Journal: The Journal of Biological Chemistry
Article Title: The cell surface hyaluronidase TMEM2 regulates cell adhesion and migration via degradation of hyaluronan at focal adhesion sites
doi: 10.1016/j.jbc.2021.100481
Figure Lengend Snippet: Association of transmembrane protein 2 (TMEM2) with integrins via interactions between the extracellular domains. A and B , targeting of TMEM2 to focal adhesions (FAs) does not require the cytoplasmic domain of TMEM2. In this experiment, mCherry-mTMEM2 ( full length ) and mCherry-mTMEM2/Δcyto (Δ cyto ) cells were analyzed for their in situ hyaluronan (HA) degradation activities. To allow specific analysis of the activity of the full-length mouse TMEM2 and its Δcyto deletion mutant, expression of endogenous human TMEM2 was silenced by siRNA treatment prior to the assay. A , in situ HA degradation assays were performed on substrate immobilized with FA-HA, as described in section. Note that the pattern of in situ HA degradation is indistinguishable between mCherry-mTMEM2/Δcyto and mCherry-mTMEM2 cells. The scale bar represents 10 μm. B , immunostaining for vinculin in mCherry-mTMEM2 and mCherry-mTMEM2/Δcyto cells on the FA-HA substrate. Note that the sites of HA degradation colocalize with vinculin-immunoreactive puncta in both mCherry-mTMEM2/Δcyto and mCherry-mTMEM2 cells. The scale bar represents 2 μm. C – E , TMEM2 associates with integrins via extracellular interactions. C , cell surface–expressed TMEM2 is coimmunoprecipitated with integrin α5β1. mCherry-mTMEM2 cells were treated with the membrane-impermeable crosslinker 3',3'-dithiobis(sulfosuccinimidyl propionate), and the lysates from these cells were immunoprecipitated with anti-mCherry antibody, followed by immunoblotting analysis with antibodies to α5, β1, and mCherry. D , the extracellular domain of TMEM2 directly binds α5β1. Binding between TMEM2 extracellular domain (ECD) and the α5β1 ECD heterodimer was analyzed by a pull-down assay. E , the ECD of TMEM2 directly binds αLβ2 (lymphocyte function-associated antigen-1). Binding between TMEM2 ECD and the αLβ2 ECD heterodimer was analyzed by a pull-down assay.
Article Snippet: Two micrograms of recombinant heterodimer of integrin ECD (α5β1; R&D Systems: 3230-A5-050; αLβ2; R&D Systems: 3868-AV-050) were applied to the TMEM2–ECD–bound and control unbound resin and incubated in HBSS ++ overnight at 4 °C.
Techniques: In Situ, Activity Assay, Mutagenesis, Expressing, Immunostaining, Membrane, Immunoprecipitation, Western Blot, Binding Assay, Pull Down Assay
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