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

完整细胞中 IFN-γ-R1/R2 预关联的直接证据

• 批准号: 7373132
• 负责人: SIDNEY PESTKA
• 金额: $ 0.07万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7373132
• 关键词: DIRECT; EVIDENCE; PREASSOCIATION; IFN; GAMMA

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.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。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，IFN-γ R1和IFN-γ R2的相互作用之前，IFN-γ与其细胞受体复合物在完整的细胞中的preassociated。IFN-γ R1和IFN-γ R2在其COOH末端用蓝色荧光蛋白（BFP）和绿色荧光蛋白（GFP）标记。两种GFP融合蛋白均在无蛋白水解降解的情况下表达。采用760 nm钛蓝宝石激光器，通过双光子激发过程激发BFP。当IFN-γ R1/BFP和IFN-γ R2/GFP在COS-1细胞中共表达时，观察到大量的GFP发射，而在BFP不表达的情况下，观察不到由760 nm激发的GFP荧光。这些数据表明，在不存在配体的正常条件下，IFN-γ R1和IFN-γ R2非常接近。当用IFN-γ处理COS-1细胞时，GFP荧光显著减弱，而BFP荧光增加，表明两条链之间的平均距离增加。这些数据支持结构模型，其中预先形成的IFN-γ受体复合物的细胞内结构域在IFN-γ处理后分散开。这种构象变化可能是后续信号组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。