Constitutive Activation of Fibroblast Growth Factor Receptor-2 by a Point Mutation Associated with Crouzon Syndrome

The fibroblast growth factor receptors (FGFRs) are a family of ligand-activated, membrane-spanning tyrosine kinases. Mutations in several human FGFR genes have been identified as playing a role in certain disorders of bone growth and development. One of these, Crouzon syndrome, an autosomal dominant disorder causing craniosynostosis, has been associated with mutations in the human FGFR-2 gene. We report here that microinjection of Xenopus embryos with RNA encoding an FGFR-2 protein bearing a Cys332® Tyr mutation (FGFR-2CS) found in Crouzon syndrome results in fibroblast growth factor (FGF)-independent induction of mesoderm in animal pole explants. Wild-type FGFR-2 did not induce mesoderm when injected at similar doses. The effects of the mutant receptor were blocked by co-expression of dominant negative mutants of either Raf or Ras. Analysis of the mutant receptor protein expressed in Xenopus oocytes indicates that it forms covalent homodimers, does not bind radiolabeled FGF, and has increased tyrosine phosphorylation. These results indicate that FGFR-2CS forms an intermolecular disulfide bond resulting in receptor dimerization and ligand-independent activation that may play a role in the etiology of Crouzon syndrome. The fibroblast growth factor receptors (FGFRs) are a family of ligand-activated, membrane-spanning tyrosine kinases. Mutations in several human FGFR genes have been identified as playing a role in certain disorders of bone growth and development. One of these, Crouzon syndrome, an autosomal dominant disorder causing craniosynostosis, has been associated with mutations in the human FGFR-2 gene. We report here that microinjection of Xenopus embryos with RNA encoding an FGFR-2 protein bearing a Cys332® Tyr mutation (FGFR-2CS) found in Crouzon syndrome results in fibroblast growth factor (FGF)-independent induction of mesoderm in animal pole explants. Wild-type FGFR-2 did not induce mesoderm when injected at similar doses. The effects of the mutant receptor were blocked by co-expression of dominant negative mutants of either Raf or Ras. Analysis of the mutant receptor protein expressed in Xenopus oocytes indicates that it forms covalent homodimers, does not bind radiolabeled FGF, and has increased tyrosine phosphorylation. These results indicate that FGFR-2CS forms an intermolecular disulfide bond resulting in receptor dimerization and ligand-independent activation that may play a role in the etiology of Crouzon syndrome. INTRODUCTIONThe fibroblast growth factors (FGFs) 1The abbreviations used are: FGFfibroblast growth factorFGFRFGF receptorBSAbovine serum albumin. are a family of polypeptide mitogens that currently consists of nine members(1Burgess W.H. Maciag T. Annu. Rev. Biochem. 1989; 58: 575-606Crossref PubMed Google Scholar, 2Wilkie A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The FGFs mediate a variety of biological processes including angiogenesis, wound healing, migration, mitogenesis, neuronal survival, and mesoderm induction(2Wilkie A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 3Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-40Crossref PubMed Scopus (1169) Google Scholar). These biological effects are mediated via binding to four members of a family of high affinity membrane-spanning tyrosine kinase receptors(2Wilkie A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 3Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-40Crossref PubMed Scopus (1169) Google Scholar). The FGFs have also been shown to bind to lower affinity cell surface heparan sulfate proteoglycans(1Burgess W.H. Maciag T. Annu. Rev. Biochem. 