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ADAMs Selectively Modulate Integrin-mediated Cell Migration
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We found that ADAMs inhibit integrin-mediated cell migration in patterns dictated by the integrin binding profiles of their isolated disintegrin domains. ADAMs were expressed in Chinese hamster ovary cells that express defined integrins (α4β1, α5β1, or both), and cell migration on full-length fibronectin https://ethereumcode.io/ or on its α4β1 or α5β1 binding fragments was studied. Here, we tested whether ADAMs can selectively regulate integrin-mediated cell migration.
After three washes, adherent cells were stained with crystal violet and solubilized with 1% SDS. After 45-min incubation on ice, cells were washed twice with PBS, resuspended in 0.3 ml of 2% paraformaldehyde in PBS, and analyzed on a FACSCalibur benchtop analyzer at the Flow Cytometry Core Facility at University of Virginia. Primary antibody (mouse anti-human α4, HP2/1), diluted to 20 μg/ml in blocking buffer (PBS/fetal bovine serum/bovine serum albumin), was incubated with cells for 45 min at 4°C. An experiment with cells transfected to express intracellular GFP showed that the biotinylation protocol did not label intracellular proteins (our unpublished data).
CHO-α4GFP cells transiently expressing ADAM12, ADAM17, ADAM19, or ADAM33 were compared for their relative ability to inhibit wound closure after 12 h on the CS-1 region of FN. In contrast, although some of the ADAM12-transfected cells extended membrane protrusions, these protrusions were less robust and did not lead to effective cell body translocation (Movie 2B). Analysis of movies showed that mock transfected cells displayed broad lamellipodia at the leading edge and the cells moved persistently in the direction of the protrusion (Movie 2A). As seen in Figure 7, mock-transfected cells migrated persistently at a rate of ∼10 μm/h. Twenty-four hours after transfection, cells were labeled with the membrane-impermeable EZ-Link NHS-LC-biotin (Pierce Chemical), and avidin precipitation and immunoblotting were conducted as described in Materials and Methods. CHO-α4/GFP cells were transfected with 4 μg of DNA (per 6-cm dish) encoding the indicated 6Xmyc-tagged constructs.