The 3′UTR of the α6 integrin message regulates localization of α6β4 integrin heterodimers

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Highlights

  • The 3’UTR of the α6 integrin message contains a zipcode involved in targeting α6β4 integrin heterodimers to sites of matrix adhesion in lung epithelial cells.

  • In epidermal keratinocytes depleted in endogenous α6 integrin protein, the protein product of α6 integrin message lacking a 3’UTR interacts with β1 integrin and not β4 integrin.

  • In epidermal keratinocytes depleted in endogenous α6 integrin protein, re-expression of α6β4 integrin heterodimers only occurs when exogenously expressed α6 integrin mRNA contains its 3’UTR.

Abstract

The α6β4 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 α6 integrin 3′ untranslated region (UTR) in α6β4 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 α6 integrin protein with or without portions of its 3′UTR. In infected A549 cells, we detected α6β4 integrin heterodimers containing the product of the adenovirus, regardless of whether the α6 integrin 3′UTR was present. However, only those α6 integrin proteins whose messages contained bases 4770–5633 of the α6 integrin 3′UTR were targeted to matrix adhesion sites. Moreover, overexpression of the full length α6 integrin 3′UTR, minus the coding sequence, in A549 cells disrupts the localization of endogenous α6β4 integrin heterodimers. Following infection of iHEKs with the same adenovirus, the induced α6 integrin protein localizes to HDs regardless of whether its message possessed a 3′UTR. In sharp contrast, in α6 integrin depleted iHEKs, restoring α6 integrin expression using the coding sequence alone via adenoviral transduction resulted in α6 integrin preferentially forming α6β1 rather than α6β4 integrin heterodimers. α6β4 integrin was only observed in knocked down cells following infection of adenovirus encoding the α6 integrin coding sequence with its 3′UTR. In summary, our data indicate that the α6 integrin 3′UTR is a key regulator of α6β4 integrin heterodimer assembly and incorporation at sites of cell-matrix adhesion.

Introduction

Cell adhesion and migration are critical processes for normal cell functioning, and these processes are aided by cell-matrix adhesion protein complexes: hemidesmosomes (HDs) and focal adhesions (FAs). The core of these complexes contains integrin heterodimers which interact with both the extracellular matrix and other cytoplasmic elements [1]. Each integrin heterodimer is a transmembrane, non-covalently bound complex that contains one α subunit and one β subunit. There are 18 α subunits and 8 β subunits which can form a variety of heterodimers, for example α2β1, α3β1, αvβ1, and α2β5. However, only 24 different heterodimers have been identified [2]. The α6β4 integrin heterodimer is unusual, not only because of the large size of the β4 integrin cytoplasmic tail, but also because it is only found in HDs and HD-related matrix adhesions, rather than in FAs [3]. HDs adhere epithelial cells to the extracellular matrix in several tissues including the skin, cornea, bladder, and some glands. In contrast, HD-related adhesions are found in certain alveolar cells in the lung and gut epithelial cells where they are presumed to mediate adhesion to the underlying connective tissue [[4], [5], [6]].

The α6 integrin subunit has two possible β subunit partners: β1 and β4 integrin. In cells which express both β1 and β4 integrin, α6 integrin preferentially pairs with β4, but how this is controlled is unclear [7,8]. Moreover, the targeting mechanism of α6β4 integrin to HD and HD-related matrix adhesion sites over other substrate attachment sites, including FAs, is also not understood. In this study, we have identified the 3′ untranslated region (UTR) of the α6 integrin messenger RNA (mRNA) as a key regulator of the localization of α6β4 integrin heterodimers as well as the formation of α6β4 versus α6β1 integrin heterodimers. In this regard, the 3′UTRs of mRNAs are known to regulate the shuttling of mRNAs to their site of translation by a sequence known as a zipcode. Although zipcode sequences can be located within coding regions of mRNA, generally, they are located within the 3′UTR [9]. For example, the zipcode in the β-actin message directs it to the site of FAs where it is translated [10]. Likewise, zipcodes in vinculin, actinin-4, and α3 integrin mRNAs direct their translation to the FA [11,12]. In this study we identified a region of the 3′UTR of α6 integrin mRNA which has zipcode characteristics and demonstrate the importance of the α6 integrin 3′UTR in regulating assembly and localization of α6β4 integrin heterodimers.

Section snippets

Cell culture conditions

A549s were cultured in HyClone MEM/EBSS media (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 2% l-glutamine, and 1% penicillin/streptomycin mixture. Immortalized human epithelial keratinocytes (iHEKs) were cultured in defined K-SFM keratinocyte media (DKSFM) (Thermo Fisher, Waltham, MA) supplemented with keratinocyte supplement and 1% penicillin/streptomycin mixture. Cells were maintained at 37 °C in 8% humidified CO2. iHEKs, exhibiting

Results and discussion

A zipcode within the α6 integrin 3′UTR is required for α6β4 heterodimer localization in A549 cells.

Zipcodes are sequences within 3′UTRs of mRNAs that direct their localization and subsequent translation. For example, the zipcode of α3 integrin was demonstrated to be necessary for α3 integrin protein translation and incorporation into FAs [12]. We therefore posited that the α6 integrin 3′UTR might be an important determinant of targeting α6β4 integrin heterodimers to HDs and HD-related

Conflicts of interest

No conflict of interests is noted by any of the listed authors.

Acknowledgements

We would like to thank Kevin Hamill of the University of Liverpool for helpful discussion. This work was begun while the senior author was a faculty member at the Feinberg School of Medicine, Northwestern University, Chicago, IL.

References (18)

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