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 bones 
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 
. The ACH phenotype is characterized by short stature, bowed legs, and shortened arms and legs 
. 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 
. FGFR3 dimerization brings the two kinase domains in close proximity such that the two kinase domains can cross-phosphorylate and activate each other 
. 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 
. Thus, multiple physical interactions regulate FGFR3 activation, and a question arises as to which of these interactions is affected by the ACH mutation.
Published studies of the effect of the ACH mutation on FGFR3 signaling demonstrate that the mutation increases ligand-independent activation 
. 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 activation. 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 dimer 
A definitive conclusion about the effect of the mutation on dimerization cannot be drawn from this study 
, however, because cross-linking gels are difficult to quantify due to the non-specific nature of the cross-linker and because cross-linking propensities depend not only on dimerization, but also on structure. In particular, since the ACH mutation is believed to induce a structural change in the unliganded dimer 
, the cross-linking efficiencies for the wild-type and the mutant may be different. In this case, chemical cross-linking cannot be used as a reliable assay to compare dimerization. Thus, despite extensive research in the field, it is not yet known if the ACH mutation alters the dimerization propensity of FGFR3.
A rigorous test of the hypothesis that the ACH mutation increases FGFR3 dimerization requires an experimental methodology that yields dimerization constants and dimerization free energies for membrane proteins. While measurements of association constants are routinely performed for soluble proteins, the development of techniques that are applicable to membrane proteins is still in its infancy. Challenges arise because membrane proteins are difficult to overexpress and purify; yet, knowledge of exact protein concentrations is required for quantitative dimerization measurements 
. For glycoproteins such as RTKs, non-mammalian expression systems are unsuitable, as they lack the appropriate post-translational modification machinery. We have shown, however, that all of these challenges can be overcome if measurements are carried out in vesicles from mammalian plasma membranes using a FRET-based method, Quantitative Imaging FRET (QI-FRET), which yields association constants for membrane proteins (and RTKs in particular) without the need for their purification 
. The RTKs are produced in mammalian cells, and thus they are post-translationally glycosylated prior to their delivery to the plasma membrane. Experiments are carried out in plasma membrane-derived vesicles, which bud off cells upon treatments that disrupt the cytoskeleton 
. The QI-FRET method yields the FRET efficiency E
, as well as the concentration of donors and acceptors, CD
, in each plasma membrane-derived vesicle, and thus yields association constants and dimerization free energies 
We have used this method previously to demonstrate that the effect of FGFR3 extracellular domains on ligand-independent FGFR3 dimerization energetics is repulsive and on the order of 1 kcal/mole 
. Here, we use the QI-FRET method to measure and compare the dimerization propensities of wild-type and mutant FGFR3 constructs in the plasma membrane of HEK293T cells, thus assaying directly the effect of the ACH mutation on FGFR3 dimerization. As the achondroplasia mutation affects ligand-independent FGFR3 signaling, here we focus on ligand-independent dimerization.