EM LOCALIZATION OF PTP1B & RTK & PTP1B INTERACTIONS

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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