FMS TRANSFORMATION/CSF-1 RECEPTOR SIGNAL TRANSDUCTION

集落刺激因子1受体（CSF-1R），由c-fms编码 原癌基因，表现出内在的，配体依赖性，蛋白酪氨酸 激酶（PTK）活性。细胞外基质中Leu-301突变 人CSF-1R的结构域揭示了其致癌性，并诱导组成性 受体PTK活性。原则上，激活人类FMS中的突变 可能导致骨髓增生性疾病的病因， 包括白血病和骨髓增生异常综合征，但没有真正的 迄今为止已经鉴定了密码子301处的激活突变。 具体目标#1是使用无偏见的方法来寻找额外的 激活人类FMS基因突变。化学诱变FMS 将"靶盒"依次再克隆到亲本载体中， 转染NIH/3T3细胞。转化细胞病灶 将被分离出来，他们的FMS基因的相关部分将被 在靶盒扩增后直接测序， 聚合酶链反应我们希望能找到其他的活化方法 可能导致肿瘤的突变 单核吞噬细胞系列细胞的转化。的 多个激活突变的位置应该限制 CSF-1R内决定其配体非依赖性的结构基序 activation. CSF-1R的信号转导涉及与细胞因子的相关性。 自身磷酸化受体与促有丝分裂素的"下游"效应物 反应受体定位位点的选择性诱变 自磷酸化消除了它与这些蛋白质偶联的能力， 产生信号转导部分缺陷的突变体。 Tyr-809突变解除CSF-1R诱导c-myc的能力 表达并大大降低CSF-1刺激的促有丝分裂性。在 相反，配体刺激的CSF-1R [Phe-809]突变体表现出野生型， 型水平的PTK活性，结合磷脂酰肌醇3-激酶， 诱导c-fos、c-jun和junB的活性， 动力学外源性c-myc基因导入NIH/3T3细胞的研究 携带CSF-1R [Phe-809]的小鼠在血清中恢复CSF-1诱导的有丝分裂原性， 自由培养基。在具体目标#2下，我们建议克隆假定的 与CSF-1R相互作用的细胞效应蛋白 侧接PTyr-809，阐明其功能，并研究其对 CSF-1应答基因。后面的实验侧重于机制 控制可能在癌症中受到干扰的正常生长 细胞

The colony-stimulating factor 1 receptor (CSF-1R), encoded by the c-fms proto-oncogene, exhibits intrinsic, ligand-dependent, protein tyrosine kinase (PTK) activity. Mutations at Leu-301 within the extracellular domain of human CSF-1R unmask its oncogenicity, and induce constitutive receptor PTK activity. In principle, activating mutations in human FMS might contribute to the etiology of myeloid proliferative disorders, including leukemias and myelodysplastic syndromes, but no bona fide activating mutations at codon 301 have thus far been identified. Specific Aim #1 is to use an unbiased approach to search for additional activating mutations in human FMS gene. Chemically mutagenized FMS "target cassettes" will be recloned en masse into a parental vector which will be transfected into NIH/3T3 cells. Foci of transformed cells will be isolated, and the relevant portions of their FMS genes will be directly sequenced after amplification of the target cassette by polymerase chain reaction. We expect to catalog alternative activating mutations that may potentially contribute to the neoplastic transformation of cells of the mononuclear phagocyte series. The locations of multiple activating mutations should circumscribe structural motifs within CSF-1R that determine its ligand-independent activation. Signal transduction by CSF-1R involves the association of the autophosphorylated receptor with "downstream" effectors of the mitogenic response. Selective mutagenesis of mapped sites of receptor autophosphorylation abrogate its ability to couple to these proteins, generating mutants that are partially defective in signal transduction. Mutation of Tyr-809 uncouples the ability of CSF-1R to induce c-myc expression and greatly reduces CSF-1 stimulated mitogenicity. In contrast, the ligand-stimulated CSF-1R[Phe-809] mutant exhibits wild- type levels of PTK activity, binds to a phosphatidylinositol 3-kinase, and induces the activity of c-fos, c-jun and junB with unperturbed kinetics. Introduction of an exogenous c-myc gene into NIH/3T3 cells bearing CSF-1R[Phe-809] restores CSF-1 induced mitogenicity in serum- free medium. Under Specific Aim #2, we propose to clone the putative cellular effector protein that interacts with CSF-1R in the region flanking PTyr-809, elucidate its function, and study its regulation of CSF-1-responsive genes. The latter experiments focus on mechanisms controlling normal growth which are likely to be perturbed in cancer cells.