Fibroblast Growth Factor Receptor 3 Transmembrane Domain Research Articles

Structure of FGFR3 Transmembrane Domain Dimer: Implications for Signaling and Human Pathologies

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.

The Role of Two SmXXXSm Motifs in the Dimerization of the FGFR3 Transmembrane Domain

Fibroblast growth factor receptor 3 (FGFR3) is a single pass membrane protein and a member of the receptor tyrosine kinase (RTK) family of proteins. FGFR3 has three distinct domains: the ligand binding extracellular domain, the cytosolic kinase domain, and the transmembrane (TM) domain. Previous work with the isolated FGFR3 TM domains has shown that it has the ability to self-dimerize. The TM domain of FGFR3 has two adjacent SmXXXSm motifs in the sequence, where the Sm residues are Ala374, Ser378 and Gly382. The SmXXXSm motif has been shown to play an important role in helix- helix interaction in other transmembrane proteins such as GpA and BNIP3. Currently, we are using site directed mutagenesis and ToxR activity assays to analyze the importance of this sequence motif for the interaction between the helices. Single mutations were made at three specific points, changing the small amino acids (Ala374, Ser378, and Gly382) to five different amino acids (Ala, Cys, Gly, Ile, and Ser). Relative activity measurements were made comparing the activity of the mutant protein to that of the wild type protein. Through these comparative measurements we can assess their role in helix-helix interaction.

Bacterial Synthesis and Purification of Normal and Mutant Forms of Human FGFR3 Transmembrane Segment

The fibroblast growth factor receptor 3 (FGFR3) is a protein belonging to the family of receptor tyrosine kinases. FGFR3 plays an important role in human skeletal development. Mutations in this protein, including Gly380Arg or Ala391Glu substitutions in the transmembrane (TM) region, can cause different disorders in bone development. The determination of the spatial structure of the FGFR3 TM domain in a normal protein and in a protein with single Gly380Arg and Ala391Glu mutations is essential in order to understand the mechanisms that control dimerization and signal transduction by receptor tyrosine kinases. The effective system of expression of eukaryotic genes in bacteria and the purification protocol for the production of milligram amounts of both normal TM fragments of FGFR3 and those with single pathogenic mutations Gly380Arg and Ala391Glu, as well as their(15)N- and [(15)N,(13)C]-isotope-labelled derivatives, were described. Each peptide was produced inEscherichia coliBL21(DE3)pLysS cells as a C-terminal extension of thioredoxin A. The purification protocol involved immobilized metal affinity chromatography and cation- and anion-exchange chromatography, as well as the fusion protein cleavage with the light subunit of human enterokinase. The efficiency of the incorporation of target peptides into DPC/SDS and DPC/DPG micelles was confirmed using NMR spectroscopy. The described methodology of production of the native FGFR3 TM domain in norma and with single Gly380Arg and Ala391Glu mutations enables one to study their spatial structure using high-resolution heteronuclear NMR spectroscopy.

Molecular Dynamics Simulations of the Transmembrane Helix of the FGFR3 Receptor in POPC and DPPC

Fibroblast growth factor receptor 3 (FGFR3) is a receptor tyrosine kinase that negatively regulates bone growth. Elevated FGFR3 activity results in achondroplasia, the most common form of human dwarfism. In the majority (∼98%) of cases the underlying mutation is G380R in the FGFR3 transmembrane domain. We have used coarse-grained molecular dynamics simulations to study the dimerization behaviour of wild-type, heterodimer, and mutant homodimer 33-residue transmembrane FGFR3 constructs in both POPC and DPPC bilayers. FGFR3 dimers are stable once formed in POPC, but dissociations are observed in DPPC. All three FGFR3 constructs exhibit bimodal helix crossing angle distributions, in contrast to the strong preference for right-handed crossing in glycophorin A (GpA) control simulations. We present evidence for a primary FGFR3 dimer interface and a less stable secondary interface. The latter is more pronounced for mutant than wild-type constructs in POPC, but not in DPPC. The helix crossing angle is right-handed at the secondary dimer interface for both heterodimer and mutant homodimer FGFR3 constructs in POPC. G370, A374, and R397 are prevalent FGFR3 dimer contacts, while the same analysis procedure on GpA control simulations selects the most important interfacial residues established by experiment. We suggest subtle differences, relative to wild-type, in the dimerization properties of G380R FGFR3 transmembrane domains.

