The cytoplasmic domain of β4 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 α6β4 binding to laminin-5. Primary β4-null keratinocytes, obtained from a newborn suffering from lethal junctional epidermolysis bullosa, were stably transduced with retroviruses carrying a full-length β4 cDNA or a β4 cDNA with phenylalanine substitutions at Tyr-1422 and Tyr-1440. Hemidesmosome assembly was evaluated on organotypic skin cultures. β4-corrected keratinocytes were indistinguishable from normal cells in terms of α6β4 expression, the localization of hemidesmosome components, and hemidesmosome structure and density, suggesting full genetic and functional correction of β 4-null keratinocytes. In cultures generated from β 4Y1422F/Y1440F keratinocytes, β4 mutants as well as α6 integrin, HD1/plectin, and BP180 were not concentrated at the dermal-epidermal junction. Furthermore, the number of hemidesmosomes was strikingly reduced as compared with β 4-corrected keratinocytes. The rare hemidesmosomes detected in β4Y1422F/Y1440F cells were devoid of subbasal dense plates and of inner cytoplasmic plaques with keratin filament insertion. Collectively, our data demonstrate that the β4 tyrosine activation motif is not required for the localization of α 6β4 at the keratinocyte plasma membrane but is essential for optimal assembly of bona fide hemidesmosomes.
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