Effect of the G375C and G346E achondroplasia mutations on FGFR3 activation

Two mutations in FGFR3, G380R and G375C are known to cause achondroplasia, the most common form of human dwarfism. The G380R mutation accounts for 98% of the achondroplasia cases, and thus has been studied extensively. Here we study the effect of the G375C mutation on the phosphorylation and the cross-linking propensity of full-length FGFR3 in HEK 293 cells, and we compare the results to previously published results for the G380R mutant. We observe identical behavior of the two achondroplasia mutants in these experiments, a finding which supports a direct link between the severity of dwarfism phenotypes and the level and mechanism of FGFR3 over-activation. The mutations do not increase the cross-linking propensity of FGFR3, contrary to previous expectations that the achondroplasia mutations stabilize the FGFR3 dimers. Instead, the phosphorylation efficiency within un-liganded FGFR3 dimers is increased, and this increase is likely the underlying cause for pathogenesis in achondroplasia. We further investigate the G346E mutation, which has been reported to cause achondroplasia in one case. We find that this mutation does not increase FGFR3 phosphorylation and decreases FGFR3 cross-linking propensity, a finding which raises questions whether this mutation is indeed a genetic cause for human dwarfism.     

Chimeras of the native form or achondroplasia mutant (G375C) of human fibroblast growth factor receptor 3 induce ligand-dependent differentiation of PC12 cells.

Mutations in the gene for human fibroblast growth factor receptor 3 (hFGFR3) cause a variety of skeletal dysplasias, including the most common genetic form of dwarfism, achondroplasia (ACH). Evidence indicates that these phenotypes are not due to simple haploinsufficiency of FGFR3 but are more likely related to a role in negatively regulating skeletal growth. The effects of one of these mutations on FGFR3 signaling were examined by constructing chimeric receptors composed of the extracellular domain of human platelet-derived growth factor receptor beta (hPDGFR beta) and the transmembrane and intracellular domains of hFGFR3 or of an ACH (G375C) mutant. Following stable transfection in PC12 cells, which lack platelet-derived growth factor (PDGF) receptors, all clonal cell lines, with either type of chimera, showed strong neurite outgrowth in the presence of PDGF but not in its absence. Antiphosphotyrosine immunoblots showed ligand-dependent autophosphorylation, and both receptor types stimulated strong phosphorylation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase, an event associated with the differentiative response of these cells. In addition, ligand-dependent phosphorylation of phospholipase Cgamma and Shc was also observed. All of these responses were comparable to those observed from ligand activation, such as by nerve growth factor, of the native PC12 cells used to prepare the stable transfectants. The cells with the chimera bearing the ACH mutation were more rapidly responsive to ligand with less sustained MAPK activation, indicative of a preactivated or primed condition and consistent with the view that these mutations weaken ligand control of FGFR3 function. However, the full effect of the mutation likely depends in part on structural features of the extracellular domain. Although FGFR3 has been suggested to act as a negative regulator of long-bone growth in chrondrocytes, it produces differentiative signals similar to those of FGFR1, to which only positive effects have been ascribed, in PC12 cells. Therefore, its regulatory effects on bone growth likely result from cellular contexts and not the induction of a unique FGFR3 signaling pathway.     

Ligand activation leads to regulated intramembrane proteolysis of fibroblast growth factor receptor 3

Fibroblast growth factor receptor 3 (FGFR3) is a major negative regulator of bone growth that inhibits the proliferation and differentiation of growth plate chondrocytes. Activating mutations of its c isoform cause dwarfism in humans; somatic mutations can drive oncogenic transformation in multiple myeloma and bladder cancer. How these distinct activities arise is not clear. FGFR3 was previously shown to undergo proteolytic cleavage in the bovine rib growth plate, but this was not explored further. Here, we show that FGF1 induces regulated intramembrane proteolysis (RIP) of FGFR3. The ectodomain is proteolytically cleaved (S1) in response to ligand-induced receptor activation, but unlike most RIP target proteins, it requires endocytosis and does not involve a metalloproteinase. S1 cleavage generates a C-terminal domain fragment that initially remains anchored in the membrane, is phosphorylated, and is spatially distinct from the intact receptor. Ectodomain cleavage is followed by intramembrane cleavage (S2) to generate a soluble intracellular domain that is released into the cytosol and can translocate to the nucleus. We identify the S1 cleavage site and show that γ-secretase mediates the S2 cleavage event. In this way we demonstrate a mechanism for the nuclear localization of FGFR3 in response to ligand activation, which may occur in both development and disease.     