Physical Basis behind Achondroplasia, the Most Common Form of Human Dwarfism

Fibroblast growth factor receptor 3 (FGFR3) is a receptor tyrosine kinase that plays an important role in long bone development. The G380R mutation in FGFR3 transmembrane domain is known as the genetic cause for achondroplasia, the most common form of human dwarfism. Despite many studies, there is no consensus about the exact mechanism underlying the pathology. To gain further understanding into the physical basis behind the disorder, here we measure the activation of wild-type and mutant FGFR3 in mammalian cells using Western blots, and we analyze the activation within the frame of a physical-chemical model describing dimerization, ligand binding, and phosphorylation probabilities within the dimers. The data analysis presented here suggests that the mutation does not increase FGFR3 dimerization, as proposed previously. Instead, FGFR3 activity in achondroplasia is increased due to increased probability for phosphorylation of the unliganded mutant dimers. This finding has implications for the design of targeted molecular treatments for achondroplasia.

The Physical Basis Behind Achondroplasia, the Most Common Form of Human Dwarfism

Fibroblast growth factor receptor 3 (FGFR3) is a receptor tyrosine kinase which plays an important role in long bone development. The Gly380Arg mutation in FGFR3 transmembrane domain has been linked to achondroplasia, the most common form of human dwarfism. However, the exact mechanism underlying the pathology is under debate. One hypothesis is that the mutation stabilizes the active FGFR3 dimer in the plasma membrane. To test this hypothesis, here we measure the activation of wild type and mutant FGFR3 in mammalian cells, and analyze the activation using a physical-chemical model accounting for dimerization, ligand binding and phosphorylation probabilities. Our results demonstrate that the achondroplasia mutation does not increase the dimerization propensity of FGFR3. Instead, the data suggest that the mutation induces a structural change in the unliganded dimer. We propose that this structural change is a cause for pathogenesis in achondroplasia.

Effect of Short Transmembrane Peptides on the Activation and Dimerization of an FGFR3 Pathogenic Mutant

Mutations in the transmembrane domains of receptor tyrosine kinases (RTKs) are implicated in many human diseases. For example, fibroblast growth factor receptor 3 (FGFR3) carrying an A391E mutation in the transmembrane (TM) region is associated with bladder cancer and Crouzon syndrome with acanthosis nigricans. Previous work has shown that the isolated FGFR3 TM domains dimerize in detergent micelles and in lipid bilayers. Therefore, we are exploring whether the TM domain of FGFR3 can inhibit the pathogenic effects of the A391E mutation. Our preliminary data show that the activation level of a chimeric Neu_FGFR3 receptor carrying the A391E mutation could be inhibited by co-expressing short mutant FGFR3 TM peptides. We also observe a decrease in dimerization propensity of the receptors due to the co-expression. Currently, we are investigating the specificity and the efficiency of the inhibition by using TM peptides from other RTKs. The TM peptides which can specifically and efficiently inhibit the activation and dimerization of FGFR3 mutants are possible candidates for future therapies for diseases linked to FGFR3 TM domain mutations.

Studies of Receptor Tyrosine Kinase Transmembrane Domain Interactions: The EmEx-FRET Method

The energetics of transmembrane (TM) helix dimerization in membranes and the thermodynamic principles behind receptor tyrosine kinase (RTK) TM domain interactions during signal transduction can be studied using Förster resonance energy transfer (FRET). For instance, FRET studies have yielded the stabilities of wild-type fibroblast growth factor receptor 3 (FGFR3) TM domains and two FGFR3 pathogenic mutants, Ala391Glu and Gly380Arg, in the native bilayer environment. To further our understanding of the molecular mechanisms of deregulated FGFR3 signaling underlying different pathologies, we determined the effect of the Gly382Asp FGFR3 mutation, identified in a multiple myeloma cell line, on the energetics of FGFR3 TM domain dimerization. We measured dimerization energetics using a novel FRET acquisition and processing method, termed "emission-excitation FRET (EmEx-FRET)," which improves the precision of thermodynamic measurements of TM helix association. The EmEx-FRET method, verified here by analyzing previously published data for wild-type FGFR3 TM domain, should have broad utility in studies of protein interactions, particularly in cases when the concentrations of fluorophore-tagged molecules cannot be controlled.

