Abstract

This project proposes to study the mechanism whereby expression of the eucaryotic growth factor, platelet-derived growth factor (PDGF), results in oncogenic transformation. PDGF is a well-characterized mitogen for cells of mesenchymal origin. However, expression of one subunit of PDGF, as acquired by simian sarcoma virus, can result in oncogenic transformation. Four areas of research are proposed. (1) Structure-function studies: The sis oncogene, homologous to the PDGF-A gene, will be mutagenized to assess which portions of the protein are required for biological activity. These mutants will be assayed for transformation using retrovirus vectors, and their protein expression will be characterized. Deletion mutants will be constructed in the N-terminal and C-terminal coding regions. Point mutants will be constructed using oligonucleotide site-directed mutagenesis in the N-terminal signal sequence, and also in potential sites of glycosylation, disulfide formation, and post-translational proteolytic processing. (2) In vitro studies of sis gene: The phage SP6 promoter will be used to synthesize RNA in vitro, which will be translated using rabbit reticulocyte lysate. Tryptic peptide maps of the PDGF-related proteins sysnthesized in vitro will be determined, and compared with in vivo synthesized proteins. Coupled transcription/translation/membrane vesicle processing will be used to study signal sequence cleavage, glycosylation, and post-translational proteolytic processing. (3) Fusions of sis with membrane anchor sequences: A major issue is whether the sis gene product must be secreted to function in an autocrine fashion. Therefore, the PDGF coding region will be fused with typical membrane anchor sequences, e.g., from the vesicular stomatitis virus glycoprotein gene. The hybrid genes will be assayed for fibroblast transformation. The localization within the cell of the hybrid proteins will be determined. The hybrid proteins also will be assayed for interaction with the PDGF-receptor. (4) Cloning the gene encoding subunit B of PDGF: DNA probes derived form the sis gene have allowed cloning the cellular gene for the A subunit of PDGF, but not for the B subunit. Using oligonucleotide probes based on PDGF-B peptide sequences, the PDGF-B gene will be isolated from a genomic library and characterized. The cloned gene will be used to determine if PDGF-B expression results in fibroblast transformation by the sis gene, perhaps by formation of PDGF A:B heterodimers. (J)

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA040573-02
Application #
3180722
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1985-07-01
Project End
1988-06-30
Budget Start
1986-07-01
Budget End
1987-06-30
Support Year
2
Fiscal Year
1986
Total Cost
Indirect Cost

  Institution

Name
University of California San Diego
Department
Type
Schools of Arts and Sciences
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093

  Publications

Tavormina, P L; Bellus, G A; Webster, M K et al. (1999) A novel skeletal dysplasia with developmental delay and acanthosis nigricans is caused by a Lys650Met mutation in the fibroblast growth factor receptor 3 gene. Am J Hum Genet 64:722-31
Chen, L I; Webster, M K; Meyer, A N et al. (1997) Transmembrane domain sequence requirements for activation of the p185c-neu receptor tyrosine kinase. J Cell Biol 137:619-31
Webster, M K; Donoghue, D J (1997) Enhanced signaling and morphological transformation by a membrane-localized derivative of the fibroblast growth factor receptor 3 kinase domain. Mol Cell Biol 17:5739-47
Webster, M K; Donoghue, D J (1996) Constitutive activation of fibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia. EMBO J 15:520-7
Webster, M K; D'Avis, P Y; Robertson, S C et al. (1996) Profound ligand-independent kinase activation of fibroblast growth factor receptor 3 by the activation loop mutation responsible for a lethal skeletal dysplasia, thanatophoric dysplasia type II. Mol Cell Biol 16:4081-7
Galvin, B D; Hart, K C; Meyer, A N et al. (1996) Constitutive receptor activation by Crouzon syndrome mutations in fibroblast growth factor receptor (FGFR)2 and FGFR2/Neu chimeras. Proc Natl Acad Sci U S A 93:7894-9
Meyer, A N; Xu, Y F; Webster, M K et al. (1994) Cellular transformation by a transmembrane peptide: structural requirements for the bovine papillomavirus E5 oncoprotein. Proc Natl Acad Sci U S A 91:4634-8
Hart, K C; Xu, Y F; Meyer, A N et al. (1994) The v-sis oncoprotein loses transforming activity when targeted to the early Golgi complex. J Cell Biol 127:1843-57
Maher, D W; Strawn, L M; Donoghue, D J (1993) Alanine mutagenesis of conserved residues in the platelet-derived growth factor family: identification of residues necessary for dimerization and transformation. Oncogene 8:533-41
Xu, Y F; Meyer, A N; Webster, M K et al. (1993) 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 123:549-60

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