Using FRET to Probe the Spatial Distributions of CD4, CXCR4 and CCR5 on Membrane

HIV通过病毒包膜（Env）糖蛋白与细胞表面蛋白CD 4和趋化因子受体CXCR 4或CCR 5的顺序相互作用进入细胞。 病毒包膜gp 120-gp 41异源二聚体以三聚体形式缔合以在病毒表面上形成刺突。 结构研究表明，一个成熟的HIV颗粒包含72个刺突，两个刺突之间的距离为21-22 nm，并且刺突形成直径为14 nm的球形突起。 CD 4与gp 120的结合导致尖峰的构象变化，允许gp 120与趋化因子受体相互作用。 gp 120与CD 4和趋化因子受体的连接引发了进一步的结构变化，使gp 41插入靶细胞膜。 在目前的模型中，一个gp 120与一个CD 4和一个趋化因子受体相互作用。 来自免疫电子显微镜的证据表明，在接触HIV之前，CD 4、CXCR 4和CCR 5各自形成独立的微簇，它们之间的距离约为10 nm。 基于结构和免疫电镜研究，我们估计，当HIV进入复合物形成时，CD 4和趋化因子受体之间的距离在10 nm以内。 我们的假设是，当gp 120三聚体结合CD 4分子时，gp 120-CD 4复合物诱导趋化因子受体与三聚体结合形成生产性HIV进入复合物。 使用免疫染色显示了由gp 120诱导的CD 4和CXCR 4或CCR 5的共定位。然而，由于免疫染色的分辨率限制，CD 4和趋化因子受体之间的时空排列和物理相互作用仍然不清楚。 我们已经采用荧光共振能量转移（FRET）与CFP或YFP融合的CD 4，CXCR 4和CCR 5的嵌合体之间的探测它们的膜分布和可视化的分子相互作用的CD 4和趋化因子受体在活细胞。 我们还使用光漂白后的荧光恢复（FRAP）来测量CD 4和CCR 5的横向扩散，它们各自与活细胞膜中的CFP或YFP融合，以探测它们的空间分布。 我们发现gp 120诱导CD 4-YFP和CCR 5-CFP之间的FRET增加，表明HIV包膜蛋白促进细胞膜上CD 4和CCR 5之间的结合。 质膜由各种脂质和蛋白质的复杂组装体组成，这些脂质和蛋白质分布在不同脂质微环境的区域中，称为脂筏或非筏微域。 脂筏被定义为富含胆固醇、鞘糖脂和鞘磷脂的微区。 脂质和非脂质筏微结构域都含有多种蛋白质，这些蛋白质通过配体、受体和信号组分之间复杂的蛋白质-蛋白质相互作用在信号转导中发挥关键作用。 为了检查脂筏微环境是否是gp 120诱导的CD 4和CCR 5之间的关联所必需的，我们通过用MRCD消耗胆固醇来破坏质膜上的这些微环境，并测量CD 4-YFP和CCR 5-CFP之间的FRET。 有趣的是，我们发现消耗胆固醇对gp 120促进的CD 4和CCR 5之间的联系没有影响。 利用FRAP测量，我们表明，CD 4是更移动的比CCR 5在质膜上。 然而，当CD 4和CCR 5都在膜上表达时，它们的迁移率变得相似，这表明即使在没有gp 120的情况下，膜上这两种受体之间也存在一些相互作用。 我们正在完成FRET和FRAP实验，这可能有助于深入了解HIV进入过程中CD 4和趋化因子受体之间的分子相互作用。

HIV enters cells through sequential interactions of the viral envelope (Env) glycoprotein with the cell surface protein CD4 and a chemokine receptor CXCR4 or CCR5. The viral envelope gp120-gp41 heterodimers associate in a trimer to form spikes on the viral surface. Structural studies suggest that a mature HIV particle contains 72 spikes and the distance between two spikes is 21-22 nm, and a spike forms a knob with a diameter of 14 nm. Binding of CD4 to gp120 leads to conformational changes in a spike that allows the gp120 to interact with a chemokine receptor. Ligation of gp120 to CD4 and a chemokine receptor triggers further structural changes that allow the gp41 to insert into the target cell membrane. In the current model, one gp120 interacts with one CD4 and one chemokine receptor. Evidence from immunoelectron microscopy demonstrated that before contacting HIV, CD4, CXCR4 and CCR5 each form independent microclusters that are separated by a distance of about 10 nm. Based on structural and immuno-EM studies, we estimate that the distance between CD4 and the chemokine receptors is well within 10nm when a HIV entry complex is formed. Our favored hypothesis is that when gp120 trimers bind CD4 molecules, gp120-CD4 complexes induce the association of chemokine receptors with the trimers to form productive HIV entry complexes. Colocalization of CD4 and CXCR4 or CCR5 induced by gp120 has been shown using immuno-staining. However, due to resolution limitations of immuno-staining, the temporal and spatial arrangement and physical interactions between CD4 and the chemokine receptors are still unclear. We have employed fluorescence resonance energy transfer (FRET) between chimeras of CD4, CXCR4 and CCR5 fused with CFP or YFP to probe their membrane distribution and to visualize molecular interactions of CD4 and the chemokine receptors in living cells. We have also used fluorescence recovery after photobleaching (FRAP) to measure lateral diffusion of CD4 and CCR5 that each fussed with CFP or YFP in living cell membrane to probe their spatial distribution. We found that gp120 induced FRET increase between CD4-YFP and CCR5-CFP, suggesting that HIV envelope protein promotes association between CD4 and CCR5 on the cell membrane. The plasma membranes consist of a complex assembly of various lipids and proteins that are distributed in regions of distinct lipid microenvironments, known as lipid raft or non-raft microdomains. Lipid rafts are defined as microdomains that are enriched in cholesterol, glycosphingolipid and sphingomyelin. Both lipid and non-lipid raft microdomains contain multiple proteins that play critical roles in signal transduction via complex protein-protein interactions between ligands, receptors, and signaling components. To examine whether lipid raft microenvironment is essential for gp120-induced association between CD4 and CCR5, we disrupted these microenvironments on the plasma membrane by depleting cholesterol with MRCD and measured FRET between CD4-YFP and CCR5-CFP. We found interestingly that depleting cholesterol had no effect on gp120-promoted association between CD4 and CCR5. Using FRAP measurement, we showed that CD4 is more mobile than CCR5 on the plasma membrane. However, when both CD4 and CCR5 were expressed on the membrane, their mobility became similar, suggesting some interactions between these two receptors on the membrane even in the absence of gp120. We are in the process to complete FRET and FRAP experiments, which may provide insight into molecular interactions between CD4 and the chemokine receptors during HIV entry.