EM LOCALIZATION OF PTP1B & RTK & PTP1B INTERACTIONS

PTP1B 的电子显微镜定位

• 批准号: 7358054
• 负责人: BENJAMIN G. NEEL
• 金额: $ 0.2万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7358054
• 关键词: EM; LOCALIZATION; PTP1B; RTK; INTERACTIONS

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Protein tyrosine phosphatase-1B (PTP1B) is a major negative regulator of several growth factor and cytokine signaling pathways, including the insulin, EGF, PDGF and leptin receptors. Previous studies in our laboratory have shown, using immunofluorescence and, more recently, FRET techniques that PTP1B is located on the surface of the ER, and that the PTP1B/RTK interaction (at least for EGFR and PDGFR) takes place on the ER surface as endocytic vesicles transit into the cell. Because of antibody limitations, it has not been possible thus far to see if PTP1B is generally distributed in the ER (or even to confirm that PTP1B is actually on the ER by immunoelectron microscopy) or to visualize at the EM level the RTK/PTP1B interaction. We propose two general types of experiment. First, by tagging PTP1B and using FLASHER technology, to perform EM localization of PTP1B in PTP1B knockout fibroblasts reconstituted with appropriately tagged PTP1B. Second, by tagging the EGFR and IR with the cysteine-based tag, and using a substrate trapping mutant of PTP1B with a FRET-compatible fluorochrome protein, to confirm the PTP1B/RTK interaction by FRET and then localize it ultrastucturally by EM using the FLASHER approach at various times following growth factor stimulation. This project is only now starting, with an initial exchange of information concerning the type of tetracysteine tag to use in these studies. We are also coordinating a visit of the postdoctoral fellow, Fawaz Haj, to the facility, upon completion of the first set of recombinant fusion proteins

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。蛋白酪氨酸磷酸酶-1B (PTP1B) 是多种生长因子和细胞因子信号传导途径的主要负调节因子，包括胰岛素、EGF、PDGF 和瘦素受体。我们实验室之前的研究表明，使用免疫荧光和最近的 FRET 技术，PTP1B 位于 ER 表面，并且 PTP1B/RTK 相互作用（至少对于 EGFR 和 PDGFR）发生在 ER 表面，如下所示内吞囊泡转运进入细胞。由于抗体的限制，迄今为止还不可能了解 PTP1B 是否普遍分布在 ER 中（甚至无法通过免疫电子显微镜确认 PTP1B 实际上位于 ER 上）或在 EM 水平上可视化 RTK/PTP1B 相互作用。我们提出两种一般类型的实验。首先，通过标记PTP1B并使用FLASHER技术，在用适当标记的PTP1B重组的PTP1B敲除成纤维细胞中进行PTP1B的电镜定位。其次，通过使用基于半胱氨​​酸的标签标记 EGFR 和 IR，并使用具有 FRET 兼容荧光染料蛋白的 PTP1B 底物捕获突变体，通过 FRET 确认 PTP1B/RTK 相互作用，然后使用 FLASHER 通过 EM 将其超微结构定位在生长因子刺激后的不同时间进行处理。该项目现在才刚刚开始，初步交换了有关这些研究中使用的四半胱氨酸标签类型的信息。在第一组重组融合蛋白完成后，我们还正在协调博士后研究员 Fawaz Haj 对该设施的访问