Abstract
Thanatophoric dysplasia type II (TDII) is a neonatal lethal skeletal dysplasia caused by a recurrent Lys-650-->Glu 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 beta10-beta11 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-650-->Glu 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.
Full Text
The Full Text of this article is available as a PDF (469.2 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Andersen P. E., Jr, Hauge M. Congenital generalised bone dysplasias: a clinical, radiological, and epidemiological survey. J Med Genet. 1989 Jan;26(1):37–44. doi: 10.1136/jmg.26.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bargmann C. I., Hung M. C., Weinberg R. A. Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185. Cell. 1986 Jun 6;45(5):649–657. doi: 10.1016/0092-8674(86)90779-8. [DOI] [PubMed] [Google Scholar]
- Bellus G. A., Hefferon T. W., Ortiz de Luna R. I., Hecht J. T., Horton W. A., Machado M., Kaitila I., McIntosh I., Francomano C. A. Achondroplasia is defined by recurrent G380R mutations of FGFR3. Am J Hum Genet. 1995 Feb;56(2):368–373. [PMC free article] [PubMed] [Google Scholar]
- Bellus G. A., McIntosh I., Smith E. A., Aylsworth A. S., Kaitila I., Horton W. A., Greenhaw G. A., Hecht J. T., Francomano C. A. A recurrent mutation in the tyrosine kinase domain of fibroblast growth factor receptor 3 causes hypochondroplasia. Nat Genet. 1995 Jul;10(3):357–359. doi: 10.1038/ng0795-357. [DOI] [PubMed] [Google Scholar]
- Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Coffin J. D., Florkiewicz R. Z., Neumann J., Mort-Hopkins T., Dorn G. W., 2nd, Lightfoot P., German R., Howles P. N., Kier A., O'Toole B. A. Abnormal bone growth and selective translational regulation in basic fibroblast growth factor (FGF-2) transgenic mice. Mol Biol Cell. 1995 Dec;6(12):1861–1873. doi: 10.1091/mbc.6.12.1861. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ellis L., Clauser E., Morgan D. O., Edery M., Roth R. A., Rutter W. J. Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose. Cell. 1986 Jun 6;45(5):721–732. doi: 10.1016/0092-8674(86)90786-5. [DOI] [PubMed] [Google Scholar]
- Hanks S. K., Quinn A. M. Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. Methods Enzymol. 1991;200:38–62. doi: 10.1016/0076-6879(91)00126-h. [DOI] [PubMed] [Google Scholar]
- Hubbard S. R., Wei L., Ellis L., Hendrickson W. A. Crystal structure of the tyrosine kinase domain of the human insulin receptor. Nature. 1994 Dec 22;372(6508):746–754. doi: 10.1038/372746a0. [DOI] [PubMed] [Google Scholar]
- Jaye M., Schlessinger J., Dionne C. A. Fibroblast growth factor receptor tyrosine kinases: molecular analysis and signal transduction. Biochim Biophys Acta. 1992 Jun 10;1135(2):185–199. doi: 10.1016/0167-4889(92)90136-y. [DOI] [PubMed] [Google Scholar]
- Johnson D. E., Williams L. T. Structural and functional diversity in the FGF receptor multigene family. Adv Cancer Res. 1993;60:1–41. doi: 10.1016/s0065-230x(08)60821-0. [DOI] [PubMed] [Google Scholar]
- Keegan K., Johnson D. E., Williams L. T., Hayman M. J. Isolation of an additional member of the fibroblast growth factor receptor family, FGFR-3. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1095–1099. doi: 10.1073/pnas.88.4.1095. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Keegan K., Meyer S., Hayman M. J. Structural and biosynthetic characterization of the fibroblast growth factor receptor 3 (FGFR-3) protein. Oncogene. 1991 Dec;6(12):2229–2236. [PubMed] [Google Scholar]
- Kitayama H., Kanakura Y., Furitsu T., Tsujimura T., Oritani K., Ikeda H., Sugahara H., Mitsui H., Kanayama Y., Kitamura Y. Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines. Blood. 1995 Feb 1;85(3):790–798. [PubMed] [Google Scholar]
- MacDonald I. M., Hunter A. G., MacLeod P. M., MacMurray S. B. Growth and development in thanatophoric dysplasia. Am J Med Genet. 1989 Aug;33(4):508–512. doi: 10.1002/ajmg.1320330420. [DOI] [PubMed] [Google Scholar]
- Mohammadi M., Dikic I., Sorokin A., Burgess W. H., Jaye M., Schlessinger J. Identification of six novel autophosphorylation sites on fibroblast growth factor receptor 1 and elucidation of their importance in receptor activation and signal transduction. Mol Cell Biol. 1996 Mar;16(3):977–989. doi: 10.1128/mcb.16.3.977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mohammadi M., Dionne C. A., Li W., Li N., Spivak T., Honegger A. M., Jaye M., Schlessinger J. Point mutation in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenesis. Nature. 1992 Aug 20;358(6388):681–684. doi: 10.1038/358681a0. [DOI] [PubMed] [Google Scholar]