The localization of FGFR3 mutations causing thanatophoric dysplasia type I differentially affects phosphorylation, processing and ubiquitylation of the receptor

Recurrent missense fibroblast growth factor receptor 3 (FGFR3) mutations have been ascribed to skeletal dysplasias of variable severity including the lethal neonatal thanatophoric dysplasia types I (TDI) and II (TDII). To elucidate the role of activating mutations causing TDI on receptor trafficking and endocytosis, a series of four mutants located in different domains of the receptor were generated and transiently expressed. The putatively elongated X807R receptor was identified as three isoforms. The fully glycosylated mature isoform was constitutively but mildly phosphorylated. Similarly, mutations affecting the extracellular domain (R248C and Y373C) induced moderate constitutive receptor phosphorylation. By contrast, the K650M mutation affecting the tyrosine kinase 2 (TK2) domain produced heavy phosphorylation of the nonglycosylated and mannose-rich isoforms that impaired receptor trafficking through the Golgi network. This resulted in defective expression of the mature isoform at the cell surface. Normal processing was rescued by tyrosine kinase inhibitor treatment. Internalization of the R248C and Y373C mutant receptors, which form stable disulfide-bonded dimers at the cell surface was less efficient than the wild-type, whereas ubiquitylation was markedly increased but apparently independent of the E3 ubiquitin-ligase casitas B-lineage lymphoma (c-Cbl). Constitutive phosphorylation of c-Cbl by the K650M mutant appeared to be related to the intracellular retention of the receptor. Therefore, although mutation K650M affecting the TK2 domain induces defective targeting of the overphosphorylated receptor, a different mechanism characterized by receptor retention at the plasma membrane, excessive ubiquitylation and reduced degradation results from mutations that affect the extracellular domain and the stop codon.     

FGFR3 heterodimerization in achondroplasia, the most common form of human dwarfism

The G380R mutation in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) causes achondroplasia, the most common form of human dwarfism. Achondroplasia is a heterozygous disorder, and thus the affected individuals express both wild-type and mutant FGFR3. Yet heterodimerization in achondroplasia has not been characterized thus far. To investigate the formation of FGFR3 heterodimers in cellular membranes, we designed an FGFR3 construct that lacks the kinase domain, and we monitored the formation of inactive heterodimers between this construct and wild-type and mutant FGFR3. The formation of the inactive heterodimers depleted the pool of full-length receptors capable of forming active homodimers and ultimately reduced their phosphorylation. By analyzing the effect of the truncated FGFR3 on full-length receptor phosphorylation, we demonstrated that FGFR3 WT/G380R heterodimers form with lower probability than wild-type FGFR3 homodimers at low ligand concentration. These results further our knowledge of FGFR3-associated bone disorders.     

Extracellular point mutations in FGFR2 result in elevated ERK1/2 activation and perturbation of neuronal differentiation

