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Submitted on September 29, 2002
Department of Molecular Genetics, Microbiology and Immunology, Robert Wood Johnson Medical School-UMDNJ, Piscataway, New Jersey 08854
Corresponding Author: Pestka{at}umdnj.edu
Our experiments were designed to test the hypothesis that the cell surface interferon gamma receptor chains are preassembled rather than associated by ligand; and to assess the molecular changes on ligand binding. To accomplish this, we used fluorescence resonance energy transfer (FRET), a powerful spectroscopic technique that has been used to determine molecular interactions and distances between the donor and acceptor. However, current commercial instruments do not provide sufficient sensitivity nor full spectra to provide decisive results of interactions between proteins labeled with blue and green fluorescent proteins (BFP and GFP) in living cells. In our experiments, we used the BFP and GFP pair, attached a monochrometer and CCD camera to a modified confocal microscope, reduced background fluorescence with the use of two-photon excitation and focused on regions of single cells to provide clear spectra of fluorescence resonance energy transfer. In contrast to the prevailing view, the results demonstrate that the receptor chains are preassociated and that the intracellular domains move apart on binding the ligand IFN-g. Application of this technology should lead to new rapid methods for high throughput screening and delineation of the interactome of cells.
Revised on October 5, 2002
Accepted on October 5, 2002
Seeing the light: Preassembly and ligand-induced changes of the interferon Gamma receptor complex in cells
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