The Extracellular Domain of Fibroblast Growth Factor Receptor 3 Inhibits Ligand-Independent Dimerization

Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
Science Signaling (Impact Factor: 6.28). 11/2010; 3(150):ra86. DOI: 10.1126/scisignal.2001195
Source: PubMed


Dysregulation of the ligand-independent dimerization of receptor tyrosine kinases (RTKs), which is the first step in the activation of RTKs, leads to various pathologies. A mechanistic understanding of the dimerization process is lacking, and this lack of basic knowledge is one bottleneck in the development of effective RTK-targeted therapies. For example, the roles and relative contributions of the different domains of RTKs to receptor dimerization are unknown. Here, we used quantitative imaging Förster resonance energy transfer (QI-FRET) to determine the contribution of the extracellular domain of fibroblast growth factor receptor 3 (FGFR3) to the dimerization of the receptor. We provide evidence that the contribution of the extracellular domain of FGFR3 to dimerization is repulsive in the absence of ligand and is on the order of ~1 kcal/mol. The repulsive contribution of the extracellular domain is similar in magnitude, but opposite in sign, to the contribution of pathogenic single-amino acid mutations to RTK signaling, and is therefore likely to be important for biological function. Together, these results highlight the fine balance in the domain interactions that regulate RTK dimerization and signaling.

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    • "Comparison of the stabilities of the EC + TM and TM dimers (− 3.4 and − 5.2 kcal/mol, respectively) demonstrates that the deletion of the EC domain stabilizes the dimer by 1.8 kcal/mol, with the positive sign indicating that the contribution is inhibitory. This result is similar to our previous measurements in vesicles produced via chemical vesiculation using formaldehyde and DTT [31] [32] [33], which yielded ~ 1 kcal/mol for the inhibitory EC domain contribution [27]. In Fig. S2, the FRET efficiency for TM-JM-YFP/ mCherry construct does not depend on the concentrations , which suggests that the construct is 100% dimer over the concentration range we study. "
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    ABSTRACT: Receptor Tyrosine Kinases (RTKs) conduct biochemical signals upon dimerization in the membrane plane. While RTKs are generally known to be activated in response to ligand binding, many of these receptors are capable of forming unliganded dimers that are likely important intermediates in the signaling process. All 58 RTKs consist of an extracellular domain, a transmembrane (TM) domain, and an intracellular domain which includes a juxtamembrane (JM) sequence and a kinase domain. Here we investigate directly the effect of the JM domain on unliganded dimer stability of FGFR3, a receptor that is critically important for skeletal development. The data suggest that FGFR3 unliganded dimers are stabilized by receptor-receptor contacts that involve the JM domains. The contribution is significant, as it is similar in magnitude to the stabilizing contribution of a pathogenic mutation and the repulsive contribution of the extracellular domain. Furthermore, we show that the effects of the JM domain and a TM pathogenic mutation on unliganded FGFR3 dimer stability are additive. We observe that the JM-mediated dimer stabilization occurs when the JM domain is linked to FGFR3 TM domain and not simply anchored to the plasma membrane. These results point to a coordinated stabilization of the unliganded dimeric state of FGFR3 by its JM and TM domains via a mechanism that is distinctly different from the case of another well studied receptor, EGFR. Copyright © 2015. Published by Elsevier Ltd.
    Journal of Molecular Biology 02/2015; 427(8). DOI:10.1016/j.jmb.2015.02.013 · 4.33 Impact Factor
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    • "All images were taken at a resolution of 512 × 512 pixels with a pixel dwell time of 1.68 μs, the shortest possible for our set-up. Standard solutions of purified eYFP and mCherry of known concentrations were imaged for calibration purposes, as described in detail previously [40] [43] [44] "
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    ABSTRACT: Here, we study the homodimerization of the transmembrane domain of Neu, as well as an oncogenic mutant (V664E), in vesicles derived from the plasma membrane of mammalian cells. For the characterization, we use a Fӧrster resonance energy transfer (FRET)-based method termed Quantitative Imaging-FRET (QI-FRET), which yields the donor and acceptor concentrations in addition to the FRET efficiencies in individual plasma membrane-derived vesicles. Our results demonstrate that both the wild-type and the mutant are 100% dimeric, suggesting that the Neu TM helix dimerizes more efficiently than other RTK TM domains in mammalian membranes. Furthermore, the data suggest that the V664E mutation causes a very small, but statistically significant change in dimer structure. This article is part of a Special Issue entitled: Interfacially active peptides and proteins.
    Biochimica et Biophysica Acta 03/2014; 1838(9). DOI:10.1016/j.bbamem.2014.03.001 · 4.66 Impact Factor
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    • "The conformation of the unliganded RTK EC domains likely prevents the transition to the fully active dimer state (Endres et al., 2013). Indeed, the FGFR3 EC domain has been found to inhibit dimerization (by $1 kcal/mol) in the absence of ligand, whereas the TM domain interaction has been shown to stabilize the FGFR3 dimer (by $À4 kcal/mol) (Li and Hristova, 2010; Chen et al., 2010). Based on the above arguments, we propose that the FGFR3 unliganded dimer is stabilized by the heptad motif contacts shown in Figure 2C, in addition to possible stabilization due to kinase domain and Grb2 interactions. "
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    ABSTRACT: Fibroblast growth factor receptor 3 (FGFR3) transduces biochemical signals via lateral dimerization in the plasma membrane, and plays an important role in human development and disease. Eight different pathogenic mutations, implicated in cancers and growth disorders, have been identified in the FGFR3 transmembrane segment. Here, we describe the dimerization of the FGFR3 transmembrane domain in membrane-mimicking DPC/SDS (9/1) micelles. In the solved NMR structure, the two transmembrane helices pack into a symmetric left-handed dimer, with intermolecular stacking interactions occurring in the dimer central region. Some pathogenic mutations fall within the helix-helix interface, whereas others are located within a putative alternative interface. This implies that although the observed dimer structure is important for FGFR3 signaling, the mechanism of FGFR3-mediated transduction across the membrane is complex. We propose an FGFR3 signaling mechanism that is based on the solved structure, available structures of isolated soluble FGFR domains, and published biochemical and biophysical data.
    Structure 10/2013; 21(11). DOI:10.1016/j.str.2013.08.026 · 5.62 Impact Factor
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