Two independent gain-of-function point mutations (S252W and P253R) in the extracellular region of the FGFR2 (fibroblast growth factor receptor 2) increase the binding affinity for the growth factor. The effect of this enhanced growth factor binding by these mutants is expected to be an increase in activation of regular signalling pathways from FGFR2 as a result of more receptors being engaged by ligand at any given time. Using PC12 (pheochromocytoma) cells as a model cell system we investigated the effect of these mutations on protein phosphorylation including the receptor, the activation of downstream signalling pathways and cell differentiation. Our results show that the effects of both of these extracellular mutations have unexpected intracellular phenotypes and cellular responses. Receptor phosphorylation was altered in both the ligand-stimulated and unstimulated states. The mutants also resulted in differential phosphorylation of a number of intracellular proteins. Both mutations resulted in enhanced ERK1/2 (extracellular-signalregulated kinase1/2) activation. Although ERK1/2 activation is believed to transduce signals resulting in cell differentiation, this response was abrogated in the cells expressing the mutant receptors. The results of the present study demonstrate that single extracellular point mutations in the FGFR2 have a profound effect on intracellular signalling and ultimately on cell fate.     

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.     

Identification of tyrosine residues in constitutively activated fibroblast growth factor receptor 3 involved in mitogenesis, Stat activation, and phosphatidylinositol 3-kinase activation

Fibroblast growth factor receptor 3 (FGFR3) mutations are frequently involved in human developmental disorders and cancer. Activation of FGFR3, through mutation or ligand stimulation, results in autophosphorylation of multiple tyrosine residues within the intracellular domain. To assess the importance of the six conserved tyrosine residues within the intracellular domain of FGFR3 for signaling, derivatives were constructed containing an N-terminal myristylation signal for plasma membrane localization and a point mutation (K650E) that confers constitutive kinase activation. A derivative containing all conserved tyrosine residues stimulates cellular transformation and activation of several FGFR3 signaling pathways. Substitution of all nonactivation loop tyrosine residues with phenylalanine rendered this FGFR3 construct inactive, despite the presence of the activating K650E mutation. Addition of a single tyrosine residue, Y724, restored its ability to stimulate cellular transformation, phosphatidylinositol 3-kinase activation, and phosphorylation of Shp2, MAPK, Stat1, and Stat3. These results demonstrate a critical role for Y724 in the activation of multiple signaling pathways by constitutively activated mutants of FGFR3.     

Interaction of fibroblast growth factor receptor 3 and the adapter protein SH2-B. A role in STAT5 activation

Fibroblast growth factor receptor 3 (FGFR3) influences a diverse array of biological processes, including cell growth, differentiation, and migration. Activating mutations in FGFR3 are associated with multiple myeloma, cervical carcinoma, and bladder cancer. To identify proteins that interact with FGFR3 and which may mediate FGFR3-dependent signaling, a yeast two-hybrid screen was employed using the cytoplasmic kinase domain of FGFR3 as bait. We identified the adapter protein SH2-B as an FGFR3-interacting protein. Coimmunoprecipitation experiments demonstrate binding of the SH2-B beta isoform to FGFR3 in 293T cells. Tyrosine phosphorylation of SH2-B beta was observed when coexpressed with activated FGFR3 mutants such as the weakly activated mutant N540K or the strongly activated mutant K650E, both associated with human developmental syndromes. The extent of tyrosine phosphorylation of SH2-B beta correlates with receptor activation, suggesting that FGFR3 activation mediates tyrosine phosphorylation of SH2-B beta. Furthermore, two tyrosine phosphorylation sites of FGFR3, Tyr-724 and Tyr-760, are required for optimal binding of the Src homology-2 (SH2) domain of SH2-B beta. We also demonstrate the phosphorylation and nuclear translocation of Stat5 by activated FGFR3, which increases in response to overexpression of SH2-B beta. Taken together, our results identify SH2-B beta as a novel FGFR3 binding partner that mediates signal transduction.     