1989; 58: 575-606Crossref PubMed Google Scholar, 2Wilkie A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The prototype FGF receptor (FGFR) is comprised of an extracellular domain made up of three immunoglobulin (Ig)-like domains designated IgI-IgIII, a hydrophobic membrane-spanning region, and a cytoplasmic tyrosine kinase domain(2Wilkie A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 3Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-40Crossref PubMed Scopus (1169) Google Scholar). The amino acid sequences of individual members of the FGFR family are highly conserved among vertebrate species(2Wilkie A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The IgIII domain of FGFR1-3 is encoded by three exons and is generated by alternative splicing of IgIIIa with one of two alternative exons designated IgIIIb and IgIIIc(4Johnson D.E. Lu J. Chen H. Werner S. Williams L.T. Mol. Cell. Biol. 1991; 11: 4627-4634Crossref PubMed Scopus (356) Google Scholar, 5Chellaiah A.T. McEwen D.G. Werner S. Xu J. Ornitz D.M. J. Biol. Chem. 1994; 269: 11620-11627Abstract Full Text PDF PubMed Google Scholar). This alternative splicing generates receptor isoforms with varying ligand binding specificities(6Zimmer Y. Givol D. Yayon A. J. Biol. Chem. 1993; 268: 7899-7903Abstract Full Text PDF PubMed Google Scholar, 7Cheon H.-G. Larochelle W.J. Bottaro D.P. Burgess W.H. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 989-993Crossref PubMed Scopus (64) Google Scholar, 8Miki T. Bottaro D.P. Fleming T.P. Smith C.L. Burgess W.H. Chan A.M.L. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 246-250Crossref PubMed Scopus (652) Google Scholar). Like the FGFs themselves, the FGFRs have unique but overlapping spatiotemporal patterns of expression during vertebrate development(9Orr-Urtreger A. Givol D. Yayon A. Yarden Y. Lonai P. Development. 1991; 113: 1419-1434Crossref PubMed Google Scholar, 10Friesel R. Brown S.A.N. Development. 1992; 116: 1051-1058PubMed Google Scholar, 11McDonald F.J. Heath J.K. Dev. Genet. 1994; 15: 148-154Crossref PubMed Scopus (20) Google Scholar). The unique patterns of expression of both FGFs and their receptors during vertebrate development suggest that each may have a specialized function. Recent experimental evidence indicates that when FGFR function is disrupted by genetic manipulation, major defects in embryonic development occur(12Amaya E. Musci T.J. Kirschner M.W. Cell. 1991; 66: 257-270Abstract Full Text PDF PubMed Scopus (922) Google Scholar, 13Peters K. Werner S. Liao X. Wert S. Whitsett J. Williams L. EMBO J. 1994; 13: 3296-3301Crossref PubMed Scopus (377) Google Scholar, 14Werner S. Weinberg W. Liao X. Peters K.G. Blessing M. Yuspa S.H. Weiner R.L. Williams L.T. EMBO J. 1993; 12: 2635-2643Crossref PubMed Scopus (217) Google Scholar, 15Yamaguchi T.P. Harpal K. Henkemeyer M. Rossant J. Genes & Dev. 1994; 8: 3032-3044Crossref PubMed Scopus (622) Google Scholar, 16Deng C. Wynshaw-Boris A. Shen M.M. Daugherty C. Ornitz D.M. Leder P. Genes & Dev. 1994; 8: 3045-3057Crossref PubMed Scopus (625) Google Scholar).Within the last year, several mutations have been identified in FGFR genes that appear to be the cause of several human disorders of bone growth and development(2Wilkie A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). One of these, Crouzon syndrome, is characterized by craniosynostosis, an abnormality of skull development in which the sutures of the growing bones fuse prematurely(17Reardon W. Winter R.M. Rutland P. Pulleyn L.J. Jones B.M. Malcolm S. Nat. Genet. 1994; 8: 98-103Crossref PubMed Scopus (607) Google Scholar). A variety of mutations in exons IgIIIa and IgIIIc of FGFR-2 have been identified in Crouzon syndrome(17Reardon W. Winter R.M. Rutland P. Pulleyn L.J. Jones B.M. Malcolm S. Nat. Genet. 1994; 8: 98-103Crossref PubMed Scopus (607) Google Scholar, 18Rutland P. Pulleyn L.J. Reardon W. Baraister M. Hayward R. Jones B. Malcolm S. Winter R.M. Oldridge M. Slaney S.F. Poole M.D. Wilkie A.O.M. Nat. Genet. 1995; 9: 173-176Crossref PubMed Scopus (386) Google Scholar, 19Jabs E.W. Li X. Scott A.F. Meyers C. Chen W. Eccles M. Mao J. Charnas L.R. Jackson C.E. Jaye M. Nat. Genet. 1994; 8: 275-279Crossref PubMed Scopus (406) Google Scholar). These mutations may either directly (Cys342® Tyr/Cys342® Arg/Cys342® Ser/Cys342® Phe; Cys278® Phe; Tyr328® Cys; Ser347® Cys; Ser354® Cys) or indirectly (Ser267® Pro; Gln289® Pro; Tyr340 His) result in the creation of a free cysteine residue that could result in covalent dimerization resulting in ligand-independent activation of the mutant receptor(2Wilkie A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Here we report that mutation of Cys332® Tyr of Xenopus FGFR-2, analogous to the Cys342® Tyr mutation most commonly found in Crouzon syndrome, promotes activation of the mutant receptor in the absence of ligand.EXPERIMENTAL PROCEDURESMaterialsC4-Raf cDNA and N17-Ras cDNA were gifts of Dr. U. Rapp (National Cancer Institute) and Dr. T. Sargent (NIH), respectively. Affinity-purified rabbit antibodies to FGFR were prepared as described previously(20Brown S.A.N. Friesel R. Biochem. Biophys. Res. Commun. 