Quantitative Fluorescence Microscopy Reveals Fibroblast Growth Factor Receptor 5 Signaling Complex Formation

Abstract

Fibroblast growth factor receptors (FGFRs) are transmembrane tyrosine kinase receptors that modulate many cellular processes including adhesion and survival. FGFR5 is a non-canonical family member that lacks a kinase domain and is presumed to be a negative regulator of signaling. However, we previously showed that overexpression of FGFR5 results in activation of downstream signaling targets, thereby challenging our understanding of this receptor. In particular, it was unclear whether FGFR5 and FGFR1 interact on the plasma membrane to form a signaling complex. We previously used quantitative fluorescence microscopy, including number and brightness analysis (N&B) and homo-forster resonance energy transfer (homoFRET), to study the dynamics and the stoichiometry of the FGFR1 signaling complex. We therefore created a library of fluorescent protein tagged FGFR1 and FGFR5 constructs to conduct similar studies of receptor membrane aggregation. FGFR5 has a unique cytoplasmic C-terminus that includes a histidine-rich zinc binding region as well as a putative SH2-binding motif. HomoFRET and anisotropy imaging revealed that FGFR5 forms homo-dimers while a C-terminal deficient isoform (FGFR5ΔC) does not, suggesting that the C-terminus drives the interaction. We validated these findings using N&B analysis of Venus-tagged FGFR5ΔC observing no change in relative brightness in the presence or absence of non-Venus tagged FGFR5ΔC (i.e. “dark receptor”). Finally, we measured changes in anisotropy of Cerulean-tagged FGFR5 when co-expressed with an excess dark FGFR1 (i.e. mCherry-tagged FGFR1). We observed an increase in FGFR5 anisotropy indicative of dimer to monomer transition, suggesting that FGFR5 also forms heterodimers with FGFR1. Currently, we are investigating the effect of FGFs and scaffolding proteins on modulating the FGFR5-FGFR1 heterocomplex. These studies will allow us to better understand the regulatory mechanisms that govern FGFR-signaling and the downstream cellular processes.