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.

我们计划在HIV进入复合体的形成过程中监测活细胞中CD4和趋化因子受体的时间和空间安排。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递。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。为了在活细胞中观察这两种趋化因子受体及其与CD4的相互作用，我们将CD4、CXCR4和CCR5分别与C端的青色荧光蛋白(CFP)和黄色荧光蛋白(YFP)融合，并在HEK 293细胞中表达。我们已经通过监测受体激活时的信号事件证实了标记的CXCR4和CCR5的功能。我们发现，CXCR4-CFP和CCR5-CFP根据其配体的不同而触发钙离子的升高。