Cell responses to FGFR3 signalling: growth, differentiation and apoptosis

FGFR3 is a receptor tyrosine kinase (RTK) of the FGF receptor family, known to have a negative regulatory effect on long bone growth. Fgfr3 knockout mice display longer bones and, accordingly, most germline-activating mutations in man are associated with dwarfism. Somatically, some of the same activating mutations are associated with the human cancers multiple myeloma, cervical carcinoma and carcinoma of the bladder. How signalling through FGFR3 can lead to either chondrocyte apoptosis or cancer cell proliferation is not fully understood. Although FGFR3 can be expressed as two main splice isoforms (IIIb or IIIc), there is no apparent link with specific cell responses, which may rather be associated with the cell type or its differentiation status. Depending on cell type, differential activation of STAT proteins has been observed. STAT1 phosphorylation seems to be involved in inhibition of chondrocyte proliferation while activation of the ERK pathway inhibits chondrocyte differentiation and B-cell proliferation (as in multiple myeloma). The role of FGFR3 in epithelial cancers (bladder and cervix) is not known. Some of the cell specificity may arise via modulation of signalling by crosstalk with other signalling pathways. Recently, inhibition of the ERK pathway in achondroplastic mice has provided hope for an approach to the treatment of dwarfism. Further understanding of the ability of FGFR3 to trigger different responses depending on cell type and cellular context may lead to treatments for both skeletal dysplasias and cancer.     

The cytoplasmic tyrosine kinase Pyk2 as a novel effector of fibroblast growth factor receptor 3 activation

Activating mutations within fibroblast growth factor receptor 3 (FGFR3), a receptor tyrosine kinase, are responsible for human skeletal dysplasias including achondroplasia and the neonatal lethal syndromes thanatophoric dysplasia types I and II. Several of these same FGFR3 mutations have also been identified somatically in human cancers, including multiple myeloma, bladder carcinoma, and cervical cancer. The molecular pathways exploited by FGFR3 to stimulate abnormal proliferation during neoplasia are unclear. The nonreceptor protein-tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2) has been shown previously to regulate apoptosis in multiple myeloma cells. Here we describe a novel interaction between FGFR3 and Pyk2, mediated by the juxtamembrane domain of FGFR3 and the kinase domain of Pyk2. Within the FGFR family, Pyk2 also interacted significantly with FGFR2. Overexpression of Pyk2 alone led to its spontaneous activation and tyrosine phosphorylation, resulting in activation of Stat5B, indicated by the reporter GFP-Stat5B. These effects were completely dependent upon Tyr(402), the autophosphorylation site of Pyk2, which allows recruitment of Src family members for further activating phosphorylations at other sites on Pyk2. In the presence of activated FGFR3, the activation of Pyk2 itself became independent of Tyr(402), indicating that FGFR3 activation circumvents the requirement for c-Src recruitment at Tyr(402) of Pyk2. We also examined the role of the tyrosine phosphatase Shp2 in antagonizing Pyk2 activation. Taken together, these results suggest that signaling pathways regulated by FGFR3 may converge with Pyk2-dependent pathways to provide maximal activation of Stat5B.     

A single-base change in the tyrosine kinase II domain of ovine FGFR3 causes hereditary chondrodysplasia in sheep

Ovine hereditary chondrodysplasia, or spider lamb syndrome (SLS), is a genetic disorder that is characterized by severe skeletal abnormalities and has resulted in substantial economic losses for sheep producers. Here we demonstrate that a non-synonymous T>A transversion in the highly conserved tyrosine kinase II domain of a positional candidate gene, fibroblast growth factor receptor 3 (FGFR3), is responsible for SLS. We also demonstrate that the mutant FGFR3 allele has an additive effect on long-bone length, calling into question the long-standing belief that SLS is inherited as a strict monogenic, Mendelian recessive trait. Instead, we suggest that SLS manifestation is determined primarily by the presence of the mutant FGFR3 allele, but it is also influenced by an animal's genetic background. In contrast to FGFR3 mutations causing dwarfism in humans, this single-base change is the only known natural mutation of FGFR3 that results in a skeletal overgrowth phenotype in any species.     

