FRET Probe of Spatial Distributions of CD4, CXCR4, CCR5

CD4、CXCR4、CCR5 空间分布的 FRET 探针

• 批准号: 6987103
• 负责人: Tian Jin
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6987103
• 关键词: CD4 molecule; HIV envelope protein gp120; biological signal transduction; bioluminescence; cell line; cell surface receptors; chemokine receptor; confocal scanning microscopy; fluorescence resonance energy transfer; green fluorescent proteins; host organism interaction; human immunodeficiency virus 1; membrane proteins; receptor binding; tissue /cell culture; virus infection mechanism

We plan to monitor temporal and spatial arrangements of CD4 and chemokine receptors in living cells during the formation of HIV entry complexes. It is not clear if there are molecular interactions between CD4 and the chemokine receptors CXCR4 and CCR5 during HIV entry and if so if such interactions affect HIV entry and/or chemokine receptor signaling. We plan to address these issues by applying fluorescence resonance energy transfer (FRET) imaging to monitor the interactions of CD4 and the chemokine receptors in living cells. We have made progress in establishing cell lines for FRET imaging analyses. To visualize these two chemokine receptors and their interactions with CD4 in living cells, we have fused CD4, CXCR4 and CCR5 each with both cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) at their C-termini and expressed them in HEK 293 cells. We have confirmed functionalities of tagged CXCR4 and CCR5 by monitoring signaling events upon receptor activation. We showed that CXCR4-CFP and CCR5-CFP trigger Ca2+ increases in response to their ligands.

我们计划监测 HIV 进入复合物形成过程中活细胞中 CD4 和趋化因子受体的时间和空间排列。目前尚不清楚在 HIV 进入过程中 CD4 与趋化因子受体 CXCR4 和 CCR5 之间是否存在分子相互作用，如果存在，这种相互作用是否会影响 HIV 进入和/或趋化因子受体信号传导。我们计划通过应用荧光共振能量转移 (FRET) 成像来监测活细胞中 CD4 和趋化因子受体的相互作用来解决这些问题。我们在建立用于 FRET 成像分析的细胞系方面取得了进展。为了可视化这两种趋化因子受体及其与活细胞中 CD4 的相互作用，我们将 CD4、CXCR4 和 CCR5 分别与青色荧光蛋白 (CFP) 和黄色荧光蛋白 (YFP) 在其 C 末端融合，并在 HEK 293 中表达它们细胞。我们通过监测受体激活时的信号事件，确认了标记的 CXCR4 和 CCR5 的功能。我们发现 CXCR4-CFP 和 CCR5-CFP 响应其配体而触发 Ca2+ 增加。