1993; 193: 1116-1122Crossref PubMed Scopus (4) Google Scholar). A murine monoclonal antibody to phosphotyrosine, PY20, was obtained from Transduction Laboratories. Recombinant FGF-1 was a gift from Dr. W. Burgess (Holland Laboratory).In Vitro MutagenesisA BamHI fragment encoding the entire open reading frame of Xenopus FGFR-2 (10Friesel R. Brown S.A.N. Development. 1992; 116: 1051-1058PubMed Google Scholar) was subcloned into pTZ19U (Bio-Rad). Mutagenesis of Cys332 to Tyr in Xenopus FGFR-2 was performed by the method of Kunkel et al.(21Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4886) Google Scholar) using the mutagenic primer 5′-TCCAGCTATATAAGTATATTCCCC-3′ to yield FGFR-2CS. The presence of the Cys332® Tyr mutation and the absence of other mutations were confirmed by sequence analysis.Plasmid ConstructionAll constructs for in vitro transcription were cloned into the BglII site of the SP64T or SP64T3 vectors (gifts of Dr. D. Melton, Harvard University). Synthesis of capped mRNA for microinjection was performed with SP6 RNA polymerase using a Message Machine kit (Ambion).Embryo InjectionsEggs were collected from Xenopus laevis females and fertilized in vitro as described previously(22Moon R.T. Christian J.L. Technique (Phila.). 1989; 1: 76-89Google Scholar). Embryos were dejellied 30-60 min after fertilization with 2% cysteine, pH 8.0, and maintained at 17°C. At the two-cell stage, embryos were transferred to 1 × MMR (5 pH 1 and and of two-cell embryos was injected in the animal pole with of the of and RNA pole was from embryos J. of Xenopus Scholar) and in × MMR 1 serum and in the presence or absence of FGF-1 at were collected at or for mRNA and for mRNA RNA was and by RNA as P. Biochem. PubMed Scopus Google Scholar, G.M. W. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, R. Mol. Cell. Biol. 1991; 11: PubMed Scopus Google Scholar). for RNA were to a Xenopus R. Mol. Cell. Biol. 1991; 11: PubMed Scopus Google and were collected and to Xenopus in and Scholar). were by and maintained in 1 × Xenopus in and Scholar) 1 and at were injected with of RNA at the and as described for or were in pH and with 1 of to and for min at × of protein were with an of × and by were and were as R. Burgess W.H. Maciag T. Mol. Cell. Biol. 1989; 9: PubMed Scopus Google Scholar). antibodies were with antibodies and Vitro were prepared as described and of protein were with an FGFR S.A.N. Friesel R. Biochem. Biophys. Res. Commun. 1993; 193: 1116-1122Crossref PubMed Scopus (4) Google Scholar). were with and kinase performed as S.A.N. Friesel R. Biochem. Biophys. Res. Commun. 1993; 193: 1116-1122Crossref PubMed Scopus (4) Google Scholar). were and were by S.A.N. Friesel R. Biochem. Biophys. Res. Commun. 1993; 193: 1116-1122Crossref PubMed Scopus (4) Google were injected with of either FGFR-2 or FGFR-2CS RNA and for to oocytes either FGFR-2 or FGFR-2CS were in of 1 × 1 and in the presence of × prepared as described R. Burgess W.H. T. Maciag T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were at for 1 by with binding were with for min at as described R. Burgess W.H. T. Maciag T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were prepared as described and were by E. Musci T.J. Kirschner M.W. Development. 1993; Google by were by mutations have been identified in the IgIII domain of human FGFR-2 in with Crouzon syndrome(17Reardon W. Winter R.M. Rutland P. Pulleyn L.J. Jones B.M. Malcolm S. Nat. Genet. 1994; 8: 98-103Crossref PubMed Scopus (607) Google Scholar, 18Rutland P. Pulleyn L.J. Reardon W. Baraister M. Hayward R. Jones B. Malcolm S. Winter R.M. Oldridge M. Slaney S.F. Poole M.D. Wilkie A.O.M. Nat. Genet. 1995; 9: 173-176Crossref PubMed Scopus (386) Google Scholar, A.O.M. Slaney S.F. Oldridge M. Poole M.D. Hayward Pulleyn L. Rutland P. Nat. Genet. 