Transient dimerization and interaction with ERGIC-53 occur in the fibroblast growth factor receptor 3 early secretory pathway

The fibroblast growth factor receptor 3 (FGFR3) secretory pathway includes N-linked glycosylation in the endoplasmic reticulum where a stringent quality control system ensures that only correctly folded receptor reaches the cell surface from where mature-functional FGFR3 signals upon ligand-mediated dimerization. We have previously shown that the increased kinase activity associated with FGFR3 bearing the thanatophoric dysplasia type II (TDII) mutation hampers its maturation, enabling the receptor to signal from the endoplasmic reticulum. Here we investigate if this biosynthetic disturbance could be explained by premature dimerization of the receptor. Our observations show that a limited fraction of the immature high-mannose, mutant receptor dimerizes in the early secretory pathway, as does the immature wild type FGFR3. In contrast, the mature fully glycosylated wild type receptor reaches the cell surface as monomer suggesting that dimerization is a transient event. The kinase activity of mutant FGFR3 is not required for dimerization to occur, although it increases dimerization efficiency. Furthermore, mutant FGFR3 trans-phosphorylates the immature wild type receptor indicating that dimerization occurs in the endoplasmic reticulum. Visualization of protein interaction inside the secretory pathway confirms receptor dimerization. In addition, it shows that both wild type and TDII FGFR3 interact with the mannose-specific lectin ERGIC-53. We conclude that transient dimerization is an obligatory step in FGFR3 biosynthesis acting as a pre-assembly quality control mechanism. Furthermore, the TDII/ERGIC-53 complex formation may function as a checkpoint for FGFR3 sorting downstream the endoplasmic reticulum. These findings have implications for understanding the pathogenesis of FGFR3-related disorders.     

Cellular signaling by fibroblast growth factor receptors

The 22 members of the fibroblast growth factor (FGF) family of growth factors mediate their cellular responses by binding to and activating the different isoforms encoded by the four receptor tyrosine kinases (RTKs) designated FGFR1, FGFR2, FGFR3 and FGFR4. Unlike other growth factors, FGFs act in concert with heparin or heparan sulfate proteoglycan (HSPG) to activate FGFRs and to induce the pleiotropic responses that lead to the variety of cellular responses induced by this large family of growth factors. A variety of human skeletal dysplasias have been linked to specific point mutations in FGFR1, FGFR2 and FGFR3 leading to severe impairment in cranial, digital and skeletal development. Gain of function mutations in FGFRs were also identified in a variety of human cancers such as myeloproliferative syndromes, lymphomas, prostate and breast cancers as well as other malignant diseases. The binding of FGF and HSPG to the extracellular ligand domain of FGFR induces receptor dimerization, activation and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of the receptor molecule. A variety of signaling proteins are phosphorylated in response to FGF stimulation including Shc, phospholipase-Cgamma, STAT1, Gab1 and FRS2alpha leading to stimulation of intracellular signaling pathways that control cell proliferation, cell differentiation, cell migration, cell survival and cell shape. The docking proteins FRS2alpha and FRS2beta are major mediators of the Ras/MAPK and PI-3 kinase/Akt signaling pathways as well as negative feedback mechanisms that fine-tune the signal that is initiated at the cell surface following FGFR stimulation.     

Intracellular retention, degradation, and signaling of glycosylation-deficient FGFR2 and craniosynostosis syndrome-associated FGFR2C278F

Fibroblast growth factors (FGFs) and their receptors (FGFRs) are known to play a critical role in a variety of fundamental processes, including wound healing, angiogenesis, and development of multiple organ systems. Mutations in the FGFR gene family have been linked to a series of syndromes (the craniosynostosis syndromes) whose primary phenotype involves aberrant development of the craniofacial skeleton. Craniosynostosis syndrome-linked FGFR mutations have been shown to be gain of function in terms of receptor activation and have been presumed to result in increased levels of FGF/FGFR signaling. Unfortunately, studies attempting to link expression of mutant FGFRs with changes in cellular phenotype have yielded conflicting results. In an effort to better understand the biochemical consequences of these mutations on receptor function, here we have investigated the effect of the FGFR2C278F mutation of Crouzon craniosynostosis syndrome on receptor trafficking, ubiquitination, degradation, and signaling. We find that FGFR2C278F exhibits diminished glycosylation, increased degradation, and limited cellular sublocalization in the osteoblastic cell line, MC3T3E1(C4). Additionally, we show that trafficking and autoactivation of wild type FGFR2 is glycosylation-dependent. Both FGFR2C278F and unglycosylated wild type FGFR2 signal through phospholipase Cgamma in a ligand-independent manner as well as exhibit dramatically increased binding to the adaptor protein, Frs2. These findings suggest that autoactive FGFR2 can signal from intracellular compartments. Based upon our results, we propose that the functional signaling of craniosynostosis mutant, autoactive receptors is limited in some cell types by protective cellular responses, such as increased trafficking to lysosomes and proteasomes for degradation.     

