Quantitative Fluorescence Microscopy Reveals FGFR5 Higher Order Oligomerization
Regeenes, Romario 1 ; Silva, Pamuditha 1 ; Kilkenny, Dawn 1 ; Rocheleau, Jon 1, 2, 3
1. Institute of Biomaterials and Biomedical Engineering, University of Toronto; 2. Department of Physiology, University of Toronto; 3. Toronto General Research Institute, University Health Network, Canada
Introduction: Fibroblast growth factor receptors (FGFR) are a family of tyrosine kinase membrane receptors that initiate intracellular signaling cascades upon dimerization. Unlike canonical FGFR1, FGFR5 does not have intracellular kinase domains. The kinase-deficient structure of FGFR5 would suggest it cannot initiate intracellular signaling like classical FGFRs; however, R5-induced activation of MAPK signaling has been observed. Furthermore, both FGFR1 and FGFR5 have affinity for FGF2. Therefore, it has been postulated that FGFR5 modulates FGFR1 signaling post-ligand binding.
Materials and Methods: To examine these FGFR interactions, we used Number and Brightness analysis (N&B) and fluorescence-anisotropy based homo-Förster resonance energy transfer (homoFRET). In this study, we adapted the dark receptor concept from Number & Brightness to study the interaction between Venus- or Cerulean-tagged FGFR5 and FGFR1.
Results: Fluorescence-anisotropy based homoFRET using FGFR5-Cerulean revealed FGFR5 exists at the cell surface as a homodimer. However, FGFR5-Venus produced anisotropy values that were lower than the tandem Venus-Dimer control, suggesting FGFR5 is a higher order oligomer. Furthermore, N&B revealed a truncated variant of FGFR5 lacking the unique intracellular domain (R5?C) forms higher-order aggregation, as did co-expression of R5?C with wild type FGFR5. This data suggests FGFR5 aggregation is driven either an interaction in the transmembrane or extracellular domains, similar to classical FGFR aggregation. Furthermore, both techniques suggest FGFR5 forms heterodimers with FGFR1. Interestingly, the addition of FGF2 ligand resulted in a R5-Venus aggregation, but no change in the R5-R1 complex.
Conclusion: These data suggest FGFR5 natively exists as a higher order oligomer but also as complexes with FGFR1. These findings shed new light into the signaling mechanisms of FGFRs that can be used to develop novel therapeutics to treat a number of diseases.