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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。关于干扰素γ (ifn - γ)受体复合物在被ifn - γ激活之前的结构存在间接和矛盾的证据。我们观察到的生物物理证据表明，在完整细胞中，ifn - γ与其细胞受体复合物相互作用之前，ifn - γ - 1和ifn - γ - 2是预先相关的。IFN-gammaR1和IFN-gammaR2在cooh末端分别用蓝色荧光蛋白（BFP）和绿色荧光蛋白（GFP）标记。两种GFP融合蛋白的表达均无蛋白水解降解。采用760nm钛蓝宝石激光器，通过双光子激发工艺激发BFP。当IFN-gammaR1/BFP和IFN-gammaR2/GFP在COS-1细胞中共表达时，可以观察到大量的GFP发射，而在不表达BFP的情况下，没有观察到760nm处激发的GFP荧光。这些数据表明，在没有配体的正常条件下，IFN-gammaR1和IFN-gammaR2是非常接近的。当使用ifn - γ处理COS-1细胞时，GFP荧光明显减弱，而BFP荧光增强，表明两条链之间的平均距离增加。这些数据支持结构模型，即预形成的ifn - γ受体复合物的细胞内结构域在ifn - γ处理后分散开来。这种构象变化可能是后续信号成分进入以启动和维持信号转导的先决条件。我们正在研究各种细胞内和受体成分的荧光及其通过荧光能量转移的相互作用。

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.