Neutron Diffraction Studies of Fluid Bilayers with Transmembrane Proteins: Structural Consequences of the Achondroplasia Mutation

Achondroplasia, the most common form of human dwarfism, is due to a G380R mutation in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) in >97% of the studied cases. While the molecular mechanism of pathology induction is under debate, the structural consequences of the mutation have not been studied. Here we use neutron diffraction to determine the disposition of FGFR3 transmembrane domain in fluid lipid bilayers, and investigate whether the G380R mutation affects the topology of the protein in the bilayer. Our results demonstrate that, in a model system, the G380R mutation induces a shift in the segment that is embedded in the membrane. The center of the hydrocarbon core-embedded segment in the mutant is close to the midpoint between R380 and R397, supporting previous measurements of arginine insertion energetics into the endoplasmic reticulum. The presented results further our knowledge about basic amino-acid insertion into bilayers, and may lead to new insights into the mechanism of pathogenesis in achondroplasia.

The Achondroplasia Mutation Does Not Alter the Dimerization Energetics of the Fibroblast Growth Factor Receptor 3 Transmembrane Domain

The Gly380 --> Arg mutation in the TM domain of fibroblast growth factor receptor 3 (FGFR3) of the RTK family is linked to achondroplasia, the most common form of human dwarfism. The molecular mechanism of pathology induction is under debate, and two different mechanisms have been proposed to contribute to pathogenesis: (1) Arg380-mediated FGFR3 dimer stabilization and (2) slow downregulation of the activated mutant receptors. Here we show that the Gly380 --> Arg mutation does not alter the dimerization energetics of the FGFR3 transmembrane domain in detergent micelles or in lipid bilayers. This result indicates that pathogenesis in achondroplasia cannot be explained simply by a higher dimerization propensity of the mutant FGFR3 TM domain, thus highlighting the importance of the observed slow downregulation in phenotype induction.

FGFR3 Dimer Stabilization Due to a Single Amino Acid Pathogenic Mutation

Mutations in the transmembrane (TM) domains of receptor tyrosine kinases (RTKs) have been implicated in the induction of pathological phenotypes. These mutations are believed to stabilize the RTK dimers, and thus promote unregulated signaling. However, the energetics behind the pathology induction has not been determined. An example of a TM domain pathogenic mutation is the Ala391-->Glu mutation in fibroblast growth factor receptor 3 (FGFR3), linked to Crouzon syndrome with acanthosis nigricans, as well as to bladder cancer. Here, we determine the free energy of dimerization of wild-type and mutant FGFR3 TM domain in lipid bilayers using Förster resonance energy transfer, and we show that hydrogen bonding between Glu391 and the adjacent helix in the dimer is a feasible mechanism for dimer stabilization. The measured change in the free energy of dimerization due to the Ala391-->Glu pathogenic mutation is -1.3 kcal/mol, consistent with previous reports of hydrogen bond strengths in proteins. This is the first quantitative measurement of mutant RTK stabilization in a membrane environment. We show that this seemingly modest value can lead to a large increase in dimer fraction and thus profoundly affect RTK-mediated signal transduction.

