Physical Basis behind Achondroplasia, the Most Common Form of Human Dwarfism

Abstract

Fibroblast growth factor receptor 3 (FGFR3) is a receptor tyrosine kinase that plays an important role in long bone development. The G380R mutation in FGFR3 transmembrane domain is known as the genetic cause for achondroplasia, the most common form of human dwarfism. Despite many studies, there is no consensus about the exact mechanism underlying the pathology. To gain further understanding into the physical basis behind the disorder, here we measure the activation of wild-type and mutant FGFR3 in mammalian cells using Western blots, and we analyze the activation within the frame of a physicalchemical model describing dimerization, ligand binding, and phosphorylation probabilities within the dimers. The data analysis presented here suggests that the mutation does not increase FGFR3 dimerization, as proposed previously. Instead, FGFR3 activity in achondroplasia is increased due to increased probability for phosphorylation of the unliganded mutant dimers. This finding has implications for the design of targeted molecular treatments for achondroplasia.Fibroblast growth factor receptor 3 (FGFR3) is a receptor tyrosine kinase that consists of an extracellular domain with three immunoglobulin-like motifs, a single transmembrane (TM) 2 domain, and an intracellular split tyrosine kinase domain. Ligands (fibroblast growth factors (fgfs)) and heparin bind to the extracellular domains and stabilize the dimer, with the ligand inducing a conformational change in the extracellular domain (1). The contact stimulates catalytic activity and results in the cross-phosphorylation of the two receptors. This activates the catalytic domains for the phosphorylation of cytoplasmic substrates and triggers signaling cascades (2, 3).Mutations in FGFR3 are known to affect long bone development, which proceeds via endochondral ossification along a pathway involving differentiation of mesenchymal stem cells into cartilage, followed by bone invasion (as the chondrocytes of the cartilage die, the space is invaded by bone). FGFR3 works as a negative regulator of bone development by mediating prodifferentiation signals in chondrocytes (4 -6). FGFR3 overactivation because of mutations alters the terminal differentiation into hypertrophic chondrocytes, effectively shortening the proliferation phase. Furthermore, FGFR3 overactivation can cause cancers of the epithelium (7).The G380R mutation in FGFR3 TM domain has been linked to achondroplasia, the most common form of human dwarfism (8). The incidence rate of achondroplasia is about 1/15,000 live births (5). It is an autosomal dominant disorder that interferes with the maturation of the cartilage growth plate of long bones (6, 9). The phenotype is characterized by short stature, narrowing of the lumbar spinal canal, accentuated bowing of the middle and lower part of the back, and trident-shaped hands (5). Affected individuals often exhibit other skeletal as well as neurological complications.The physical basis underlying achondroplasia is still under debate, and multiple mechanisms may be contributi...

FGFR3 Heterodimerization in Achondroplasia, the Most Common Form of Human Dwarfism

“…At concentrations in the range 125-1000 ng/ml FGF1, we observe a plateau in the phosphorylation; the phosphorylation level does not change as more ligand is added. We have previously observed such a plateau at high ligand concentrations in transient transfection experiments (13). This plateau is rationalized by the fact that at such high ligand concentrations all receptors that are accessible to ligand have responded to it, and FGFR3 phosphorylation is at its highest level possible (13).…”

“…These dimers will be indistinguishable in many experiments, and their abundance will depend on the respective expression levels and on the dimerization propensities, which are unknown. Furthermore, the wildtype and mutant unliganded dimer structures have been proposed to be different (13,17), and thus the formation and the stability of heterodimers cannot even be predicted based on previous homodimerization studies.To investigate the formation of FGFR3 heterodimers in a cellular environment, here we designed an FGFR3 construct that lacks the kinase domain, and we monitored the formation of heterodimers between this construct and full-length wildtype and mutant FGFR3. Such full-length/truncated heterodimers will be inactive.…”

“…These dimers will be indistinguishable in many experiments, and their abundance will depend on the respective expression levels and on the dimerization propensities, which are unknown. Furthermore, the wildtype and mutant unliganded dimer structures have been proposed to be different (13,17), and thus the formation and the stability of heterodimers cannot even be predicted based on previous homodimerization studies.…”

“…The increase, which is the likely cause for pathogenesis, has been linked to an increase in the phosphorylation of the unliganded dimer (13). However, FGFR3 dimerization does not appear affected by the G380R mutation, as both the wild-type FGFR3 and the G380R mutant show very similar cross-linking and dimerization propensities (13).…”

“…In cellular studies, the G380R mutation increases FGFR3 phosphorylation in the absence of ligand and at low ligand concentration (12)(13)(14)(15). The increase, which is the likely cause for pathogenesis, has been linked to an increase in the phosphorylation of the unliganded dimer (13).…”