SPECIFIC ASSOCIATION OF CD63 WITH THE VLA-3 AND VLA-6 INTEGRINS

We screened monoclonal antibodies to cell surface proteins and selected an antibody, called 6H1, that recognizes a putative integrin-associated protein. The 6H1 monoclonal antibody (mAb) indirectly coprecipitated alpha(3) beta(1) and/or alpha(6) beta(1) but not alpha(2) beta(1), or alpha(5) beta(1) from Brij 96 detergent lysates of multiple cell lines. Large scale purification using the 6H1 mAb yielded a single protein of 45-60 kDa with an amino-terminal sequence that exactly matched CD63. Confirming that the 6H1 mAb recognized the CD63 protein, 6H1 and a known anti-CD63 mAb yielded identical coprecipitation results and identical colocalization into lysosomal granules containing cathepsin D. Furthermore, we used an established anti-CD63 mAb to detect this protein in an alpha(3) beta(1) immunoprecipitate, and also we observed VLA-3 and CD63 colocalization in cellular ''footprints.'' Notably, the cytoplasmic domain of alpha(3) was neither required nor sufficient for CD63 association, suggesting that it occurred elsewhere within the alpha(3) beta(1) complex. Knowledge of these specific CD63-alpha(3) beta(1), and CD63-alpha(6) beta(1) biochemical associations should lead to critical insights into the specialized functions of alpha(3) beta(1), alpha(6) beta(1) and CD63.