DIRECT EVIDENCE FOR PREASSOCIATION OF IFN-GAMMA-R1/R2 IN INTACT CELLS

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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. Indirect and contradictory evidence exists about the structure of the interferon gamma (IFN-gamma) receptor complex prior to its activation by IFN-gamma. We have observed biophysical evidence that IFN-gammaR1 and IFN-gammaR2 are preassociated prior to the interaction of IFN-gamma with its cellular receptor complex in intact cells. IFN-gammaR1 and IFN-gammaR2 are labeled on their COOH-termini with the blue fluorescence protein (BFP) and green fluorescence protein (GFP). Both GFP fusion proteins are expressed without proteolytic degradation. 760 nm Ti-sapphire laser is used to excite BFP through two-photon excitation process. When IFN-gammaR1/BFP and IFN-gammaR2/GFP are coexpressed in COS-1 cells, substantial GFP emission is seen, while GFP fluorescence excited by 760nm is not seen in the absence of BFP expression. These data demonstrate that IFN-gammaR1 and IFN-gammaR2 are in close proximity under normal conditions in the absence of ligand. When COS-1 cells are treated with IFN-gamma, GFP fluorescence diminishes substantially, while BFP fluorescence increases, demonstrating that the average distance between the two chains increases. These data support the structural model whereby the intracellular domains of the preformed IFN-gamma receptor complex spread apart after IFN-gamma treatment. This conformational change may be a prerequisite for the entry of subsequent signaling components to initiate and maintain signal transduction. We are examining the fluorescence of various intracellular and receptor components and their interactions by fluorescence energy transfer.