Fig. 1. A biochemical comparison of the glycosylation and tyrosine phosphorylation of the WT and mutant FGFR3s. (A) Schematic depiction of FGFR3 protein including the Ig-like domains (Ig1–3) of the extracellular domain, the transmembrane domain (TM) and the split tyrosine kinase domains (TK1 and TK2) of the intracellular domain. Also shown is the position of the mutations examined in this study. (B) FGFR3s for WT, G380R, R248C, Y373C, K650N, K650M and K650E all appeared as three discrete bands of differing mobilities (105, 115, 130 kDa) by Western blot, as a consequence of glycosylation. EndoH treatment removed mannose-rich moieties, and PNGase treatment removed all glycosylated moieties from FGFR3. (C) Tunicamycin treatment inhibited glycosylation of native WT and K650M FGFR3 proteins. (D) The levels of the native and differently glycosylated WT, G380R, R248C, Y373C, K650N, K650M and K650E FGFR3 isoforms were examined at 24 and 48 h by Western blot (upper panels). The relative levels of the native and differently glycosylated isoforms from at least five experiments were quantified at 24 h (lower graphics). The asterisks mark the significantly lower relative levels of the fully glycosylated isoforms compared with that of the WT (*p < 0.01; **p < 0.001). (E) The detection of tyrosine phosphorylation on the FGFR3s. WT, G380R, R248C, Y373C, K650N, K650M and K650E FGFR3s were immunoprecipitated by anti-FGFR3 and examined by Western blot with anti-FGFR3 or anti-phosphotyrosine. (F) WT and K650M FGFR3s were treated with Sugen5402 (tyrosine kinase inhibitor) and examined by Western blotting of anti-FGFR3 immunoprecipitations. Sugen5402 treatment reduced tyrosine phosphorylation and increased the relative level of the fully-glycosylated isoform of the K650M mutant.

Fig. 2. The intracellular mutation X807R displays similar biochemical properties to those of the K650 mutations. (A) Schematic representation of the X807R mutation with the additional read-through domain of the FGFR3 protein that includes a hydrophobic rich region. (B) The X807R FGFR3 appeared as three discrete bands of differing mobilities (approximately 128,139,153 kDa) by Western blot, as a consequence of glycosylation. EndoH treatment removed mannose-rich moieties, and PNGase treatment removed all glycosylated moieties from FGFR3. (C) The relative levels of the native and differently glycosylated X807R FGFR3s from eight experiments were quantified at 24 h, where the relative level of the lowest mobility isoform (153 kDa and marked by asterisks) was significantly lower than that of the WT receptor (**p < 0.001). (D) Tyrosine phosphorylation on the X807R FGFR3 mutation was assessed by immunoprecipitation by anti-FGFR3 and examined by Western blot with anti-FGFR3 or anti-phosphotyrosine. (E) The X807R, K650M and WT receptors detected by immunofluorescence (green) partially colocalised with the Golgi marker GM130 (red). Scale bars equal 2 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. The expression of FGFR3 is associated with elevated levels of tyrosine phosphorylated proteins. (A) Immunofluorescence detection of FGFR3 (green) and tyrosine phosphorylated proteins (red) revealed that detectable levels of tyrosine phosphorylation are only associated with FGFR3-transfected cells exemplified here by cells transiently transfected with the WT and K650M FGFR3 variants. The nuclei (blue) of non-transfected cells are marked by asterisks. (B) The levels of detectable tyrosine phosphorylated proteins varied depending upon the construct that was transfected (WT, G380R, Y373C, K650N, K650M, K650E and X807R FGFR3s). Scale bars equal 2 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. Greater association of FGFR3-positive puncta (green) in WT, K650M or X807R transfected cells, with rab6-positive puncta (red, lower nine panels) compared with calnexin (red, upper nine panels) or sec31-positive puncta (red, middle nine panels). Scale bars equal 2 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Treatments of FGFR3-transfected cells with nocodazole or BFA suggest that the tyrosine phosphorylated proteins are primarily associated with the Golgi apparatus. (A) Western analysis of transfected cells with either the WT or K650M isoforms and treated with nocodazole, shows no change in glycosylation. (B) Immunofluorescence detection of FGFR3 (green) and tyrosine phosphorylated proteins (red) of cells transfected with WT, K650M or X807R isoforms and treated with nocodazole. (C) Western analysis of transfected cells with either the WT, G380R, Y373C, K650N, K650M or K650E isoforms showing that BFA treatment inhibits glycosylation. (D) Immunofluorescence detection of FGFR3 (green) and tyrosine phosphorylated proteins (red) of cells transfected with WT, G380R, Y373C, K650N, K650M or K650E isoforms and treated with BFA. Scale bars equal 2 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

No statistically significant correlation between bone pathologies from which the FGFR3 mutations arise, and glycosylation processing defects or the level of tyrosine phosphorylation

The table lists the ranking scores for each parameter used, prior to determining the correlation coefficient r².