Abstract
The basement membrane protein laminin-332 (laminin-5) mediates both stable cell adhesion and rapid cell migration and thus has the potential to either restrain or promote tumor cell metastasis. The major cellular receptors for laminin-332 are integrin α3β1, which mediates rapid tumor cell migration, and integrin α6β4, which often mediates stable cell attachment. Tetraspanin protein CD151 interacts directly with both α3β1 and α6β4 integrins and with other tetraspanins, thereby promoting α3β1 and α6β4 association with tetraspanin-enriched microdomains on the cell surface. To explore the possibility of selectively modulating tumor cell responses to laminin-332, we re-expressed a series of CD151 mutants in epidermoid carcinoma cells with near total, RNAi-mediated silencing of endogenous CD151. The interactions of CD151 with its integrin partners or its interactions with other tetraspanins were selectively disrupted by specific mutations in the CD151 large extracellular loop (EC2 domain) or in intracellular CD151 palmitoylation sites, respectively. CD151-integrin association and CD151-tetraspanin association were both important for α3β1 integrin-dependent initial adhesion and rapid migration on laminin-332. Remarkably, however, only CD151-integrin association was required for stable, α6β4 integrin-dependent cell attachment on laminin-332. In addition, we found that a QRD amino acid motif in the CD151 EC2 domain, which had been thought to be crucial for CD151-integrin interaction, is not essential for CD151-integrin association or for the ability of CD151 to promote several different integrin functions. These new data suggest potential strategies for selectively modulating migratory cell responses to laminin-332, while leaving stable cell attachment on laminin-332 intact.
Highlights
Skin epithelial integrity, wound healing, platelet aggregation, and thrombus growth and stability, as well as reduced pathological angiogenesis [1,2,3,4,5,6,7]
CD151 function in platelets has been linked to the platelet integrin ␣IIb3 [3], the basis of CD151 functions in carcinomas and in normal epithelia may be its association with its major integrin partners ␣31, ␣61, and ␣64
CD151 promotes the association of its integrin partners with other tetraspanins and tetraspanin-associated proteins in cell surface microdomains referred to as tetraspanin-enriched microdomains (TEMs)2 [15]
Summary
Antibodies and Extracellular Matrix Proteins—Anti-integrin mAbs used in this study were anti-␣3, A3-X8, and A3-IIF5 [25]; anti-␣6, A6-ELE [26]; and GoH3 (GeneTex, San Antonio, TX). After three rinses in cold PBS, cells were stained on ice for 45 min with a Cy2-conjugated goat antimouse secondary antibody (Jackson ImmunoResearch Laboratories) diluted 1:200 in blocking buffer. Cells or substrates were stained for 1 h with primary antibodies in blocking buffer and for 45 min with Cy2 goat anti-mouse antibody. Cells remaining after rinses were fixed for 30 min at room temperature with 10% formalin fixative containing 2 mM MgCl2, followed by a 20-min stain with freshly filtered 0.1% crystal violet in double distilled H2O. Cells were fixed, stained with crystal violet, destained, and analyzed as described above for adhesion assays, except that total cells input was determined using wells that were not treated with trypsin-EDTA
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