1995; 9: PubMed Scopus Google Scholar). The most identified mutation is Cys342® resulting in the of a free cysteine residue that may be to intermolecular disulfide we a mutation in a Xenopus FGFR-2 cDNA analogous to the human Cys342® Tyr of RNA from FGFR-2 or FGFR-2CS were injected into both of two-cell Xenopus At the stage, animal pole was and for K. J. Smith Development. Google Scholar). animal from embryos injected with FGFR-2CS RNA in a similar to with FGF-1 injected with similar of FGFR-2 RNA and animal of of FGFR-2CS RNA results in in M. and R. E. mesoderm induction and animal have been shown to be blocked by the expression of a dominant negative Raf or dominant negative M. 1992; PubMed Scopus Google Scholar, Williams L.T. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). with co-expression of either a dominant negative Raf or dominant negative with FGFR-2CS animal also mesoderm induction by for the expression of the Xenopus of is expressed in the mesoderm of embryos and has been shown to be a for mesoderm induction by growth factors as B.M. D. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). Wild-type FGFR-2 or FGFR-2CS were injected into the animal pole of both of two-cell were from embryos and the or RNA was and by RNA that mRNA is expressed in animal in a with FGFR-2CS of as as of FGFR-2CS RNA was to induce expression of and the was similar to that by mRNA expression was in injected with a similar of FGFR-2 RNA or in animal were not in animal FGFR-2CS and either a dominant negative or a dominant negative of of mesoderm by FGFR-2CS. Embryos at the two-cell were injected with either FGFR-2 or FGFR-2CS RNA in the dominant negative Raf and of FGFR-2CS RNA was with of RNA or N17-Ras animal were at and at either or as RNA was and mRNA expression by RNA The was with an to as an RNA embryos were injected and animal as described in A. were when embryos and RNA was and by RNA for mRNA expression two as an for RNA a for mesoderm D. Kirschner M. Cell. Full Text PDF PubMed Scopus Google is also in a by expression of FGFR-2CS as by RNA mRNA is not expressed in animal not or in injected with a similar of FGFR-2 The expression of mRNA is blocked in animal FGFR-2CS and either a dominant negative or a dominant negative by both and by the expression of and of but not FGFR-2, has the to induce mesoderm in the absence of of most Crouzon syndrome mutations identified is the creation or of cysteine in the IgIII domain of A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). and are to a disulfide bond to of the These two cysteine are conserved the FGFR of either of two could result in of the of the domain and the creation of a free cysteine The creation of a free cysteine residue either directly or indirectly a for the effects of FGFR dimerization and ligand-independent activation by of an intermolecular disulfide we FGFR-2 or FGFR-2CS RNA into Xenopus from or injected oocytes were by or and with an FGFR S.A.N. Friesel R. Biochem. Biophys. Res. Commun. 1993; 193: 1116-1122Crossref PubMed Scopus (4) Google Scholar). both FGFR-2 and FGFR-2CS as forms of and FGFR-2CS an at with the of a These protein were to with a monoclonal antibody to The of was similar to that with the FGFR similar of receptor protein were in each FGFR-2CS of the in FGFR-2CS kinase were both FGFR-2 and FGFR-2CS in vitro tyrosine kinase FGFR-2CS FGFR-2 kinase were a high of the kinase as a results by with antibodies to FGFR and These indicate that a of FGFR-2CS as a covalent with tyrosine kinase resulting in activation of the mutant a has been described for growth factor A. A. J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Mol. Cell. Biol. 1994; PubMed Scopus Google and for the mutations of the tyrosine M. A. Bottaro D.P. M. A. B. 1995; PubMed Scopus Google of FGFR-2 and FGFR-2CS expressed in Xenopus oocytes were either or injected with FGFR-2 RNA or FGFR-2CS and were prepared and to and as described and of FGFR-2 and FGFR-2CS was by with a antibody were prepared as described for A and to with a monoclonal antibody to from injected oocytes were to with an and in vitro kinase were performed as described and are each The are shown to the IgIII domain of FGFR-2 has been shown to be for ligand Y. Givol D. Yayon A. J. Biol. Chem. 1993; 268: 7899-7903Abstract Full Text PDF PubMed Google Scholar, 7Cheon H.