FRET Probe of Spatial Distributions of CD4, CXCR4, CCR5

我们计划在HIV进入复合物形成过程中监测活细胞中CD4和趋化因子受体的时空排列。目前尚不清楚在HIV进入过程中CD4和趋化因子受体CXCR4和CCR5之间是否存在分子相互作用，以及这种相互作用是否影响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CD4和活细胞中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在活细胞中与CD4的相互作用，我们将CD4、CXCR4和CCR5分别与c末端的青色荧光蛋白（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触发Ca2+增加响应于它们的配体。

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.