Assembly of the β4-Integrin Interactome Based on Proximal Biotinylation in the Presence and Absence of Heterodimerization*

Myllymäki, Satu-Marja; Kämäräinen, Ulla-Reetta; Liu, Xiaonan; Cruz, Sara Pereira; Miettinen, Sini; Vuorela, Mikko; Varjosalo, Markku; Manninen, Aki

doi:10.1074/mcp.ra118.001095

Cited by 21 publications

(24 citation statements)

References 65 publications

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“…The specificity of these β4 interactors was validated by additional BioID experiments using integrin β4– and IL2R-BirA*–expressing keratinocytes in the absence of biotin to exclude any false-positive interactors. These data are in line with a previous study in which some FA components, such as tensin-3, were identified as interactors of integrin β4 ( Myllymäki et al, 2019 ). Moreover, some of these β4 proximity interactors were validated by Western blotting in the presence or absence of biotin ( Fig.…”

Section: Resultssupporting

confidence: 93%

Hemidesmosomes modulate force generation via focal adhesions

Wang

Zuidema

Molder

et al. 2020

Journal of Cell Biology

106

100

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Hemidesmosomes are specialized cell-matrix adhesion structures that are associated with the keratin cytoskeleton. Although the adhesion function of hemidesmosomes has been extensively studied, their role in mechanosignaling and transduction remains largely unexplored. Here, we show that keratinocytes lacking hemidesmosomal integrin α6β4 exhibit increased focal adhesion formation, cell spreading, and traction-force generation. Moreover, disruption of the interaction between α6β4 and intermediate filaments or laminin-332 results in similar phenotypical changes. We further demonstrate that integrin α6β4 regulates the activity of the mechanosensitive transcriptional regulator YAP through inhibition of Rho–ROCK–MLC– and FAK–PI3K–dependent signaling pathways. Additionally, increased tension caused by impaired hemidesmosome assembly leads to a redistribution of integrin αVβ5 from clathrin lattices to focal adhesions. Our results reveal a novel role for hemidesmosomes as regulators of cellular mechanical forces and establish the existence of a mechanical coupling between adhesion complexes.

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Section: Resultssupporting

confidence: 93%

Hemidesmosomes modulate force generation via focal adhesions

Wang

Zuidema

Molder

et al. 2020

Journal of Cell Biology

106

100

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“…We have identified these bands in a recent study using mass spectroscopy as full-length β4-(β4-FL) and α6-subunits, respectively ( Fig. 1B) [23]. The additional <150 kD fragment, precipitating with an antibody binding to the C-terminus of β4-integrin, was identified as a truncated form of β4-integrin (β4-C) that was lacking most of its extracellular domain.…”

Section: Resultsmentioning

confidence: 90%

Cell surface expression of integrin β4-subunit in the absence of α6-subunit

Myllymäki

Manninen

2019

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Laminin-rich basement membrane (BM) guides epithelial cell polarity, regulates epithelial cell behavior and maintains the integrity of epithelial tissues. αβ1-and α6β4-integrins both contribute to laminin adhesion and signaling via the assembly of integrin adhesion complexes that help to orient the apico-basal polarity axis. β4-integrin differs from other integrin subunits due to its large cytoplasmic domain that connects to cellular intermediate filament (IF) networks in specialized adhesions called hemidesmosomes (HD). β4-integrin is only known to form a heterodimer with the α6-subunit. In normal tissues, β4-integrin is expressed in cells that also express the α6-subunit. However, in most cells analyzed, β4-integrin is expressed in large excess over α6-integrin and in some tumor cells, β4-integrin appears to promote tumorigenic signaling despite loss of HDs formation. The fate of free β4-subunit and its potential functions in cells have not been extensively studied. Here, we have studied subcellular localization and potential surface delivery of β4-integrin in the absence of its heterodimer partner α6. We provide evidence that a significant fraction of β4-subunit can reach the cell surface without α6-subunit. We also report that β4 is cleaved at its extracellular domain to produce a membrane-bound proteolytic product with an intact cytoplasmic domain. The processed β4-integrin did not co-precipitate with α6-subunit. Taken together, our data suggest that β4-integrin might have functions that are independent of heterodimer formation.

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“…Scale bars: 10 μm. (D) Venn diagram comparing the α6β4 interactome in keratinocytes with that of β4 in MDCK cells ( Myllymäki et al, 2019 ). Numbers of overlapping and specific proteins are presented.…”

Section: Resultsmentioning

confidence: 99%

“…In simple epithelial tissues and cell lines, such as intestinal and mammary gland cells, integrin α6β4 typically resides in type II HDs ( Uematsu et al, 1994 ; Fontao et al, 1997 ). The group of Aki Manninen used BioID to identify 91 significant cytoplasmic proximity interactors of α6β4 in Madin Darby Canine kidney epithelial cells ( Myllymäki et al, 2019 ) . We compared these interactors with the interactors identified for α6β4 in keratinocytes and found that only 30 proteins were common between the two data sets ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…However, while in the keratinocytes all CMSC members were identified, in the kidney cells only LL5α/β and ERC1 were identified. This relatively small overlap in the two interactomes might be explained by the use of different cell lines and/or the subcellular localization of integrin α6β4 in type I versus type II HDs ( Myllymäki et al, 2019 ). However, it could also have a technical reason, since it has been shown that proteomes identified by different hands and methods can differ considerably ( Horton et al, 2016 ).…”

Section: Discussionmentioning

confidence: 99%

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Comparative interactomics analysis reveals potential regulators of α6β4 distribution in keratinocytes

et al. 2020

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The integrin α6β4 and cytoskeletal adaptor plectin are essential components of type I and type II hemidesmosomes (HDs). We recently identified an alternative type II HD adhesion complex that also contains CD151 and the integrin α3β1. Here, we have taken a BioID proximity labeling approach to define the proximity protein environment for α6β4 in keratinocytes. We identified 37 proteins that interacted with both α6 and β4, while 20 and 78 proteins specifically interacted with the α6 and β4 subunits, respectively. Many of the proximity interactors of α6β4 are components of focal adhesions (FAs) and the cortical microtubule stabilizing complex (CMSC). Though the close association of CMSCs with α6β4 in HDs was confirmed by immunofluorescence analysis, CMSCs have no role in the assembly of HDs. Analysis of the β4 interactome in the presence or absence of CD151 revealed that they are strikingly similar; only 11 different interactors were identified. One of these was the integrin α3β1, which interacted with α6β4 more strongly in the presence of CD151 than in its absence. These findings indicate that CD151 does not significantly contribute to the interactome of α6β4, but suggest a role of CD151 in linking α3β1 and α6β4 together in tetraspanin adhesion structures.

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Assembly of the β4-Integrin Interactome Based on Proximal Biotinylation in the Presence and Absence of Heterodimerization*

Cited by 21 publications

References 65 publications

Hemidesmosomes modulate force generation via focal adhesions

Hemidesmosomes modulate force generation via focal adhesions

Cell surface expression of integrin β4-subunit in the absence of α6-subunit

Comparative interactomics analysis reveals potential regulators of α6β4 distribution in keratinocytes

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