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

Abstract

Fibroblast activation protein alpha (FAPα) 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 FAPα 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 FAPα binding partner dipeptidyl-peptidase IV (DPPIV) corroborated the proteomic strategy. Reverse co-IPs validated the FAPα 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 FAPα interaction partner. Many newly identified FAPα 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 FAPα to lipid raft structures. In density gradient centrifugation, FAPα co-fractionates with caveolin-1. Immunofluorescence optical sectioning microscopy of FAPα and lipid raft markers further corroborates recruitment of FAPα to lipid rafts and invadopodia. FAPα 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.

<p>Fibroblast Activation Protein (FAP) Overexpression Induces Epithelial–Mesenchymal Transition (EMT) in Oral Squamous Cell Carcinoma by Down-Regulating Dipeptidyl Peptidase 9 (DPP9)</p>

“…26 Yang et al 14 inhibited FAP with Talabostat does not reduce levels of its downstream CCL-2 secretion. Knopf et al 25 suggested that FAP directly binds to erlin-2, stomatin, and caveolin-1. However, it is not clear whether this binding is due to FAP enzyme activity pattern or nonenzyme activity pattern.…”

Identification of Novel Natural Substrates of Fibroblast Activation Protein-alpha by Differential Degradomics and Proteomics

“…Peptides were eluted in a linear gradient with increasing concentration of 5 mM KH 2 PO 1 M KCl 25% acetonitrile pH 2.7; 10 -14 fractions were collected and desalted using self-packed C18 STAGE tips (Empore; 3 M, St Paul, MN) (34). Differential proteomics samples were pre-fractionated by SDS-PAGE as described previously (31). For nanoflow-LC-MS/MS analysis, samples for quantitative proteomic comparison were analyzed on a Qstar Elite (AB Sciex, Framingham, MA) and TAILS samples on an Orbitrap XL (Thermo Scientific GmbH, Bremen, Germany) mass spectrometer.…”

Significance of prohibitin domain family in tumorigenesis and its implication in cancer diagnosis and treatment

“…Some PHB1 proteins have been reported to interact with low density detergent-insoluble lipid raft domains in the plasma membrane 26 , 51 , acting as transmembrane adapters to activate downstream signals 14 , 52 , 53 . Previous studies also identified that PHB1 on plasma membrane acts as a viral receptor protein to facilitate virus entry into host cells 8 , 54 , 55 .…”