Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on. ERM (ezrin/radixin/moesin) proteins provide a regulated linkage between membrane-associated proteins and the actin cytoskeleton. Previous work has shown that ezrin can exist in a dormant monomeric state in which the N-terminal FERM domain is tightly associated with the C-ERMAD (carboxyl-terminal ERM association domain), masking binding sites for at least some ligands, including F-actin and the scaffolding protein EBP50. Activation of ezrin requires relief of the intramolecular association, and this is believed to involve phosphorylation of threonine 567.

Studies have therefore employed the T567D phosphomimetic mutant to explore the consequences of ezrin activation in vivo. Ezrin also exists as a stable dimer, in which the orientation of the two subunits is unknown, but might involve the central α-helical region predicted to form a coiled−coil. By characterization of ezrin mutants, we show that relief of the intramolecular association in the monomer results in unmasking of ligand binding sites and a significant conformational change, that the T567D mutation has a small effect on the biochemical activation of ezrin, and that the predicted coiled−coil region does not drive dimer formation. These results provide strong support for the conformational activation model of ezrin, elucidate the basis for dimer formation, and reveal that a mutant generally considered to be fully activated is not.

Fibroblast growth factors (fgfs) are widely believed to activate their receptors by mediating receptor dimerization. Here we show, however, that the FGF receptors form dimers in the absence of ligand, and that these unliganded dimers are phosphorylated. We further show that ligand binding triggers structural changes in the FGFR dimers, which increase FGFR phosphorylation.

The attempt to polymerize β‐AL using MeAl(BHT) 2 /I i Pr in a 1:1 ratio was unsuccessful, and it resulted in sluggish polymerization accompanied by dimerization (see Table S2, run 19), revealing a bimolecular cooperative activation is critical for achieving high activity.

The observed effects due to the ligands fgf1 and fgf2 are very different. The fgf2-bound dimer structure ensures the smallest separation between the transmembrane (TM) domains and the highest possible phosphorylation, a conclusion that is supported by a strong correlation between TM helix separation in the dimer and kinase phosphorylation. The pathogenic A391E mutation in FGFR3 TM domain emulates the action of fgf2, trapping the FGFR3 dimer in its most active state. This study establishes the existence of multiple active ligand-bound states, and uncovers a novel molecular mechanism through which FGFR-linked pathologies can arise. The fibroblast growth factor receptor (FGFR) family includes four receptors that bind 18 ligands called fibroblast growth factors, using heparin as a co-factor.

These receptors play important roles in all cell types, but are best known for the critical role that they play in the development of the skeletal system. Many pathogenic mutations of FGFR genes are linked to skeletal, cranial and other developmental abnormalities in humans. Furthermore, FGFR overexpression and mutations have been reported in a variety of cancers,.FGF receptors are single-pass membrane proteins, with N-terminal extracellular (EC) domains consisting of three immunoglobulin-like subdomains (D1, D2 and D3), a transmembrane (TM) domain consisting of a single α-helix, and an intracellular (IC) region encompassing a tyrosine kinase domain. FGFRs transduce biochemical signals via lateral dimerization in the plasma membrane. Receptor dimerization is necessary for activation, as it brings the two tyrosine kinase domains into close proximity, allowing them to cross-phosphorylate each other on tyrosines in their activation loops.

This activates the kinases, which then bind adaptor proteins and phosphorylate cytoplasmic substrates, triggering downstream signalling cascades that control cell growth and differentiation.High-resolution crystal structures of isolated FGFR EC domains in the presence of different fgfs have provided detailed views of ligand–receptor and receptor–receptor interactions in the EC portion, as well as the role of the co-factor heparin,. However, there is little mechanistic understanding of how conformational changes are transmitted from the EC domains through the TM domains to the kinase domains, in response to ligand binding. Different fgf ligands can elicit distinctly different biological responses, but the mechanism behind the specificity is unknown. To gain insight into these issues, here we study the dimerization of FGFR1, FGFR2 and FGFR3, as well as the response of these receptors to the ligands fgf1 and fgf2. Our results show that ligand binding to unliganded FGFR dimers triggers a switch to ligand-specific configurations of the TM helices, which in turn increase receptor phosphorylation.

