ERBB RECEPTOR CONTROL OF BREAST CANCER GROWTH

ERBB 受体控制乳腺癌生长

• 批准号: 2698169
• 负责人: ANNE W. HAMBURGER
• 金额: $ 22.13万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2001-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2698169
• 关键词: active sites; biological signal transduction; breast neoplasms; cell growth regulation; cell line; enzyme inhibitors; epidermal growth factor; growth factor receptors; molecular cloning; neoplastic cell; protein kinase C; receptor binding; receptor expression; site directed mutagenesis; transfection; yeast two hybrid system

A crucial role for Epidermal Growth Factor Receptor (erbB) family members in development of breast cancer has been demonstrated over the past 15 years. We have recently cloned a novel protein, EBP1, which interacts with the cytoplasmic domain of erbB3. Treatment of cells with the erbB-3 ligand, heregulin (HRG) induces the translocation of EBP1 from the cytoplasm to the nucleus. Overexpression of EBP1 inhibits proliferation of human breast cancer cells. We propose to study the physiological function of erbB-3EBP1 interactions in human breast cancer cells. Understanding the regulation and consequences of these interactions could lead to a better understanding of erbB-3 signal transduction and to the definition of new therapeutic targets in breast cancer. First, to define the function of erbB-3-EBP1 interactions, we will a) determine HRG-mediated interactions of EBP1 with other erbB receptors and the dependence of such interactions on the expression of erbB-3 heterodimeric binding partners using the yeast-two hybrid system and engineered murine cells expressing defined combinations of erbB receptors 2) establish the colocalization of EBP1 and erbB-3 by confocal microscopy before and after HRG treatment, c) determine the region of EBP1 required for erbB-3 binding and the effects of expression of different regions of EBP1 on HRG-mediated signalling using a series of deletion and single site mutants of EBP1, d) determine the effects of EBP1 overexpression on basal and HRG-induced cell growth and differentiation using stable transfectants with inducible expression. Second, we will determine the role of Protein Kinase C (PKC) in modulating EBP1 activity. The sites of phosphorylation after HRG treatment will be determined using tryptic phosphopeptide mapping and phosphamino acid analysis. Effects of PKC inhibitors on EBP1 function will be assessed in intact cells. Potential PKC sites will be mutated and effects of such mutations on EBP1 function defined. Third, we will define the significance of EBP1 nuclear localization. We will a) determine if EBP1 is part of a multiprotein nuclear localization complex by isolating EBP1 interacting proteins using the yeast two-hybrid system b) define the role of EBP1 and its accessory transcriptional activators using GAL4 DNA binding domain fusions c) identify preferred DNA binding sites for EBP1 using a degenerate oligonucleotide strategy.

表皮生长因子受体（erbB）家族的关键作用 在乳腺癌的发展过程中， 过去15年 我们最近克隆了一种新的蛋白质EBP 1， 与erbB 3的胞质结构域相互作用。处理细胞 erbB-3配体heregulin（HRG）诱导EBP 1易位 从细胞质到细胞核。 EBP 1过表达抑制 人乳腺癌细胞的增殖。 我们建议研究 erbB-3EBP 1相互作用在人乳腺癌中的生理功能 细胞了解这些的规则和后果 相互作用可以更好地理解erbB-3信号 转导和乳腺癌新治疗靶点的定义 癌 首先，为了定义erbB-3-EBP 1相互作用的功能，我们将a） 确定HRG介导的EBP 1与其他erbB受体的相互作用 以及这种相互作用对erbB-3表达的依赖性 使用酵母双杂交系统的异二聚体结合配偶体， 表达确定的erbB组合的工程鼠细胞 受体2）通过共聚焦显微镜建立EBP 1和erbB-3的共定位。 在HRG处理之前和之后进行显微镜检查，c）确定 erbB-3结合所需的EBP 1和表达的影响 EBP 1不同区域对HRG介导的信号传导的影响， EBP 1的缺失突变体和单位点突变体，d）确定 EBP 1过表达对基础和HRG诱导的细胞生长的影响， 使用具有诱导型表达的稳定转染子进行分化。 其次，我们将确定蛋白激酶C（PKC）在 调节EBP 1活性。 HRG后磷酸化位点 使用胰蛋白酶磷酸肽图谱确定治疗， 磷酸氨基酸分析PKC抑制剂对EBP 1功能的影响 将在完整细胞中进行评估。 潜在的PKC位点将发生突变 并定义了这些突变对EBP 1功能的影响。 三是 明确EBP 1核定位的意义。 我们将（a） 确定EBP 1是否是多蛋白核定位复合物的一部分 通过使用酵母双杂交系统分离EBP 1相互作用蛋白， B）定义EBP 1及其辅助转录激活因子的作用 c）鉴定优选的DNA结合 使用简并寡核苷酸策略的EBP 1位点。