The ®broblast growth factor receptor (FGFR) family members mediate a number of important cellular processes, and are mutated or overexpressed in several forms of human cancer. Mutation of Lys650?Glu in the activation loop of the FGFR3 kinase domain causes the lethal human skeletal disorder thanatophoric dysplasia type II (TDII) and is also found in patients with multiple myeloma, bladder and cervical carcinomas. This mutation leads to constitutive activation of FGFR3. To compare the signaling activity of FGFR family members, this activating mutation was generated in FGFR1, FGFR3, and FGFR4. We show that the kinase domains of FGFR1, FGFR3, and FGFR4 containing the activation loop mutation, when targeted to the plasma membrane by a myristylation signal, can transform NIH3T3 cells and induce neurite outgrowth in PC12 cells. Phosphorylation of Shp2, PLC-g, and MAPK was also stimulated by all three`TDII-like' FGFR derivatives. Additionally, activation of Stat1 and Stat3 was observed in cells expressing the activated FGFR derivatives. Finally, we demonstrate that FGFR1, FGFR3, and FGFR4 derivatives can stimulate PI-3 kinase activity. Our comparison of these activated receptor derivatives reveals a signi®cant overlap in the panel of eector proteins used to mediate downstream signals. This also represents the ®rst demonstration that activation of FGFR4, in addition to FGFR1 and FGFR3, can induce cellular transformation. Moreover, our results suggest that Stat activation by FGFRs is important in their ability to act as oncogenes. Oncogene (2000) 19, 3309 ± 3320.

Ligand binding to receptor tyrosine kinases (RTKs) regulates receptor dimerization and activation of the kinase domain. To examine the role of the transmembrane domain in regulation of RTK activation, we have exploited a simplified transmembrane motif, [VVVEVVV] n , previously shown to activate the Neu receptor. Here we demonstrate rotational linkage of the transmembrane domain with the kinase domain, as evidenced by a periodic activation of Neu as the dimerization motif is shifted across the transmembrane domain. These results indicate that activation requires a specific orientation of the kinase domains with respect to each other. Results obtained with platelet-derived growth factor receptor-␤ suggest that this rotational linkage of the transmembrane domain to the kinase domain may be a general feature of RTKs. These observations suggest that activating mutations in RTK transmembrane and juxtamembrane domains will be limited to those residues that position the kinase domains in an allowed rotational conformation.

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).

Fibroblast growth factor receptor 3 (FGFR3) mutations are frequently involved in human developmental disorders and cancer. Activation of FGFR3, through mutation or ligand stimulation, results in autophosphorylation of multiple tyrosine residues within the intracellular domain. To assess the importance of the six conserved tyrosine residues within the intracellular domain of FGFR3 for signaling, derivatives were constructed containing an N-terminal myristylation signal for plasma membrane localization and a point mutation (K650E) that confers constitutive kinase activation. A derivative containing all conserved tyrosine residues stimulates cellular transformation and activation of several FGFR3 signaling pathways. Substitution of all nonactivation loop tyrosine residues with phenylalanine rendered this FGFR3 construct inactive, despite the presence of the activating K650E mutation. Addition of a single tyrosine residue, Y724, restored its ability to stimulate cellular transformation, phosphatidylinositol 3-kinase activation, and phosphorylation of Shp2, MAPK, Stat1, and Stat3. These results demonstrate a critical role for Y724 in the activation of multiple signaling pathways by constitutively activated mutants of FGFR3. INTRODUCTIONThe fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases (RTKs) mediates growth, differentiation, and cellular migration in a diverse range of cell types. These receptors function as high-affinity binding proteins for the FGF family of ligands, of which there are nearly 20 members (Szebenyi and Fallon, 1999). Ligand binding to the extracellular domain of FGFRs results in receptor dimerization and transphosphorylation of tyrosine residues in the intracellular domain.Many human disorders have been linked to mutations in FGFR3 (reviewed in Webster and Donoghue, 1997b;Burke et al., 1998). For example, mutations in the extracellular domain of FGFR3 such as R248C and G370C result in thanatophoric dysplasia type I (TDI), a lethal skeletal disorder. Mutations in the FGFR3 transmembrane domain can also lead to constitutive receptor activation, such as the G380R mutation responsible for most cases of achondroplasia, or skeletal dwarfism. Mutations in the FGFR3 kinase domain are responsible for developmental disorders that range in severity from the relatively mild hypochondroplasia (N540K) to the neonatal lethal disorder TDII (K650E). A different mutation at this same position, K650M, leads to a second syndrome, SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans), which involves skeletal malformations, CNS disturbances, and skin dysplasia (Tavormina et al., 1999).An exciting link between FGFR3 and cancer has recently been uncovered. The G375C and K650E mutations involved in skeletal malformation syndromes have also been identified in some patients with multiple myeloma, a proliferative B cell disorder (Chesi et al., 1997;Richelda et al., 1997). The K650E kinase domain mutation, as well as the R248C, S249C, and G370C extracellular domain mutati...