Molecular Analysis of a Drosophila Growth Factor

果蝇生长因子的分子分析

基本信息

批准号：
9421740
负责人：
Amanda Simcox
金额：
$ 30.5万
依托单位：
Ohio State University Research Foundation -DO NOT USE
依托单位国家：
美国
项目类别：
Continuing grant
财政年份：
1995
资助国家：
美国
起止时间：
1995-02-15 至 1998-01-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=9421740&HistoricalAwards=false
关键词：
Molecular Analysis Drosophila Growth Factor

项目摘要

9421740 Simcox A signal transduction pathway involving intracellular Ras is highly conserved and used by a number of receptor-tyrosine kinases including the Drosophila EGF receptor (DER). The long term goal of this work is to understand how signal transduction mediated by ubiquitous DER results in a tissuespecific response. DER has multiple roles in development and is involved in cell survival, differentiation and proliferation. Tissue-specific signaling via the receptor is likely to involve localized aomponents, including ligands. The candidate ligand defective dorsal discs (ddd) is the focus of this proposal. The ddd activity is required for ventral patterning in the embryo and growth of the imaginal wing and haltere discs; ddd transcripts are spatially localized in these tissues. The genetic characterization of ddd shows it functions nonautonomously and interacts with DER. DER is epistatic to ddd which suggests ddd acts upstream of the receptor. Both the nonautonomy and the data also support this idea. The predicted ddd protein has an EGF-like domain, which is characteristic of EGF receptor ligands. Ddd also has an Iglike domain. The neuregulins share this combination of domains. These ligands bind vertebrate Neu, an EGF-receptor relative. Interestingly, DER is equally similar to Neu and the EGF receptor. Thus, the genetic and molecular evidence suggest Ddd is a DER ligand. Here it is proposed to extend the molecular-genetic characterization of ddd and test the hypothesis that Ddd is a DER ligand. The objectives are: (1) To determine the phenotypic effects of expression of ddd transgenes. Constructs based on ddd cDNAs will be used to rescue ddd mutants and to determine the developmental consequence of ectopic ddd expression. (2) To describe the tissue and cell distribution of Ddd throughout development. Polyclonal antisera directed against a Ddd-fusion protein will be used in antibody staining in animals of all developmental stages. The temporal and spatial expression dom ain and subcellular or extracellular location of Ddd will be determined. Ddd expression in other disc mutants will be examined. (3) To test the hypothesis that Ddd is a DER ligand using cell-culture experiments. The ability of Ddd and DER to interact molecularly will be tested in ligandbinding and immunoprecipitation assays. The ability of Ddd to activate DER will be tested by monitoring receptor autophosphorylation. The highly conserved EGF receptor pathway regulates cell multiplication and differentiation. Not surprisingly, genes in the pathway have oncogenic forms. Thus this research on ddd, a candidate ligand for the Drosophila EGF receptor, which is related to both Neu and the EGF receptor, will contribute to the understanding of abnormal, as well as normal, growth control and pattern formation.

9421740 SIMCOX A涉及细胞内RA的信号转导途径是高度保守的，并由包括果蝇EGF受体（DER）在内的许多受体 - 酪氨酸激酶使用。这项工作的长期目标是了解无处不在的信号转导如何导致组织特异性响应。 DER在发育中具有多重作用，并参与细胞存活，分化和增殖。通过受体的组织特异性信号传导可能涉及包括配体在内的局部大容量。候选配体有缺陷的背盘（DDD）是该提案的重点。 DDD活性是在胚胎和想象翼和吊带盘的生长中腹侧图案所必需的。 DDD转录本在这些组织中被空间定位。 DDD的遗传表征表明它非自主的功能并与DER相互作用。 DER是对DDD的代表性的，这表明DDD在受体的上游作用。非自治和数据都支持这一想法。预测的DDD蛋白具有EGF样结构域，这是EGF受体配体的特征。 DDD还具有一个Igike域。神经结合蛋白共享这种域的组合。这些配体结合脊椎动物NEU，EGF受体亲戚。有趣的是，DER与NEU和EGF受体同样相似。因此，遗传和分子证据表明DDD是一种der配体。在这里，提议扩展DDD的分子遗传学特征，并检验DDD是一种配体的假设。目标是：（1）确定DDD转基因表达的表型效应。基于DDD cDNA的构建体将用于挽救DDD突变体，并确定异位DDD表达的发育后果。（2）在整个发育过程中描述DDD的组织和细胞分布。针对DDD融合蛋白的多克隆抗血清将用于所有发育阶段的动物的抗体染色。将确定DDD的时间和空间表达DOM AIN以及细胞外部或细胞外位置。将检查其他圆盘突变体中的DDD表达。（3）测试了使用细胞培养实验的DDD是DDD的假设。 DDD和DER相互作用分子的能力将在配体和免疫沉淀测定中进行测试。 DDD激活DER的能力将通过监测受体自磷酸化测试。高度保守的EGF受体途径调节细胞繁殖和分化。毫不奇怪，途径中的基因具有致癌形式。因此，与果蝇EGF受体的候选配体DDD有关，与NEU和EGF受体有关，将有助于理解异常以及正常情况，即生长控制和模式形成。