-G. Larochelle W.J. Bottaro D.P. Burgess W.H. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 989-993Crossref PubMed Scopus (64) Google Scholar). The Cys332® Tyr mutation of in to a free cysteine residue of intermolecular disulfide may also the of IgIII resulting in ligand ligand binding and were performed Xenopus oocytes with either FGFR-2 or FGFR-2CS injected with FGFR-2 RNA a major of injected with FGFR-2CS RNA in binding to oocytes These indicate that mutation of Cys332 results in a of ligand and activation of the mutant receptor is These are with mutation of an residue in which has also been shown to ligand M. Xu M. K. J. Biol. Chem. 1992; Full Text PDF PubMed Google of to FGFR-2 and FGFR-2CS. injected with either FGFR-2 or FGFR-2CS RNA were with and to and as described and The of the is shown to the that creation of a cysteine residue in the IgIII domain of FGFR-2 results in the of an intermolecular disulfide bond and ligand-independent activation of These results indicate a for the dominant effects in Crouzon syndrome and other mutations in FGFR A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). is that most of FGFR mutations are and the of in may the to which individual mutations the the in among with mutations may indicate that other genes may be that the effects of A of each FGFR as described may which may for Xenopus has to be a for the of mutant in J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). has been FGF receptor and function E. Musci T.J. Kirschner M.W. Cell. 1991; 66: 257-270Abstract Full Text PDF PubMed Scopus (922) Google Scholar, E. Musci T.J. Kirschner M.W. Development. 1993; Google Scholar, H. M. K. A. Williams L. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). the induction of mesoderm in Xenopus animal by a FGFR-2 does not of the that to craniosynostosis, it does an to the of the mutations associated with is the to a the of RNA injected and the biological effects This of may be of the FGFR mutations identified are that mutants may be and of expression may be to of the the etiology of Crouzon syndrome to be results that a mutation associated with syndrome results in activation of FGFR-2 with biological for vertebrate development. INTRODUCTIONThe fibroblast growth factors (FGFs) 1The abbreviations used are: FGFfibroblast growth factorFGFRFGF receptorBSAbovine serum albumin. are a family of polypeptide mitogens that currently consists of nine members(1Burgess W.H. Maciag T. Annu. Rev. Biochem. 1989; 58: 575-606Crossref PubMed Google Scholar, 2Wilkie A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The FGFs mediate a variety of biological processes including angiogenesis, wound healing, migration, mitogenesis, neuronal survival, and mesoderm induction(2Wilkie A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 3Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-40Crossref PubMed Scopus (1169) Google Scholar). These biological effects are mediated via binding to four members of a family of high affinity membrane-spanning tyrosine kinase receptors(2Wilkie A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 3Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-40Crossref PubMed Scopus (1169) Google Scholar). The FGFs have also been shown to bind to lower affinity cell surface heparan sulfate proteoglycans(1Burgess W.H. Maciag T. Annu. Rev. Biochem. 1989; 58: 575-606Crossref PubMed Google Scholar, 2Wilkie A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The prototype FGF receptor (FGFR) is comprised of an extracellular domain made up of three immunoglobulin (Ig)-like domains designated IgI-IgIII, a hydrophobic membrane-spanning region, and a cytoplasmic tyrosine kinase domain(2Wilkie A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 3Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-40Crossref PubMed Scopus (1169) Google Scholar). The amino acid sequences of individual members of the FGFR family are highly conserved among vertebrate species(2Wilkie A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The IgIII domain of FGFR1-3 is encoded by three exons and is generated by alternative splicing of IgIIIa with one of two alternative exons designated IgIIIb and IgIIIc(4Johnson D.E. Lu J. Chen H. Werner S. Williams L.T. Mol. Cell. Biol. 1991; 11: 4627-4634Crossref PubMed Scopus (356) Google Scholar, 5Chellaiah A.T. McEwen D.G. Werner S. Xu J. Ornitz D.M. J. Biol. Chem. 1994; 269: 11620-11627Abstract Full Text PDF PubMed Google Scholar). This alternative splicing generates receptor isoforms with varying ligand binding specificities(6Zimmer Y. Givol D. Yayon A. J. Biol. Chem. 1993; 268: 7899-7903Abstract Full Text PDF PubMed Google Scholar, 7Cheon H.