Seeing the light: preassembly and ligand-induced changes of the interferon gamma receptor complex in cells.
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.
Krause
Christopher D
CD
Department of Molecular Genetics, Microbiology and Immunology, Robert Wood Johnson Medical School-University of Medicine and Dentistry of New Jersey, Piscataway 08854-5635, USA.
Mei
Erwen
E
Xie
Junxia
J
Jia
Yiwei
Y
Bopp
Martin A
MA
Hochstrasser
Robin M
RM
Pestka
Sidney
S
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism*; D008024:Ligands; D008164:Luminescent Proteins / Q000737:chemistry; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D017471:Receptors, Interferon / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D008024:Ligands; D008164:Luminescent Proteins; D017471:Receptors, Interferon; C409504:blue fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins; D007371:Interferon-gamma