The families of fibroblast growth factor receptors (FGFRs) consist of four closely related members, FGFR1-FGFR4. They contain 2 or 3 immunoglobulin-like extracellular domains, a transmembrane domain, and a cytoplasmic tyrosine kinase domain. In contrast to the role of the other FGFRs in stimulation of cell proliferation, the role of FGFR3 is controversial. It has been shown that congenital deficiency of FGFR3 leads to skeletal overgrowth while in multiple myeloma FGFR3 is overexpressed as a consequence of a chromosome translocation which promotes myeloma cell proliferation and prevents apoptosis. In addition FGFR3 acts as an oncogene in bladder cancer and cervical cancer. To elucidate the fuction of wild type FGFR3 and some mutations, found in several tumor types, we designed two pairs of PCR primers to generate Y373C and K650E mutations by using pcDNA3.1-FGFR3wt plasmid as a template. We then transfected FGFR3wt, FGFR3Y373C and FGFR3K650E into U937 histocytic lymphoma cells, which do not overexpress FGFR3. Two days after transfection, 400ug/ml G418 was added to select the transfectants. Three weeks after G418 selection, the cells were harvested and FGFR3 overexpression was validated by RT-PCR and Western-blot. Specific antibodies to detect phosphorylated-ERK, phosphorylated-STAT1, and p21 proteins were employed and examined by Western-blot. There was dramatic decrease of p-ERK in the FGFR3Y373C and FGFR3K650E transfected cells compared with the control (transfected with vector). There was no significant change for p-STAT1 and p21, which were reported to cause growth arrest in chondrocytes. Two days after serum starvation, cells were stimulated with αFGF at 10ng/mL and 50ng/mL in the presence of 10ug/mL heparin for 10 minutes at 37°C. Stimulation caused rapid and dose dependent phosphorylation of ERK in FGFR3wt, but not in FGFR3Y373C and FGFR3K650E transfected cells. Our data indicates that the role of FGFR3 may be cell type-specific and specific mutations of FGFR3 significantly change the signal transduction pathways. Detailed studies of different signal transduction pathways with transfected FGFR3wt, FGFR3Y373C and FGFR3K650E into other cell lines are currently ongoing.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]