Synthesis and initial characterization of FGFR3 transmembrane domain: consequences of sequence modifications

Receptor Tyrosine Kinases (RTKs) conduct biochemical signals via lateral dimerization in the plasma membrane, and defects in their dimerization lead to unregulated signaling and disease. RTK transmembrane (TM) domains are proposed to play an important role in the process, underscored by the finding that single amino acids mutations in the TM domains can induce pathological phenotypes. Therefore, many important questions pertaining to the mode of signal transduction and the mechanism of pathology induction could be answered by studying the chemical–physical basis behind RTK TM domain dimerization and the interactions of RTK TM domains with lipids in model bilayer systems. As a first step towards this goal, here we report the synthesis of the TM domain of fibroblast growth factor receptor 3 (FGFR3), an RTK that is crucial for skeletal development. We have used solid phase peptide synthesis to produce two peptides: one corresponding to the membrane embedded segment and the naturally occurring flanking residues at the N- and C-termini (TM wt), and a second one in which the flanking residues have been substituted with diLysines at the termini (TM KK). We have demonstrated that the hydrophobic FGFR3 TM domain can be synthesized for biophysical studies with high yield. The protocol presented in the paper can be applied to the synthesis of other RTK TM domains. As expected, the Lys flanks decrease the hydrophobicity of the TM domain, such that TM KK elutes much earlier than TM wt during reverse phase HPLC purification. The Lysines have no effect on peptide solubility in SDS and on peptide secondary structure, but they abolish peptide dimerization on SDS gels. These results suggest that caution should be exercised when modifying RTK TM domains to render them more manageable for biophysical studies.

Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis and Förster Resonance Energy Transfer Suggest Weak Interactions between Fibroblast Growth Factor Receptor 3 (FGFR3) Transmembrane Domains in the Absence of Extracellular Domains and Ligands

Lateral dimerization of membrane proteins has evolved as a means of signal transduction across the plasma membrane for all receptor tyrosine kinases (RTKs). The transmembrane (TM) domains of RTKs are proposed to play an important role in the dimerization process. We have investigated whether the TM domains of one RTK, fibroblast growth factor receptor 3 (FGFR3), dimerize in lipid vesicles in the absence of the extracellular domains and ligands. We have performed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with peptides produced via solid-phase peptide synthesis that correspond to the TM domain of FGFR3. We have carried out Forster resonance energy transfer (FRET) measurements using two donor-acceptor pairs, fluorescein/rhodamine and Cy3/Cy5, as a function of peptide concentration and donor-to-acceptor mole ratios. Our results suggest that FGFR3 TM domains form sequence-specific dimers in lipid bilayers. However, the dimerization propensity of FGFR3 TM domain is much weaker than the dimerization propensity of glycophorin A (GpA), the well-characterized "membrane dimer standard". We discuss our findings in the context of cell signaling across the plasma membrane and diseases or disorders that occur due to single amino acid mutations in the TM domain of FGFR3.

Phenotypic expression of the fibroblast growth factor receptor 3 (FGFR3) mutation P250R in a large craniosynostosis family.

The craniosynostosis syndromes are a heterogeneous group of sporadic, autosomal dominant disorders with significant clinical overlap. Recently, we described a large family with autosomal dominant craniosynostosis suggestive of Saethre-Chotzen syndrome,...

Common mutations in the gene encoding fibroblast growth factor receptor 3 account for achondroplasia, hypochondroplasia and thanatophoric dysplasia.

The mapping of the achondroplasia locus to the short arm of chromosome 4 and the subsequent identification of a recurrent missense mutation (Gly380Arg) in the gene encoding fibroblast growth factor receptor 3 (FGFR-3) has been followed by the detection of common FGFR-3 mutations in two clinically related disorders: thanatophoric dysplasia (TD; types I and II) and hypochondroplasia. The relative clinical homogeneity of achondroplasia was substantiated by demonstration of its genetic homogeneity: 100% of patients examined exhibited mutations in the transmembrane domain of FGFR-3. Although most cases of hypochondroplasia were accounted for by a recurrent missense substitution (Asn540Lys) in the first tyrosine kinase domain of FGFR-3, a significant proportion (40%) of the patients in the present study did not possess this Asn540Lys mutation. Furthermore, in three families with hypochondroplasia, the genetic defect was not linked to the FGFR-3 locus, thus supporting the clinical heterogeneity of this disease. In TD, a recurrent mutation located in the second tyrosine kinase domain of FGFR-3 has been detected in all TDII patients. By contrast, seven distinct mutations in three different protein domains were identified in 25 out of 26 TDI patients in this study. This suggests that TD, like achondroplasia, is a genetically homogeneous skeletal disorder.

