The alpha 6 beta 4 integrin heterodimer is an essential component of hemidesmosomes (HDs) and HD-related structures, which adhere epithelial cells to the underlying extracellular matrix. In this study, we focused on the importance of the alpha 6 integrin 3' untranslated region (UTR) in alpha 6 beta 4 integrin localization. To do so, A549 cells (a type II lung alveolar cell line) and immortalized human epidermal keratinocytes (iHEK) were infected with adenovirus encoding the entire alpha 6 integrin protein with or without portions of its 3'UTR. In infected A549 cells, we detected alpha 6 beta 4 integrin heterodimers containing the product of the adenovirus, regardless of whether the alpha 6 integrin 3'UTR was present. However, only those alpha 6 integrin proteins whose messages contained bases 4770-5633 of the alpha 6 integrin 3'UTR were targeted to matrix adhesion sites. Moreover, overexpression of the full length a6 integrin 3'UTR, minus the coding sequence, in A549 cells disrupts the localization of endogenous alpha 6 beta 4 integrin heterodimers. Following infection of iHEKs with the same adenovirus, the induced alpha 6 integrin protein localizes to HDs regardless of whether its message possessed a 3'UTR. In sharp contrast, in alpha 6 integrin depleted iHEKs, restoring alpha 6 integrin expression using the coding sequence alone via adenoviral transduction resulted in alpha 6 integrin preferentially forming alpha 6 beta 1 rather than alpha 6 beta 4 integrin heterodimers. alpha 6 beta 4 integrin was only observed in knocked down cells following infection of adenovirus encoding the alpha 6 integrin coding sequence with its 3'UTR. In summary, our data indicate that the alpha 6 integrin 3'UTR is a key regulator of alpha 6 beta 4 integrin heterodimer assembly and incorporation at sites of cell-matrix adhesion. (C) 2019 Elsevier Inc. All rights reserved.