Generation of monoclonal antibodies to integrin-associated proteins - Evidence that alpha(3)beta(1) complexes with EMMPRIN/basigin/OX47/M6

The alpha(3) beta(1) integrin forms complexes with other cell-surface proteins, including transmembrane-4 superfamily (TM4SF) proteins (e.g. CD9, CD53, CD63, CD81, and CD82). To identify additional cell-surface proteins associated with alpha(3) beta(1) integrin, a monoclonal antibody selection protocol was developed, Mice were immunized with integrin alpha(3) beta(1)-containing complexes isolated from HT1080 fibrosarcoma cells, and then 712 hybridoma clones were produced, and 95 secreted antibodies that recognized the HT1080 cell surface, Among these, 12 antibodies directly recognizing integrin alpha(3) or beta(1) subunits were eliminated, Of the remaining 83, 16 co-immunoprecipitated proteins that resembled integrins under non-stringent detergent conditions. These 16 included 15 monoclonal antibodies recognizing EMMPRIN/basigin/OX-47/M6, a 45-55-kDa transmembrane protein with two immunoglobulin domains. The EMMPRIN protein associated with alpha(3) beta(1) and alpha(6) beta(1), but not alpha(2) beta(1) or alpha(5) beta(1), as shown by reciprocal immunoprecipitation experiments, Also, association with alpha(3) beta(1) was confirmed by cell-surface cross-linking and immunofluorescence co-localization experiments, Importantly, EMMPRIN-alpha(3) beta(1) complexes appear not to contain TM4SF proteins, suggesting that they are distinct from TM4SF protein-alpha(3) beta(1) complexes.