Generation of Monoclonal Antibodies to Integrin-associated Proteins EVIDENCE THAT α3β1 COMPLEXES WITH EMMPRIN/BASIGIN/OX47/M6

Abstract The α3β1integrin 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 α3β1 integrin, a monoclonal antibody selection protocol was developed. Mice were immunized with integrin α3β1-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 α3 or β1 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 α3β1 and α6β1, but not α2β1 or α5β1, as shown by reciprocal immunoprecipitation experiments. Also, association with α3β1 was confirmed by cell-surface cross-linking and immunofluorescence co-localization experiments. Importantly, EMMPRIN-α3β1 complexes appear not to contain TM4SF proteins, suggesting that they are distinct from TM4SF protein-α3β1 complexes.