Achondroplasia, the most common genetic form of dwarfism, is an autosomal dominant disorder whose underlying mechanism is a defect in the maturation of the cartilage growth plate of long bones. Achondroplasia has recently been shown to result from a Gly to Arg substitution in the transmembrane domain of the fibroblast growth factor receptor 3 (FGFR3), although the molecular consequences of this mutation have not been investigated. By substituting the transmembrane domain of the Neu receptor tyrosine kinase with the transmembrane domains of wild‐type and mutant FGFR3, the Arg380 mutation in FGFR3 is shown to activate both the kinase and transforming activities of this chimeric receptor. Residues with side chains capable of participating in hydrogen bond formation, including Glu, Asp, and to a lesser extent, Gln, His and Lys, were able to substitute for the activating Arg380 mutation. The Arg380 point mutation also causes ligand‐independent stimulation of the tyrosine kinase activity of FGFR3 itself, and greatly increased constitutive levels of phosphotyrosine on the receptor. These results suggest that the molecular basis of achondroplasia is unregulated signal transduction through FGFR3, which may result in inappropriate cartilage growth plate differentiation and thus abnormal long bone development. Achondroplasia may be one of the number of cogenital disorders where constitutive activation of a member of the FGFR family leads to development abnormalities.

The fibroblast growth factor-receptor 3 (FGFR3) Lys650 codon is located within a critical region of the tyrosine kinase-domain activation loop. Two missense mutations in this codon are known to result in strong constitutive activation of the FGFR3 tyrosine kinase and cause three different skeletal dysplasia syndromes-thanatophoric dysplasia type II (TD2) (A1948G [Lys650Glu]) and SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans) syndrome and thanatophoric dysplasia type I (TD1) (both due to A1949T [Lys650Met]). Other mutations within the FGFR3 tyrosine kinase domain (e.g., C1620A or C1620G [both resulting in Asn540Lys]) are known to cause hypochondroplasia, a relatively common but milder skeletal dysplasia. In 90 individuals with suspected clinical diagnoses of hypochondroplasia who do not have Asn540Lys mutations, we screened for mutations, in FGFR3 exon 15, that would disrupt a unique BbsI restriction site that includes the Lys650 codon. We report here the discovery of three novel mutations (G1950T and G1950C [both resulting in Lys650Asn] and A1948C [Lys650Gln]) occurring in six individuals from five families. Several physical and radiological features of these individuals were significantly milder than those in individuals with the Asn540Lys mutations. The Lys650Asn/Gln mutations result in constitutive activation of the FGFR3 tyrosine kinase but to a lesser degree than that observed with the Lys540Glu and Lys650Met mutations. These results demonstrate that different amino acid substitutions at the FGFR3 Lys650 codon can result in several different skeletal dysplasia phenotypes.

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...

We have identified a novel fibroblast growth factor receptor 3 (FGFR3) missense mutation in four unrelated individuals with skeletal dysplasia that approaches the severity observed in thanatophoric dysplasia type I (TD1). However, three of the four individuals developed extensive areas of acanthosis nigricans beginning in early childhood, suffer from severe neurological impairments, and have survived past infancy without prolonged life-support measures. The FGFR3 mutation (A1949T: Lys650Met) occurs at the nucleotide adjacent to the TD type II (TD2) mutation (A1948G: Lys650Glu) and results in a different amino acid substitution at a highly conserved codon in the kinase domain activation loop. Transient transfection studies with FGFR3 mutant constructs show that the Lys650Met mutation causes a dramatic increase in constitutive receptor kinase activity, approximately three times greater than that observed with the Lys650Glu mutation. We refer to the phenotype caused by the Lys650Met mutation as "severe achondroplasia with developmental delay and acanthosis nigricans" (SADDAN) because it differs significantly from the phenotypes of other known FGFR3 mutations.