-G. Larochelle W.J. Bottaro D.P. Burgess W.H. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 989-993Crossref PubMed Scopus (64) Google Scholar, 8Miki T. Bottaro D.P. Fleming T.P. Smith C.L. Burgess W.H. Chan A.M.L. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 246-250Crossref PubMed Scopus (652) Google Scholar). Like the FGFs themselves, the FGFRs have unique but overlapping spatiotemporal patterns of expression during vertebrate development(9Orr-Urtreger A. Givol D. Yayon A. Yarden Y. Lonai P. Development. 1991; 113: 1419-1434Crossref PubMed Google Scholar, 10Friesel R. Brown S.A.N. Development. 1992; 116: 1051-1058PubMed Google Scholar, 11McDonald F.J. Heath J.K. Dev. Genet. 1994; 15: 148-154Crossref PubMed Scopus (20) Google Scholar). The unique patterns of expression of both FGFs and their receptors during vertebrate development suggest that each may have a specialized function. Recent experimental evidence indicates that when FGFR function is disrupted by genetic manipulation, major defects in embryonic development occur(12Amaya E. Musci T.J. Kirschner M.W. Cell. 1991; 66: 257-270Abstract Full Text PDF PubMed Scopus (922) Google Scholar, 13Peters K. Werner S. Liao X. Wert S. Whitsett J. Williams L. EMBO J. 1994; 13: 3296-3301Crossref PubMed Scopus (377) Google Scholar, 14Werner S. Weinberg W. Liao X. Peters K.G. Blessing M. Yuspa S.H. Weiner R.L. Williams L.T. EMBO J. 1993; 12: 2635-2643Crossref PubMed Scopus (217) Google Scholar, 15Yamaguchi T.P. Harpal K. Henkemeyer M. Rossant J. Genes & Dev. 1994; 8: 3032-3044Crossref PubMed Scopus (622) Google Scholar, 16Deng C. Wynshaw-Boris A. Shen M.M. Daugherty C. Ornitz D.M. Leder P. Genes & Dev. 1994; 8: 3045-3057Crossref PubMed Scopus (625) Google Scholar).Within the last year, several mutations have been identified in FGFR genes that appear to be the cause of several human disorders of bone growth and development(2Wilkie A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). One of these, Crouzon syndrome, is characterized by craniosynostosis, an abnormality of skull development in which the sutures of the growing bones fuse prematurely(17Reardon W. Winter R.M. Rutland P. Pulleyn L.J. Jones B.M. Malcolm S. Nat. Genet. 1994; 8: 98-103Crossref PubMed Scopus (607) Google Scholar). A variety of mutations in exons IgIIIa and IgIIIc of FGFR-2 have been identified in Crouzon syndrome(17Reardon W. Winter R.M. Rutland P. Pulleyn L.J. Jones B.M. Malcolm S. Nat. Genet. 1994; 8: 98-103Crossref PubMed Scopus (607) Google Scholar, 18Rutland P. Pulleyn L.J. Reardon W. Baraister M. Hayward R. Jones B. Malcolm S. Winter R.M. Oldridge M. Slaney S.F. Poole M.D. Wilkie A.O.M. Nat. Genet. 1995; 9: 173-176Crossref PubMed Scopus (386) Google Scholar, 19Jabs E.W. Li X. Scott A.F. Meyers C. Chen W. Eccles M. Mao J. Charnas L.R. Jackson C.E. Jaye M. Nat. Genet. 1994; 8: 275-279Crossref PubMed Scopus (406) Google Scholar). These mutations may either directly (Cys342® Tyr/Cys342® Arg/Cys342® Ser/Cys342® Phe; Cys278® Phe; Tyr328® Cys; Ser347® Cys; Ser354® Cys) or indirectly (Ser267® Pro; Gln289® Pro; Tyr340 His) result in the creation of a free cysteine residue that could result in covalent dimerization resulting in ligand-independent activation of the mutant receptor(2Wilkie A.O.M. Morriss-Kay G.M. Jones E.Y. Heath J.K. Curr. Biol. 1995; 5: 500-507Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Here we report that mutation of Cys332® Tyr of Xenopus FGFR-2, analogous to the Cys342® Tyr mutation most commonly found in Crouzon syndrome, promotes activation of the mutant receptor in the absence of