NAG-2, a novel transmembrane-4 superfamily (TM4SF) protein that complexes with integrins and other TM4SF proteins

Transmembrane-4 superfamily (TM4SF) proteins form complexes with integrins and other cell-surface proteins. To further characterize the major proteins present in a typical TM4SF protein complex, we raised monoclonal antibodies against proteins co-immunoprecipitated with CD81 from MDA-MB-435 breast cancer cells. Only two types of cell-surface proteins were recognized by our 35 selected antibodies. These included an integrin (alpha(6) beta(1)) and three different TM4SF proteins (CD9, CD63, and NAG-2). The protein NAG-P (novel anti gen-a) is a previously unknown 30-kDa cell-surface protein. Using an expression cloning protocol, cDNA encoding NAG-2 was isolated. When aligned with other TM4SF proteins, the deduced amino acid sequence of NAG-2 showed most identity (34%) to CD53. Flow cytometry, Northern blotting, and immunohistochemistry showed that NAG-2 is widely present in multiple tissues and cell types but is absent from brain, lymphoid cells, and platelets, Within various tissues, strongest staining was seen on fibroblasts, endothelial cells, follicular dendritic cells, and mesothelial cells. In nonstringent detergent, NAG-2 protein was co-immunoprecipitated with other TM4SF members (CD9 and CD81) and integrins (alpha(3) beta(1) and alpha(6) beta(1)). Also, two-color immunofluorescence showed that NAG-S was co-localized with CD81 on the surface of spread HT1080 cells. These results confirm the presence of NAG-S in specific TM4SF.TM4SF and TM4SF-integrin complexes.