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

Fibroblast activation protein alpha (FAP alpha) 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 alpha interactome in cancer-associated fibroblasts using the quantitative immuno-precipitation 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 alpha binding partner dipeptidyl-peptidase IV (DPPIV) corroborated the proteomic strategy. Reverse co-IPs validated the FAP alpha 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 alpha interaction partner. Many newly identified FAP alpha 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 FAP alpha to lipid raft structures. In density gradient centrifugation, FAP alpha co-fractionates with caveolin-1. Immunofluorescence optical sectioning microscopy of FAP alpha and lipid raft markers further corroborates recruitment of FAP alpha to lipid rafts and invadopodia. FAP alpha 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 into novel biological aspects of a marker protein for cancer-associated fibroblasts. (C) 2015 Elsevier B.V. All rights reserved.