Signaling pathways that trigger cellular proliferation are complex but are generally initiated by the binding of growth factors to their receptor, of which the fibroblast growth factor (FGF) is an important example. Under normal conditions, these interactions are strongly regulated and are important for homeostasis and wound healing. When this regulation breaks down, uncontrolled cell growth can occur and a legion of cancers have been closely tied to the de-regulation of FGF binding to its receptor (FGFR). Therefore, there is a need to develop sensitive assays that seek to understand the binding and regulation of the FGF-FGFR interaction under physiological conditions as one potential way to better develop effective cancer therapies. Fluorescence resonance energy transfer (FRET) has proven to be a suitable technique for such binding studies because both structural and kinetic information about FGF-FGFR interactions can be elucidated.  During FRET, respective binding proteins are site specifically labelled with donor and acceptor dyes by the use of highly fluorescent maleimide dyes that form thiol bonds only with cysteine residues of the proteins. We use recombination techniques to introduce cysteine residues at specific sites in the two proteins (FGF and FGFR) and characterize their labeling efficiencies with a range of FRET-suitable dyes. The results from UV/Vis spectroscopy, SDS-PAGE and fluorescence correlation spectroscopy (FCS) show successful labelling of FGF with maleimide-functionalized Alexa Fluor series dyes (488, 594, 546 and 647). Currently, we are focusing on mutation, expression, purification and labeling of the various extracellular binding domains of FGFR, which will be followed by investigation of ensemble and single molecule FRET between the two proteins.<br/> <br/>

Developing FRET assays to study the binding and regulation of fibroblast growth factor to its receptor

253rd National Meeting (2017)

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