- Muenke M., Schell U. Fibroblast-growth-factor receptor mutations in human skeletal disorders. Trends Genet. 1995 Aug;11(8):308–313. doi: 10.1016/s0168-9525(00)89088-5. [DOI] [PubMed] [Google Scholar]
- Nagata H., Worobec A. S., Oh C. K., Chowdhury B. A., Tannenbaum S., Suzuki Y., Metcalfe D. D. Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder. Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10560–10564. doi: 10.1073/pnas.92.23.10560. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Neilson K. M., Friesel R. E. Constitutive activation of fibroblast growth factor receptor-2 by a point mutation associated with Crouzon syndrome. J Biol Chem. 1995 Nov 3;270(44):26037–26040. doi: 10.1074/jbc.270.44.26037. [DOI] [PubMed] [Google Scholar]
- Orioli I. M., Castilla E. E., Barbosa-Neto J. G. The birth prevalence rates for the skeletal dysplasias. J Med Genet. 1986 Aug;23(4):328–332. doi: 10.1136/jmg.23.4.328. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Peters K., Ornitz D., Werner S., Williams L. Unique expression pattern of the FGF receptor 3 gene during mouse organogenesis. Dev Biol. 1993 Feb;155(2):423–430. doi: 10.1006/dbio.1993.1040. [DOI] [PubMed] [Google Scholar]
- Rimoin D. L. Histopathology and ultrastructure of cartilage in the chondrodystrophies. Birth Defects Orig Artic Ser. 1974;10(9):1–18. [PubMed] [Google Scholar]
- Rousseau F., Bonaventure J., Legeai-Mallet L., Pelet A., Rozet J. M., Maroteaux P., Le Merrer M., Munnich A. Mutations in the gene encoding fibroblast growth factor receptor-3 in achondroplasia. Nature. 1994 Sep 15;371(6494):252–254. doi: 10.1038/371252a0. [DOI] [PubMed] [Google Scholar]
- Rousseau F., Saugier P., Le Merrer M., Munnich A., Delezoide A. L., Maroteaux P., Bonaventure J., Narcy F., Sanak M. Stop codon FGFR3 mutations in thanatophoric dwarfism type 1. Nat Genet. 1995 May;10(1):11–12. doi: 10.1038/ng0595-11. [DOI] [PubMed] [Google Scholar]
- Santoro M., Carlomagno F., Romano A., Bottaro D. P., Dathan N. A., Grieco M., Fusco A., Vecchio G., Matoskova B., Kraus M. H. Activation of RET as a dominant transforming gene by germline mutations of MEN2A and MEN2B. Science. 1995 Jan 20;267(5196):381–383. doi: 10.1126/science.7824936. [DOI] [PubMed] [Google Scholar]
- Shah K., Astley R., Cameron A. H. Thanatophoric dwarfism. J Med Genet. 1973 Sep;10(3):243–252. doi: 10.1136/jmg.10.3.243. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shiang R., Thompson L. M., Zhu Y. Z., Church D. M., Fielder T. J., Bocian M., Winokur S. T., Wasmuth J. J. Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia. Cell. 1994 Jul 29;78(2):335–342. doi: 10.1016/0092-8674(94)90302-6. [DOI] [PubMed] [Google Scholar]
- Tavormina P. L., Rimoin D. L., Cohn D. H., Zhu Y. Z., Shiang R., Wasmuth J. J. Another mutation that results in the substitution of an unpaired cysteine residue in the extracellular domain of FGFR3 in thanatophoric dysplasia type I. Hum Mol Genet. 1995 Nov;4(11):2175–2177. doi: 10.1093/hmg/4.11.2175. [DOI] [PubMed] [Google Scholar]
- Tavormina P. L., Shiang R., Thompson L. M., Zhu Y. Z., Wilkin D. J., Lachman R. S., Wilcox W. R., Rimoin D. L., Cohn D. H., Wasmuth J. J. Thanatophoric dysplasia (types I and II) caused by distinct mutations in fibroblast growth factor receptor 3. Nat Genet. 1995 Mar;9(3):321–328. doi: 10.1038/ng0395-321. [DOI] [PubMed] [Google Scholar]
- Webster M. K., Donoghue D. J. Constitutive activation of fibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia. EMBO J. 1996 Feb 1;15(3):520–527. [PMC free article] [PubMed] [Google Scholar]
- Xu Y. F., Meyer A. N., Webster M. K., Lee B. A., Donoghue D. J. The v-sis protein retains biological activity as a type II membrane protein when anchored by various signal-anchor domains, including the hydrophobic domain of the bovine papilloma virus E5 oncoprotein. J Cell Biol. 1993 Nov;123(3):549–560. doi: 10.1083/jcb.123.3.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang B., Tavaré J. M., Ellis L., Roth R. A. The regulatory role of known tyrosine autophosphorylation sites of the insulin receptor kinase domain. An assessment by replacement with neutral and negatively charged amino acids. J Biol Chem. 1991 Jan 15;266(2):990–996. [PubMed] [Google Scholar]