions at Tyr-1422 and Tyr-1440. Hemidesmosome assembly was evaluated on organotypic skin cultures. beta (4)-corrected keratinocytes were indistinguishable from normal cells in terms of alpha (6)beta (4) expression, the localization of hemidesmosome components, and hemidesmosome structure and density, suggesting full genetic and functional correction of beta (4)-null keratinocytes. In cultures generated from beta (Y1422F/Y1440F)(4) keratinocytes, beta (4) mutants as well as a, integrin, HD1/plectin, and BP180 were not concentrated at the dermal-epidermal junction. Furthermore, the number of hemidesmosomes was strikingly reduced as compared with beta (4)-corrected keratinocytes. The rare hemidesmosomes detected in beta (Y1422F/Y1440F)(4) cells were devoid of sub-basal dense plates and of inner cytoplasmic plaques with keratin filament insertion. Collectively, our data demonstrate that the beta (4) tyrosine activation motif is not required for the localization of alpha (6)beta (4) at the keratinocyte plasma membrane but is essential for optimal assembly of bona fide hemidesmosomes.