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Many recombinant ADAM disintegrin domains have been shown to interact with a variety of β1 integrins, including α4β1 (Tomczuk et al., 2003; White, 2003; Bridges et al., 2004; White et al., 2005). These domains have been implicated in interactions with integrins and syndecans, respectively. Downstream of their metalloprotease domains, ADAMs contain a disintegrin domain and a cysteine-rich domain. ADAMs shed a variety of important cell surface molecules, including cytokines (e.g., tumor necrosis factor-α), growth factors (e.g., epidermal growth factor family members), and adhesion molecules (e.g., L1, selectins, and vascular cell adhesion molecule) (Blobel, 2005). The integrin α4β1 engages the CS-1 region of FN and promotes lamellipodia protrusion in a focal adhesion-independent manner (Pinco et al., 2002; Goldfinger et al., 2003; Mostafavi-Pour et al., 2003).

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ADAM12 Inhibits Integrin α4β1-mediated Random Cell Motility

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Microscopy of mock- or ADAM12-transfected CHO-α4/GFP cells in scratch wound migration assays. (A) CHO-α4/GFP cells, which express both the α4β1 and α5β1 integrins, were either mock transfected or transfected to express full-length ADAM12. Full-length ADAM12 inhibits the migration of CHO-α4/GFP cells on the α4β1 binding CS-1 region but not on the α5β1 binding CCBD of fibronectin. In contrast, expression of ADAM12 had no significant effect on the migration of the same cells (CHO-α4/GFP) on the α5β1 substrate (Figure 3C).

To produce protein with higher purity, a 6Xhistidine (His) tag was added to the 3′ end of the disintegrin domain coding sequence, using a site-directed mutagenesis kit (Stratagene, La https://aberturasaconcagua.com/ Jolla, CA). Mouse ADAM12 disintegrin domain in the vector pGEX-2T was provided by Anna Zolkiewski (Kansas State University, Manhattan, KS). Mouse anti-α4 integrin functional blocking monoclonal antibody (mAb) HP2/1 and mouse anti-α5 integrin functional blocking antibody CBL497 were obtained from Chemicon International (Temecula, CA).

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