The physical-chemical basis behind FGFR activation: Implications for human pathologies.

摘要

Fibroblast growth factor receptors (FGFRs), members of the receptor tyrosine kinase (RTK) family, play critical roles in cell signal transduction. Overall, FGFRs, like other RTKs, transduce signals via a process involving lateral dimerization, ligand binding, phosphorylation and stimulation of downstream signaling cascades. Recent decades have witnessed a progress in understanding FGFR signaling pathways. However, the mechanism behind FGFR activation is yet to be fully understood.;In this project, we seek to understand the underlying physical-chemical basis behind FGFR3 activation and to elucidate its implications for human pathology, by utilizing quantitative measurements and mathematical modeling of interactions and activation of full-length FGFR3 in mammalian cellular membranes.;We first investigated the effect of the A391E mutation on FGFR3 activation in the absence of ligand. This transmembrane domain mutation is the molecular cause for Crouzon syndrome with Acanthosis Nigricans. Here, we compared the mutant activation with the wild-type in the absence of ligand. The results show that the mutation enhances the ligand-independent activation propensity of the receptor by -1.7kcal/mol. This value is consistent with the observed strength of hydrogen bonds in biological membranes, and supports the hypothesis that the mutation causes overactivation of the receptor, possibly through a hydrogen-bonding mediated stabilization mechanism, and thus leads to the pathology.;We then addressed the specificity of the biological responses mediated by different fgf-FGFR pairs. In particular, we studied, analyzed and compared the activation of FGFR3 over a wide range of fgf1 and fgf2 concentrations. We found that while the strength of fgf2 binding to FGFR3 is lower than the strength of fgf1 binding, the fgf2-bound dimers exhibit higher phosphorylation of critical tyrosines in the activation loop. As a result, fgf1 and fgf2 elicit a similar FGFR3 response at low, but not at high, concentrations. The results demonstrate the versatility of FGFR3 response to fgf1 and fgf2, and highlight the complexity in fgf signaling.;Finally, we investigated the effect of the A391E mutation in the presence of ligand. We compared the responses of mutant and wild-type receptors to fgf1 and fgf2 over a wide range of ligand concentrations. With the assistance of mathematical modeling, we extracted parameters describing ligand-induced activation. The results show that the A391E mutation enhances FGFR3 activation through a combined mechanism involving enhanced dimerization propensity and increased phosphorylation probability, but has no effect on ligand binding. The work reveals that a single mutation might cause multiple effects that contribute to the pathology.;We successfully applied quantitative methodologies to study full-length FGFR activation in mammalian cell membranes. The ability to carry out such measurements can shed light on ligand binding specificity and on the pathology mechanism due to mutations occurring in RTKs. This research could aid the development of treatments for RTK-linked diseases and could provide guidance for targeted therapeutic inhibitor design.