We further show that a pathogenic FGFR mutant causes unregulated ligand-independent signalling by mimicking the most active ligand-bound configuration. ( a) Measured FRET in plasma membrane-derived vesicles, as a function of receptor concentration, for FGFR1 (black), FGFR2 (olive) and FGFR3 (red). Every data point represents a single vesicle. ( b) The donor concentration is plotted as a function of the acceptor concentration, for each vesicle.

( c) Dimeric fraction as a function of total receptor concentrations. The experimentally determined dimeric fractions are binned and are shown with the symbols, along with the standard errors.

Each bin contains between 5 and 50 experimental points. The solid lines are the dimerization curves, plotted for the optimized dimerization parameters in. The dimeric receptor fraction as a function of receptor concentration is shown in. From this concentration dependence we obtained, by fitting, the two-dimensional dissociation constant K diss and the structural parameter ‘intrinsic FRET’, (refs,; ).

Intrinsic FRET does not depend on the dimerization propensities, and is directly related to the distance between the fluorescent proteins. As discussed below, measurements of intrinsic FRET allow us to capture structural changes that occur on the cytoplasmic side of the receptor on ligand binding to the extracellular domains.

The values of the two-dimensional dissociation constants, K diss are 710, 111 and 24 μm −2 for FGFR1, FGFR2 and FGFR3, respectively, corresponding to dimerization free energies of −4.3±0.1, −5.4±0.1 and −6.3±0.1 kcal mol −1 (see equation (10) and; uncertainties are standard errors). The intrinsic FRET values for the unliganded FGFR1, FGFR2 and FGFR3 dimers are 0.66, 0.43 and 0.55, respectively.To evaluate the biological significance of the measured unliganded dimerization of the FGFRs, we note that physiological FGFR expression levels can be as high as ∼80 000 receptors per cell, corresponding to ∼80–100 receptors per μm 2 (ref. The experimental dimerization curves that we measured for the three receptors, shown in, predict substantial dimer populations, at least for FGFR2 ( ∼20%) and FGFR3 ( ∼50%), at receptor concentrations as low as 10 receptors per μm 2. Furthermore, we see a substantial increase in dimeric fraction with concentration, consistent with reports that FGFR overexpression is linked to cancer,. Thus, unliganded FGFR dimerization is important in physiological context. Contributions of FGFR domains to unliganded dimerizationTo determine the contribution of individual domains to the energetics of dimerization of the unliganded FGF receptors, we created two truncated versions of each receptor. In one truncated version (EC+TM), the intracellular domains were removed and the fluorescent proteins were attached to the cytoplasmic end of the TM domains via flexible, 15 residue (GGS) 5 linkers.

In the second truncated version (TM), both the IC and EC domains were removed, so that these constructs only contained the TM domains attached to the fluorescent proteins via the same flexible (GGS) 5 linkers. In earlier work, we showed that the attachment of the fluorescent protein to the TM domain via this linker does not have an effect on dimerization.The dimerization of the truncated constructs in plasma membrane-derived vesicles was characterized by QI-FRET as above. The dimerization curves for the truncated receptors are shown in, along with the results for the full-length receptors for comparison. The dimerization constants, free energies of dimerization and intrinsic FRET values for all the variants are shown in.

These results reveal several important aspects of FGFR unliganded dimerization. First, they show that the TM domains alone have a strong propensity for dimerization, with dimerization free energies between −5.2 and −6.0 kcal mol −1. Second, they demonstrate that FGFRs that lack IC domains form dimers. It has been proposed previously that the IC domain is required for FGFR dimerization in the absence of ligand. However, our results directly show that the IC domain is not necessary for FGFR dimerization.

