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Originally published In Press as doi:10.1074/mcp.M200065-MCP200 on October 5, 2002.
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Molecular & Cellular Proteomics 1:805-815, 2002.
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Seeing the Light

Preassembly and Ligand-Induced Changes of the Interferon {gamma} Receptor Complex in Cells*

Christopher D. Krause{ddagger}, Erwen Mei, Junxia Xie{ddagger}, Yiwei Jia§, Martin A. Bopp, Robin M. Hochstrasser and Sidney Pestka{ddagger},||

{ddagger} Department of Molecular Genetics, Microbiology and Immunology, Robert Wood Johnson Medical School-University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854
§ Olympus America Inc., Melville, New York 11747-3157
Regional Laser and Biomedical Technology Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104

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, 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 or the full spectra to provide decisive results of interactions between proteins labeled with blue and green fluorescent proteins in living cells. In our experiments, we used the blue fluorescent protein and green fluorescent protein pair, attached a monochrometer and charge-coupled device 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 interferon {gamma}. Application of this technology should lead to new rapid methods for high throughput screening and delineation of the interactome of cells.

|| To whom correspondence should be addressed: Dept. of Molecular Genetics, Microbiology, and Immunology, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854-5635. Tel.: 732-235-4567; Fax: 732-235-5223; E-mail: Pestka{at}umdnj.edu


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