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.
Berditchevski
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F
Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Bazzoni
G
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Hemler
M E
ME
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism*; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010980:Platelet Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism*; D011233:Precipitin Tests; D016029:Receptors, Very Late Antigen / Q000378:metabolism*; D060149:Tetraspanin 30; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C554586:CD63 protein, human; D015815:Cell Adhesion Molecules; D010980:Platelet Membrane Glycoproteins; D016029:Receptors, Very Late Antigen; D060149:Tetraspanin 30