Third, the differences in stability of the two types of truncated receptors (EC+TM and TM) suggest that the contribution of the EC domains to unliganded FGFR dimerization is destabilizing for all three receptors, by 1.4–2.3 kcal mol −1. Dimerization curves are shown for the full-length receptors (black), for truncated receptors that lack the IC domain and thus contain only the EC and TM domains (olive), and for the TM domains only (red). Data for EC+TM FGFR3 and TM FGFR3 are from ref. The measured dimeric fractions are binned and are shown with the symbols, along with the standard errors. Each bin contains between 5 and 50 experimental points. The solid lines are the best fits of a monomer–dimer equilibrium model to the single-vesicle data.

These data demonstrate that the TM domains have a strong propensity for dimerization. The EC domains, on the other hand, inhibit dimerization. The contribution of the IC domains is favourable, but it varies from zero to −3 kcal mol −1 for the three receptors. Dersu uzala subtitle download for mac. ( a) Histograms of single-vesicle intrinsic FRET values, measured for the three FGF EC+TM receptor constructs in the presence of saturating fgf1 (black) or fgf2 (olive) concentrations. Intrinsic FRET is a measure of the separation between the fluorescent proteins in the dimer. Two different intrinsic FRET values were measured for fgf1 and fgf2.

Therefore, the binding of these two ligands to the extracellular domains leads to different separation of the fluorescent proteins on the cytoplasmic side of the membrane. ( b) Western blots, reporting on the phosphorylation of the full-length receptors in the presence of saturating concentrations of fgf1 and fgf2 (5 μg ml −1). Expression of the receptors was probed with antibodies to the extracellular domains of the three receptors. Phosphorylation was assayed using antibodies against phosphorylated tyrosines in the activation loop of the three kinases (anti-phospho-Y653/4) or other phosphorylated tyrosine residues. Two bands are observed for all receptors.

Only the top bands, corresponding to the fully glycosylated mature receptors that reside primarily in the plasma membrane, were considered in our analysis. There is a difference between the phosphorylation in response to fgf1 and fgf2 for FGFR1 and FGFR3, but not for FGFR2 (see text). ( c) Relative FGFR3 phosphorylation in response to fgf1 and fgf2 is quantified and compared using a t-test.

Five independent experiments were performed in two cell lines, CHO and HEK 293T. Phosphorylation was calculated by dividing the intensities of the anti-phospho-Y bands to the intensities of the anti-receptor bands, and scaled to the fgf2 case. The difference in FGFR3 phosphorylation in response to fgf1 and fgf2 is highly statistically significant ( P. Changes in FGFR phosphorylation on fgf1 or fgf2 bindingTo investigate the biological significance of the two different ligand-bound states that we observed in the QI-FRET experiments, we compared the phosphorylation of full-length FGFR1, FGFR2 and FGFR3 at saturating fgf1 or fgf2 concentrations (5 μg ml −1) using western blotting. For detection of activated (phosphorylated) receptors we used anti-phospho-Tyr antibodies that are specific for phosphorylated tyrosines in the activation loop of the three receptors (anti-phospho-Y653/4) or other intracellular tyrosines. Typical western blot results are shown in.

Only the top bands, corresponding to the mature fully glycosylated receptors, were quantified. Comparing liganded FGFR1 and FGFR3, we consistently observe 20–40% higher phosphorylation in the presence of fgf2 than fgf1 in CHO cells. To test for statistical significance of this observation using a Student’s t-test, we performed five independent FGFR3 experiments in two different cell lines, CHO and HEK 293T. In both cell lines, activation by saturating fgf2 is 40% higher than for fgf1. The calculated P value is 0.1; ). This finding is consistent with the literature, and may be due to the fact that FGFR2 interacts with soluble adaptor proteins, such as Grb2, which can regulate its dimerization and activity.

These adapter proteins are present in the activation/western blot experiments that probe the overall biological response of the receptors to their ligands, but they are absent from the plasma membrane-derived vesicles, which do not contain cell cytoplasm. Ligand binding triggers a switch in the FGFR3 TM dimerThe only FGFR TM domain high-resolution dimer structure reported thus far is the one for the TM domain of FGFR3 (ref.