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 anti body selection protocol was developed, Mice were immunized with integr in alpha(3) beta(1)-containing complexes isolated from HT1080 fibrosar coma cells, and then 712 hybridoma clones were produced, and 95 secret ed 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 protei ns 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 d omains. 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 al pha(3) beta(1) was confirmed by cell-surface cross-linking and immunof luorescence co-localization experiments, Importantly, EMMPRIN-alpha(3) beta(1) complexes appear not to contain TM4SF proteins, suggesting th at they are distinct from TM4SF protein-alpha(3) beta(1) complexes.