A common FGFR3 mutation functions as a diagnostic marker for achondroplasia-group disorders in the Japanese population

Recent DNA studies performed by several groups have detected mutations of the gene encoding fibroblast growth factor receptor 3 (FGFR3) in patients with achondroplasia-group disorders, including achondroplasia (ACH), hypochondroplasia (HCH), and thanatophoric dysplasia (TD). For this study, we analyzed the FGFR3 gene in 31 Japanese patients with typical ACH, four with HCH, three with a condition intermediate between ACH and HCH (ACH/HCH-intermediate), and one with TD. Of the 31 typical ACH patients, 29 showed a G1138 to A transition and the other two a G1138 to C transversion, both resulting in a common Gly380Arg substitution in the transmembrane domain of FGFR3. The one TD and the four HCH patients did not display any mutations in the transmembrane domain of FGFR3. Of the three ACH/HCH-intermediate cases, one patient showed the Gly380Arg substitution and one did not, and further analysis of the second patient revealed the presence of Asn540Lys substitution. The first patient was, therefore, genotypically diagnosed as ACH and the second as HCH. Peripheral blood leukocyte DNA analysis in the remaining ACH/HCH-intermediate patient indicated an unequal ratio of mutant to normal PCR products, possibly representing a somatic mosaic for the Gly380Arg mutation. Analysis of the common FGFR3 mutation thus appears to help in the molecular diagnosis of patients with achondroplasia-group disorders.

Fibroblast growth factor receptor 3 (FGFR3) transmembrane mutation in Crouzon syndrome with acanthosis nigricans.

Crouzon syndrome, an autosomal dominant condition characterized by craniosynostosis, ocular proptosis and midface hypoplasia, is associated with mutations in fibroblast growth factor receptor 2 (FGFR2) (refs 1-3). For example, we have identified 10 different mutations in the FGFR2 extracellular immunoglobulin III (IgIII) domain in 50% (16/32) of our Crouzon syndrome patients. All mutations described so far for other craniosynostotic syndromes with associated limb anomalies--Jackson-Weiss, Pfeiffer, and Apert--also occur in the extracellular domain of FGFR2, as well as FGFR1 for Pfeiffer syndrome. In contrast, only FGFR3 mutations have been reported in dwarfing conditions--achondroplasia, thanatophoric dysplasia, and hypochondroplasia. For achondroplasia, greater than 99% of mutations occur in the FGFR3 transmembrane domain. We now report the unexpected observation of a FGFR3 transmembrane domain mutation, Ala391Glu, in three unrelated families with Crouzon syndrome and acanthosis nigricans, a specific skin disorder of hyperkeratosis and hyperpigmentation. The association of non-dwarfing and even non-skeletal conditions with FGFR3 mutations reveals the potential for a wide range of FGFR pleiotropic effects as well as locus heterogeneity in Crouzon syndrome. Our study underscores the biologic complexity of the FGFR gene family.

Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia

Achondroplasia (ACH) is the most common genetic form of dwarfism. This disorder is inherited as an autosomal dominant trait, although the majority of cases are sporadic. A gene for ACH was recently localized to 4p 16.3 by linkage analyses. The ACH candidate region includes the gene encoding fibroblast growth factor receptor 3 (FGFR3), which was originally considered as a candidate for the Huntington's disease gene. DNA studies revealed point mutations in the FGFR3 gene in ACH heterozygotes and homozygotes. The mutation on 15 of the 16 ACH-affected chromosomes was the same, a G→A transition, at nucleotide 1138 of the cDNA. The mutation on the only ACH-affected chromosome 4 without the G→A transition at nucleotide 1138 had a G→C transversion at this same position. Both mutations result in the substitution of an arginine residue for a glycine at position 380 of the mature protein, which is in the transmembrane domain of FGFR3.