Thanatophoric dysplasia type II (TDII) is a neonatal lethal skeletal dysplasia caused by a recurrentLys-6503Glu mutation within the highly conserved activation loop of the kinase domain of fibroblast growth factor receptor 3 (FGFR3). We demonstrate here that this mutation results in profound constitutive activation of the FGFR3 tyrosine kinase, approximately 100-fold above that of wild-type FGFR3. The mechanism of FGFR3 activation in TDII was probed by constructing various point mutations in the activation loop. Substitutions at position 650 indicated that not only Glu but also Asp and, to a lesser extent, Gln and Leu result in pronounced constitutive activation of FGFR3. Additional mutagenesis within the 10-11 loop region (amino acids Tyr-647 to Leu-656) demonstrated that amino acid 650 is the only residue which can activate the receptor when changed to a Glu, indicating a specificity of position as well as charge for mutations which can give rise to kinase activation. Furthermore, when predicted sites of autophosphorylation at Tyr-647 and Tyr-648 were mutated to Phe, either singly or in combination, constitutive kinase activity was still observed in response to the Lys-6503Glu mutation, although the effect of these mutations on downstream signalling was not investigated. Our data suggest that the molecular effect of the TDII activation loop mutation is to mimic the conformational changes that activate the tyrosine kinase domain, which are normally initiated by ligand binding and autophosphorylation. These results have broad implications for understanding the molecular basis of other human developmental syndromes that involve mutations in members of the FGFR family. Moreover, these findings are relevant to the study of kinase regulation and the design of activating mutations in related tyrosine kinases.Fibroblast growth factor receptors (FGFRs) play an important role in regulating biological processes, including proliferation, differentiation, angiogenesis, and embryonic development. The four members of the FGFR family are highly related at the amino acid level, with each protein having an extracellular ligand-binding domain composed of three immunoglobulin-like domains, a transmembrane spanning region, and a cytoplasmic tyrosine kinase domain that is split by the kinase insert region. In response to ligand binding by members of the fibroblast growth factor family, FGFRs dimerize, resulting in autophosphorylation of the kinase domain and interaction with and phosphorylation of effector signalling proteins (for reviews, see references 11 and 12).Recently, several human congenital skeletal disorders have been shown to result from point mutations in members of the FGFR family. For instance, a variety of mutations mapping to the region between the second immunoglobulin loop and the transmembrane domain of FGFR2 cause the highly related craniosynostosis disorders known as Crouzon, Jackson-Weiss, Apert, and Pfeiffer syndromes. Pfeiffer syndrome also results from a mutation at a similar position in FGFR1 (for a revi...
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