Abstract
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.
Highlights
2) The inhibitory effect of ECTMWT-1 on FGFR3/WT and FGFR3/A391E is significantly larger than its effect on FGFR3/G380R (Table 1 and Fig. 10)
The conclusion of these studies is that the WT/G380R heterodimers form with lower probability than the WT/WT homodimers or the WT/A391E heterodimers for FGF1 concentrations lower than 125 ng/ml
Here we find that the likelihoods for WT/WT homodimers, WT/G380R heterodimers, and WT/A391E heterodimers are indistinguishable at higher ligand concentrations
Summary
Cell Culture—Human embryonic kidney (HEK) 293 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). Western Blots—After culture in normal medium for 24 h following transfection and starvation in serum-free medium for 24 h, cells were lysed with lysis buffer (25 mM Tris-Cl, 0.5% Triton X-100, 20 mM NaCl, 2 mM EDTA, 2 mM NaVO4, and protease inhibitor from Roche Applied Science). To determine whether there was statistically significant inhibition across the range of ligand concentrations, we performed a 2 test [21] in which the 2 contribution of each point, i, is shown in Equation 1,. The 2 test was used to assess the statistical difference between different inhibition curves, a and b In this case, reduced 2 was calculated by summing the difference over the curves for each ligand concentration, i, as shown in Equation 3,. The p value for the reduced 2 was determined using a 2 table [21], with a p value Ͻ 0.05 considered significant
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
