Thermodynamics of receptor tyrosine kinase dimerization in model mammalian membranes: Determining the molecular basis behind cancers and skeletal dysplasias

Receptor tyrosine kinases are involved in signal transduction across the cellular membrane. RTKs typically pass information into cells through a dimerization process modulated by ligand recognition and binding. It is believed that ligand binding results in structural rearrangement of RTKs from an 'inactive' to an 'active' state. Fibroblast growth factor receptor 3 is an RTK involved in cell signaling, regulation, and proliferation. Mutations in FGFR3 are implicated in cancers and growth disorders such as achondroplasia and thanatophoric dysplasia, a lethal skeletal dysplasia1-12. These diseases are known to be caused by increased receptor activation. ErbB2 (HER2/Neu), another member of the RTK family, initiates signaling cascades critical for regulation of cell growth, proliferation, differentiation, and motility upon activation13-15. Overactivation of ErbB2 can lead to cancer due its function in regulating cell growth and inhibiting apoptosis. The V664E mutation in Neu is one of the first discovered oncogenic mutations in an RTK TM domain16,17. Although it is known to increase activation, the exact mechanism is unknown.;Quantitative Imaging Forster Resonance Energy Transfer (QI-FRET) was used to directly probe the interactions of wild-type and mutant versions of FGFR3 and Neu using confocal microscopy. Cell-derived plasma membrane vesicles were prepared to mimic the native environment for the expressed constructs 18,19. This methodology allowed for direct determination of the concentrations of donors and acceptors in each vesicle19. Assuming a two-state model of monomers and dimers in thermodynamic equilibrium, the strength of interaction was determined.;FGFR3 signaling is dependent upon ligand activation. Here, we investigated the effect of fgf1 on FGFR3 dimerization in live cells with two-photon microscopy. We found that the average FRET efficiency of the FGFR3 constructs changed upon ligand addition. This same method can be used to quantify changes in dimerization upon addition of other ligands. Furthermore, it can be applied to other RTKs and their ligand binding partners to assay the effect of ligands in other systems.