Multiple human skeletal and craniosynostosis disorders, including Crouzon, Pfeiffer, Jackson-Weiss, and Apert syndromes, result from numerous point mutations in the extracellular region of fibroblast growth factor receptor 2 (FGFR2). Many of these mutations create a free cysteine residue that potentially leads to abnormal disulfide bond formation and receptor activation; however, for noncysteine mutations, the mechanism of receptor activation remains unclear. We examined the effect of two of these mutations, W290G and T341P, on receptor dimerization and activation. These mutations resulted in cellular transformation when expressed as FGFR2͞Neu chimeric receptors. Additionally, in full-length FGFR2, the mutations induced receptor dimerization and elevated levels of tyrosine kinase activity. Interestingly, transformation by the chimeric receptors, dimerization, and enhanced kinase activity were all abolished if either the W290G or the T341P mutation was expressed in conjunction with mutations that eliminate the disulfide bond in the third immunoglobulin-like domain (Ig-3). These results demonstrate a requirement for the Ig-3 cysteine residues in the activation of FGFR2 by noncysteine mutations. Molecular modeling also reveals that noncysteine mutations may activate FGFR2 by altering the conformation of the Ig-3 domain near the disulfide bond, preventing the formation of an intramolecular bond. This allows the unbonded cysteine residues to participate in intermolecular disulfide bonding, resulting in constitutive activation of the receptor.Fibroblast growth factor receptors (FGFRs) form a family of at least four receptor tyrosine kinases that share several structural features, including three extracellular immunoglobulin-like domains (Ig), a single transmembrane domain, and an intracellular split tyrosine kinase domain (1, 2). Binding of fibroblast growth factors in the presence of heparan sulfate proteoglycans leads to dimerization of receptor molecules, followed by tyrosine autophosphorylation (3-5).

The ES oncoprotein of bovine papillomavirus, only 44 amino adds long, occurs as a disulfide-bonded transmembrane dimer. This remarkable oncoprotein stimulates signal transduction.through activation of the plateletderived growth factor (PDGF) receptor, and E5 exhibits limited amino acid sequence similarit with PDGF. Results presented here sugest that a key feature of the hydrophobic transmembrane domain is an amino acid side chain that participates in interhelical hydrogen bond formation. These data are reminiscent of the activated neu oncogene, in which a point mutation in the membrane domain leads to lipndindependent dimerization and activation of a receptor tyrosine kinase. Signfcantly, the tranmembrane domain of E5 can be largely replaced by the transmembrane domain from the activated neu receptor tyrosine kinase. Extensive mutageneis defines the minimal structural features required for transformation by the ES oncoprotein as, first, the ability to dimerize and, second, presentation ofa negatively charged residue at the extralular side of the membrane. The biological activity of ES mutants that lack most amino acid residues similar to PDGF snggests that ES and PDGF activate the PDGF receptor by disnct mechanisms.Bovine papillomavirus type I (BPV) typifies the fibropapilloma viruses, which transform fibroblasts in vitro and lead to development of fibrosarcomas in their host. In contrast, the human papillomaviruses infect epithelial cells and are strongly implicated in carcinomas of the cervix and respiratory tract (1). The fibroblast-transforming function of BPV is due to a small open reading frame, designated E5, encoding a 44-residue protein that forms membrane-associated disulfide-bonded dimers (2-6). Recent work has demonstrated that the E5 oncoprotein activates the platelet-derived growth factor (PDGF) P-receptor (7,8) and may display functional similarity with the human T-lymphotropic virus type I p12' protein (9).Inspection of the amino acid sequence of the E5 oncoprotein reveals two distinct domains (2-6): an extremely hydrophobic N-terminal domain, residues 1-32, and a hydrophilic C-terminal domain, residues 33-44. Several studies have led to the conclusion that the E5 oncoprotein is membraneanchored with a type II orientation, with the N terminus intracellular and the C terminus extracellular (10). In addition, the hydrophobic domain of E5 can function as a signal-anchor domain, indistinguishable from signal-anchor domains of well-characterized type II proteins such as neuraminidase, transferrin receptor, or asialoglycoprotein receptor (11).In this work, we describe the minimal structural features required for biological transforming activity by a transmembrane peptide. The results presented here suggest that E5 and PDGF activate the PDGF receptor by fundamentally distinct mechanisms. ES/neu Substitution Mutant. Degenerate oligonucleotides were synthesized that would encode a pool of E5/neu transmembrane recombinants in which the 44 residues would be derived as follows: 1-5 from E5, 6-10 from...

