The thanatophoric dysplasia type II mutation hampers complete maturation of fibroblast growth factor receptor 3 (FGFR3), which activates signal transducer and activator of transcription 1 (STAT1) from the endoplasmic reticulum
ABSTRACT The K650E substitution in the fibroblast growth factor receptor 3 (FGFR3) causes constitutive tyrosine kinase activity of the receptor and is associated to the lethal skeletal disorder, thanatophoric dysplasia type II (TDII). The underlying mechanisms of how the activated FGFR3 causes TDII remains to be elucidated. FGFR3 is a transmembrane glycoprotein, which is synthesized through three isoforms, with various degrees of N-glycosylation. We have studied whether immature FGFR3 isoforms mediate the abnormal signaling in TDII. We show that synthesis of TDII-FGFR3 presents two phosphorylated forms: the immature non-glycosylated 98-kDa peptides and the intermediate 120-kDa glycomers. The mature, fully glycosylated 130-kDa forms, detected in wild type FGFR3, are not present in TDII. Endoglycosidase H cleaves the sugars on TDII intermediates thus indicating their intracellular localization in the endoplasmic reticulum. Accordingly, TDII-FGFR3-GFP co-localizes with calreticulin in the endoplasmic reticulum. Furthermore, following TDII transfection, signal transducer and activator of transcription 1 (STAT1) is phosphorylated in the absence of FGFR3 ligand and brefeldin A does not inhibit its activation. On the contrary, the cell membrane-anchored FRS2alpha protein is not activated in TDII cells. The opposite situation is observed in stable TDII cell clones where, despite the presence of phosphorylated mature receptor, STAT1 is not activated whereas FRS2alpha is phosphorylated. We speculate that the selection process favors cells defective in STAT1 activation through the 120-kDa TDII-FGFR3, thus allowing growth of the TDII cell clones. Accordingly, apoptosis is observed following TDII-FGFR3 transfection. These observations highlight the importance of the immature TDII-FGFR3 proteins as mediators of an abnormal signaling in TDII.
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ABSTRACT: The kinase activity of the thanatophoric dysplasia type II-fibroblast growth factor receptor 3 mutant (TDII-FGFR3) hampers its maturation. As a consequence, the immature receptor activates extracellular regulated kinases (ERKs) from the endoplasmic reticulum (ER), which leads to apoptosis. On the other hand, in stable TDII-FGFR3 cells receptor biosynthesis is restored and ERKs are activated from the cell surface. To identify potential mediators of cell adaptation to the activated receptor we investigated gene products that are differently regulated in TDII and wild-type FGFR3 cells. cDNA representational difference analysis reveals Sprouty4 up regulation in the TDII-FGFR3 cells. Interestingly, Sprouty4 inhibits the TDII-FGFR3-mediated ERKs activation from the ER, but fails to suppress ERKs activation from cell surface. We conclude that cell adaptation to activated FGFR3 include Sprouty4 activity, which silences the premature receptor signaling and suppress apoptosis.FEBS letters 09/2009; 583(19):3254-8. DOI:10.1016/j.febslet.2009.09.021
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ABSTRACT: Activating mutations in fibroblast growth factor receptor 3 (FGFR3) cause several human skeletal dysplasias as a result of attenuation of cartilage growth. It is believed that FGFR3 inhibits chondrocyte proliferation via activation of signal transducers and activators of transcription (STAT) proteins, although the exact mechanism of both STAT activation and STAT-mediated inhibition of chondrocyte growth is unclear. We show that FGFR3 interacts with STAT1 in cells and is capable of activating phosphorylation of STAT1 in a kinase assay, thus potentially serving as a STAT1 kinase in chondrocytes. However, as demonstrated by western blotting with phosphorylation-specific antibodies, imaging of STAT nuclear translocation, STAT transcription factor assays and STAT luciferase reporter assays, FGF does not activate STAT1 or STAT3 in RCS chondrocytes, which nevertheless respond to a FGF stimulus with potent growth arrest. Moreover, addition of active STAT1 and STAT3 to the FGF signal, by means of cytokine treatment, SRC-mediated STAT activation or expression of constitutively active STAT mutants does not sensitize RCS chondrocytes to FGF-mediated growth arrest. Since FGF-mediated growth arrest is rescued by siRNA-mediated downregulation of the MAP kinase ERK1/2 but not STAT1 or STAT3, our data support a model whereby the ERK arm but not STAT arm of FGF signaling in chondrocytes accounts for the growth arrest phenotype.Journal of Cell Science 03/2008; 121(Pt 3):272-81. DOI:10.1242/jcs.017160
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ABSTRACT: Mutations of the Fibroblast Growth Factor Receptor 3 (FGFR3) gene have been implicated in a series of skeletal dysplasias including hypochondroplasia, achondroplasia and thanatophoric dysplasia. The severity of these diseases ranges from mild dwarfism to severe dwarfism and to perinatal lethality, respectively. Although it is considered that the mutations give rise to constitutively active receptors, it remains unclear how the different mutations are functionally linked to the severity of the different pathologies. By examining various FGFR3 mutations in a HEK cell culture model, including the uncharacterized X807R mutation, it was found that only the mutations affecting the intracellular domain, induced premature receptor phosphorylation and inhibited receptor glycosylation, suggesting that premature receptor tyrosine phosphorylation of the native receptor inhibits its glycosylation. Moreover, these mutations appeared to be associated with elevated receptor signaling in the Golgi apparatus. In conclusion, although pathological severity could not be correlated with a single factor arising from FGFR3 mutations, these results suggest that intracellular domain mutations define a distinct means by which mutated FGFR3 could disrupt bone development.Biochimica et Biophysica Acta 05/2007; 1773(4):502-12. DOI:10.1016/j.bbamcr.2006.12.010