The stromal cell-surface protease fibroblast activation protein-alpha localizes to lipid rafts and is recruited to invadopodia

23.07.2015

Knopf JD, Tholen S, Koczorowska MM, De Wever O, Biniossek ML, Schilling O.

Biochim Biophys Acta. 2015;1853(10 Pt A):2515-25.

Biochim Biophys Acta.            online article

Fibroblast activation protein alpha (FAPalpha) is a cell surface protease expressed by cancer-associated fibroblasts in the microenvironment of most solid tumors. As there is increasing evidence for proteases having non-catalytic functions, we determined the FAP? interactome in cancer-associated fibroblasts using the quantitative immunoprecipitation combined with knockdown (QUICK) method. Complex formation with adenosin deaminase, erlin-2, stomatin, prohibitin, Thy-1 membrane glycoprotein, and caveolin-1 was further validated by immunoblotting. Co-immunoprecipitation (co-IP) of the known stoichiometric FAP? binding partner dipeptidyl-peptidase IV (DPPIV) corroborated the proteomic strategy. Reverse co-IPs validated the FAPalpha interaction with caveolin-1, erlin-2, and stomatin while co-IP upon RNA-interference mediated knock-down of DPPIV excluded adenosin deaminase as a direct FAP? interaction partner. Many newly identified FAP? interaction partners localize to lipid rafts, including caveolin-1, a widely-used marker for lipid raft localization. We hypothesized that this indicates a recruitment of FAPalpha to lipid raft structures. In density gradient centrifugation, FAPalpha co-fractionates with caveolin-1. Immunofluorescence optical sectioning microscopy of FAPalpha and lipid raft markers further corroborates recruitment of FAP? to lipid rafts and invadopodia. FAPalpha is therefore an integral component of stromal lipid rafts in solid tumors. In essence, we provide one of the first interactome analyses of a cell surface protease and translate these results