Crouzon syndrome is an autosomal dominant condition primarily characterized by craniosynostosis. This syndrome has been associated with a variety of amino acid point mutations in the extracellular domain of fibroblast growth factor receptor 2 (FGFR2). FGFR2/Neu chimeras were generated by substituting the extracellular domain of Neu with that of FGFR2 containing the following Crouzon mutations: Tyr-340->His; Cys-342->Tyr; Cys-342-*Arg; Cys- The human fibroblast growth factor receptor (FGFR) family is composed of four widely expressed receptor tyrosine kinases that play important roles in growth and development (1).These receptors possess three extracellular immunoglobulinlike domains, a single transmembrane domain, and an intracellular tyrosine kinase domain (see Fig. 1B). Various mutations in three of these receptors, FGFR1, FGFR2, and FGFR3, have recently been associated with several human developmental syndromes (2-21). Mutations located in the extracellular domain of FGFR2 have been linked to one such disorder called Crouzon syndrome (2,6,13,14,19,20,22), an autosomal dominant condition that is characterized by craniosynostosis, maxillary hypoplasia, shallow orbits, and ocular proptosis.The Crouzon mutations described by Reardon et al. (2) result in single amino acid substitutions within or near the third immunoglobulin-like region, except for one mutation that generates a new donor splice site, leading to the deletion of 17 amino acids in the juxtamembrane region (20,23

Fibroblast growth factor (FGF) receptors (FGFRs) are membrane-spanning tyrosine kinase receptors that mediate regulatory signals for cell proliferation and differentiation in response to FGFs. We have previously determined that the Lys6503Glu mutation in the activation loop of the kinase domain of FGFR3, which is responsible for the lethal skeletal dysplasia thanatophoric dyplasia type II (TDII), greatly enhances the ligand-independent kinase activity of the receptor. Here, we demonstrate that expression of this construct induces a c-fos promoter construct approximately 10-fold but does not lead to proliferation or morphological transformation of NIH 3T3 cells. In contrast, the isolated kinase domain of activated FGFR3, targeted to the plasma membrane by a myristylation signal, is able to stimulate c-fos expression by 40-fold, induce proliferation of quiescent cells, and morphologically transform fibroblasts. This result suggests that the extracellular and transmembrane domains of FGFRs exert a negative regulatory influence on the activity of the kinase domain. Targeting of the activated kinase domain to either the cytoplasm or the nucleus does not significantly affect biological signaling, suggesting that signals from FGFR3 resulting in mitogenesis originate exclusively from the plasma membrane. Furthermore, our novel observation that expression of a highly activated FGFR3 kinase domain is able to morphologically transform fibroblasts suggests that dysregulation of FGFR3 has the potential to play a role in human neoplasia.Fibroblast growth factor (FGF) receptors (FGFRs) are high-affinity membrane-spanning receptors for FGFs. FGFRs are normally catalytically inactive in the absence of FGF ligands. The binding of FGF to the extracellular domain of FGFRs, in the presence of heparan sulfate proteoglycans, induces the dimerization of two receptor molecules, allowing transphosphorylation of tyrosines within the activation loop of the intracellular tyrosine kinase domains. Activation loop phosphorylation greatly enhances the ability of FGFRs to autophosphorylate as well as to phosphorylate substrates which transmit biological signals into the cell leading to cell proliferation, differentiation, angiogenesis, or embryogenesis (4,11,21,23). Although growth factor receptor-mediated signaling has traditionally been assumed to initiate from the plasma membrane, FGF-induced cell proliferation requires prolonged exposure to ligand (63), during which activated FGFRs relocalize to the perinuclear and/or nuclear compartments of the cell (35,43,44).Point mutations in different domains of three of the four highly related FGFRs have been identified as causing human developmental abnormalities, including skeletal and cranial malformation syndromes (38,40,58). Recent work suggests that the biochemical mechanism underlying these syndromes is ligand-independent activation of the FGFR tyrosine kinase activity (58), and this constitutive signal transduction is postulated to cause premature and abnormal maturation of the affected long bo...