Fibroblast activation protein (FAP) is a cell-surface serine protease that is normally overexpressed in the microenvironment of several cancers. Therefore, it is a potential target for cancer drug development and delivery. FAP exhibits prolyl endopeptidase activity and is active as a homodimer with specificity for type I collagen. Moreover, it can form a heterodimeric complex with dipeptidyl peptidases IV (DPPIV), another cell-surface serine protease in the same family. The role of the heterodimer versus homodimer in terms of degradation of extracellular matrix components remains unclear. Furthermore, the mechanism that regulates FAP homo- and heterodimerization and its relation to proteolytic activity is not completely understood. Here, we investigate key residues of small-X-small motif (Gly, Ser and Ala) in the FAP TM dimer interface that may be significant for FAP homodimerization via the AraTM assay. Furthermore, the DN-AraTM assay is used to determine if the TM domains of DPPIV and FAP are involved in heterodimerization. We compared homodimerization of the mutated FAP TM (G10L, S14L, and A18L) against wild-type FAP TM. Mutations to predicted interfacial residues reduced the GFP signal of the AraTM chimera relative to wild type, which is indicative of a reduction in dimerization, whereas predicted off-interface residues showed no significant change from wild type. The results implied that small residues in the predicted small-X<sub>3</sub>-small dimer interface affect stabilization of FAP homodimerization. Furthermore, the reduced the GFP signal in the DN-AraTM assay indicates heterodimer formation between DPPIV and FAP TM domain. Heterodimerization between FAP and DPPIV was confirmed in the full-length FAP expressed in HEK293T cells. Moreover, mutation in FAP TM and residues in FAP ectodomain in the full-length FAP expressed in HEK293T cells also reduced FAP exopeptidase activity. Collectively, these results indicate that FAP homodimerization and heterodimerization with DPPIV may play an important role in regulation of FAP activity.

Understanding the significance of fibroblast activation protein (FAP) homo- and heterodimerization in relation to proteolytic activity

249th National Meeting (2015)

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