Using FRET to Probe the Spatial Distributions of CD4, CX

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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.