Mutations that cause osteoglophonic dysplasia define novel roles for FGFR1 in bone elongation

Activating mutations in the genes for fibroblast growth factor receptors 1-3 (FGFR1-3) are responsible for a diverse group of skeletal disorders. In general, mutations in FGFR1 and FGFR2 cause the majority of syndromes involving craniosynostosis, whereas the dwarfing syndromes are largely associated with FGFR3 mutations. Osteoglophonic dysplasia (OD) is a "crossover" disorder that has skeletal phenotypes associated with FGFR1, FGFR2, and FGFR3 mutations. Indeed, patients with OD present with craniosynostosis, prominent supraorbital ridge, and depressed nasal bridge, as well as the rhizomelic dwarfism and nonossifying bone lesions that are characteristic of the disorder. We demonstrate here that OD is caused by missense mutations in highly conserved residues comprising the ligand-binding and transmembrane domains of FGFR1, thus defining novel roles for this receptor as a negative regulator of long-bone growth.     

Dwarfism in Dexter cattle is not caused by the mutations in FGFR3 responsible for achondroplasia in humans

Dexter cattle carry a genetic defect causing a dwarf phenotype in the heterozygotes (Dx +/–), while homozygotes (Dx +/+) are stillborn with extreme shortening of limbs and gross craniofacial defects and are described as 'bulldog' calves. The heterozygous phenotype has been likened to achondroplastic dwarfism in humans (ACH), which has recently been shown to be the result of mutations in the transmembrane region of the fibroblast growth factor receptor 3 (FGFR3) gene. We have sequenced the transmembrane region of bovine FGFR3 from normal Dexter cattle (Dx -/-) and bulldog calves (Dx +/+). The sequence from both is identical and therefore excludes mutations in the transmembrane region of FGFR3 as the cause of Dexter dwarfism.     

The kinase activity of fibroblast growth factor receptor 3 with activation loop mutations affects receptor trafficking and signaling

Amino acid substitutions at the Lys-650 codon within the activation loop kinase domain of fibroblast growth factor receptor 3 (FGFR3) result in graded constitutive phosphorylation of the receptor. Accordingly, the Lys-650 mutants are associated with dwarfisms with graded clinical severity. To assess the importance of the phosphorylation level on FGFR3 maturation along the secretory pathway, hemagglutinin A-tagged derivatives were studied. The highly activated SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans) mutant accumulates in its immature and phosphorylated form in the endoplasmic reticulum (ER), which fails to be degraded. Furthermore, the Janus kinase (Jak)/STAT pathway is activated from the ER by direct recruitment of Jak1. Abolishing the autocatalytic property of the mutated FGFR3 by replacing the critical Tyr-718 reestablishes the receptor full maturation and inhibits signaling. Differently, the low activated hypochondroplasia mutant is present as a mature phosphorylated form on the plasma membrane, although with a delayed transition in the ER, and is completely processed. Signaling does not occur in the presence of brefeldin A; instead, STAT1 is activated when protein secretion is blocked with monensin, suggesting that the hypochondroplasia receptor signals at the exit from the ER. Our results suggest that kinase activity affects FGFR3 trafficking and determines the spatial segregation of signaling pathways. Consequently, the defect in down-regulation of the highly activated receptors results in the increased signaling capacity from the intracellular compartments, and this may determine the severity of the diseases.