EM LOCALIZATION OF PTP1B & RTK & PTP1B INTERACTIONS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。蛋白酪氨酸磷酸酶-1B(PTP1B)是多种生长因子和细胞因子信号通路的主要负调控因子，包括胰岛素、EGF、PDGF和瘦素受体。我们实验室以前的研究表明，使用免疫荧光和最近的FRET技术，PTP1B位于内质网的表面，PTP1B/RTK的相互作用(至少对于EGFR和PDGFR)发生在内吞囊泡进入细胞时发生在内质网的表面。由于抗体的限制，到目前为止还不可能看到PTP1B是否普遍分布在内质网中(甚至通过免疫电子显微镜证实PTP1B确实在内质网上)，或者在EM水平上显示RTK/PTP1B的相互作用。我们提出了两种一般类型的实验。首先，通过标记PTP1B并使用闪存技术，在用适当标记的PTP1B重组的PTP1B基因敲除成纤维细胞中进行PTP1B的EM定位。其次，用基于半胱氨酸的标记标记EGFR和IR，并使用底物捕获突变体PTP1B和FRET相容的荧光蛋白，通过FRET确认PTP1B/RTK的相互作用，然后在生长因子刺激后的不同时间用EM使用Flasher方法对其进行超结构定位。这个项目现在才刚刚开始，初步交换了关于这些研究中使用的四半胱氨酸标签类型的信息。我们还在协调博士后研究员法瓦兹·哈吉在完成第一组重组融合蛋白后访问该设施。

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