EM LOCALIZATION OF PTP1B & RTK & PTP1B INTERACTIONS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。蛋白酪氨酸磷酸酶-1B（PTP 1B）是几种生长因子和细胞因子信号通路的主要负调节因子，包括胰岛素、EGF、PDGF和瘦素受体。我们实验室以前的研究表明，使用免疫荧光和最近的FRET技术，PTP 1B位于ER表面，并且PTP 1B/RTK相互作用（至少对于EGFR和PDGFR）发生在ER表面，因为内吞囊泡转运进入细胞。由于抗体的限制，迄今为止还不可能看到PTP 1B是否通常分布在ER中（或者甚至通过免疫电子显微镜证实PTP 1B实际上在ER上）或在EM水平上可视化RTK/PTP 1B相互作用。我们提出了两种一般类型的实验。首先，通过标记PTP 1B并使用FLASHER技术，在用适当标记的PTP 1B重建的PTP 1B敲除成纤维细胞中进行PTP 1B的EM定位。其次，通过用基于半胱氨酸的标签标记EGFR和IR，并使用PTP 1B的底物捕获突变体与FRET相容的荧光蛋白，通过FRET确认PTP 1B/RTK相互作用，然后在生长因子刺激后的不同时间使用FLASHER方法通过EM对其进行超微结构定位。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。我们还在协调博士后研究员Fawaz Haj在完成第一套重组融合蛋白后对该设施的访问

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