Direct Assessment of the Effect of the Gly380Arg
Achondroplasia Mutation on FGFR3 Dimerization Using
Quantitative Imaging FRET
Jesse Placone, Kalina Hristova*
Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland, United States of America
The Gly380Arg mutation in FGFR3 is the genetic cause for achondroplasia (ACH), the most common form of human
dwarfism. The mutation has been proposed to increase FGFR3 dimerization, but the dimerization propensities of wild-type
and mutant FGFR3 have not been compared. Here we use quantitative imaging FRET to characterize the dimerization of
wild-type FGFR3 and the ACH mutant in plasma membrane-derived vesicles from HEK293T cells. We demonstrate a small,
but statistically significant increase in FGFR3 dimerization due to the ACH mutation. The data are consistent with the idea
that the ACH mutation causes a structural change which affects both the stability and the activity of FGFR3 dimers in the
absence of ligand.
Citation: Placone J, Hristova K (2012) Direct Assessment of the Effect of the Gly380Arg Achondroplasia Mutation on FGFR3 Dimerization Using Quantitative
Imaging FRET. PLoS ONE 7(10): e46678. doi:10.1371/journal.pone.0046678
Editor: Bin He, Baylor College of Medicine, United States of America
Received June 7, 2012; Accepted September 4, 2012; Published October 9, 2012
Copyright: ? 2012 Placone and Hristova. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Institutes of Health [GM095930]. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Fibroblast growth factor receptor 3 (FGFR3) negatively
regulates long bone growth by controlling the differentiation of
chondrocytes in the growth plate . Single amino acid mutations
in FGFR3 are known to impact long bone development and to
lead to pathologies . Most of the known pathogenic mutations
in FGFR3 are gain of function mutations which over-activate the
receptor and cause premature chondrocyte differentiation. Thus,
the proliferation stage for the chondrocytes is shortened due to the
mutations, leading to a decrease in the overall length of the long
One of the best known FGFR3 mutations is the Gly380Arg
mutation in the transmembrane (TM) domain of the receptor .
This point mutation has been associated with 97% of the reported
cases for achondroplasia (ACH), the most common form of human
dwarfism [2,7]. The ACH phenotype is characterized by short
stature, bowed legs, and shortened arms and legs [8,9]. The
incidence rate of ACH is approximately one in 15,000 live births,
and most of the cases are sporadic.
Since the discovery of the Gly380Arg mutation as the genetic
cause for human dwarfism, research in the field has focused on the
effect of this mutation on FGFR3 signaling. FGFR3 is a member
of the receptor tyrosine kinase (RTK) superfamily. Thus, FGFR3
is a single pass receptor which consists of an extracellular ligand
binding domain, a TM domain and an intracellular kinase
domain, and functions via lateral dimerization in the membrane
[10–12]. FGFR3 dimerization brings the two kinase domains in
close proximity such that the two kinase domains can cross-
phosphorylate and activate each other [13,14]. This process is
regulated by ligands from the fgf family, which bind to FGFR3
extracellular domain on the cell surface in the presence of heparin
sulfates. The bound ligands are believed to stabilize the dimer,
alter its structure and enhance its activation [15–18]. Thus,
multiple physical interactions regulate FGFR3 activation, and a
question arises as to which of these interactions is affected by the
Published studies of the effect of the ACH mutation on FGFR3
signaling demonstrate that the mutation increases ligand-indepen-
dent activation [17,19–21]. However, the activation of FGFR3 at
high ligand concentrations, and the binding of ligand (fgf1) to
FGFR3, are not affected by the ACH mutation. Thus, the effect of
the mutation is restricted to ligand-independent FGFR3 activa-
tion. The cause for this increase, however, is controversial.
Webster and Donoghue hypothesized that the activity is increased
because the mutation increases FGFR3 dimerization . Their
hypothesis was based on the observation that FGFR3 activity was
increased, as compared to wild-type, when the glycine residue at
position 380 was replaced with amino acids capable of forming
hydrogen bonds. However, they did not compare the dimerization
propensities of the wild-type and the mutant.
He et al. used cross-linking of the full-length FGFR3 in
mammalian membranes to test the hypothesis that the ACH
mutation increases FGFR3 dimerization . Despite the
increased FGFR3 activation at low ligand concentration due to
the mutation, there was no discernible difference in the cross-
linking propensities of the wild-type and the mutant. Instead, the
ACH mutation was found to increase the probability for
phosphorylation of tyrosines in the kinase activation loop, and
was hypothesized to induce a structural change in the unliganded
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Effect of the G380R Mutation on FGFR3 Dimerization
PLOS ONE | www.plosone.org7 October 2012 | Volume 7 | Issue 10 | e46678