Using FRET to Probe the Spatial Distributions of CD4, CX

使用 FRET 探测 CD4、CX 的空间分布

• 批准号: 7312953
• 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/7312953
• 关键词: Using; FRET; Probe; Spatial; Distributions

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 M?OCD 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.

HIV 通过病毒包膜 (Env) 糖蛋白与细胞表面蛋白 CD4 和趋化因子受体 CXCR4 或 CCR5 的连续相互作用进入细胞。病毒包膜 gp120-gp41 异二聚体结合成三聚体，在病毒表面形成刺突。结构研究表明，一个成熟的HIV颗粒含有72个尖峰，两个尖峰之间的距离为21-22 nm，一个尖峰形成一个直径为14 nm的旋钮。 CD4 与 gp120 的结合导致尖峰构象发生变化，从而允许 gp120 与趋化因子受体相互作用。 gp120 与 CD4 和趋化因子受体的连接触发进一步的结构变化，使 gp41 插入靶细胞膜。在当前模型中，1 个 gp120 与 1 个 CD4 和 1 个趋化因子受体相互作用。免疫电镜证据表明，在接触HIV之前，CD4、CXCR4和CCR5各自形成独立的微团簇，其间隔距离约为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，CD4 与趋化因子受体之间的距离远在 10 nm 以内。我们倾向于的假设是，当 gp120 三聚体结合 CD4 分子时，gp120-CD4 复合物诱导趋化因子受体与三聚体结合，形成有效的 HIV 进入复合物。免疫染色显示 gp120 诱导的 CD4 和 CXCR4 或 CCR5 共定位。然而，由于免疫染色分辨率的限制，CD4与趋化因子受体之间的时间和空间排列以及物理相互作用仍不清楚。 我们利用与 CFP 或 YFP 融合的 CD4、CXCR4 和 CCR5 嵌合体之间的荧光共振能量转移 (FRET) 来探测它们的膜分布，并可视化 CD4 和活细胞中趋化因子受体的分子相互作用。我们还使用光漂白后荧光恢复 (FRAP) 来测量 CD4 和 CCR5 的横向扩散，这些 CD4 和 CCR5 分别与活细胞膜中的 CFP 或 YFP 融合，以探测它们的空间分布。我们发现 gp120 诱导 CD4-YFP 和 CCR5-CFP 之间的 FRET 增加，表明 HIV 包膜蛋白促进细胞膜上 CD4 和 CCR5 之间的结合。质膜由各种脂质和蛋白质的复杂组装组成，这些脂质和蛋白质分布在不同的脂质微环境区域，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结构域。脂质和非脂筏微结构域都含有多种蛋白质，这些蛋白质通过配体、受体和信号成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对于 gp120 诱导的 CD4 和 CCR5 之间的关联至关重要，我们通过用 M?OCD 消耗胆固醇来破坏质膜上的这些微环境，并测量 CD4-YFP 和 CCR5-CFP 之间的 FRET。有趣的是，我们发现消耗胆固醇对 gp120 促进的 CD4 和 CCR5 之间的关联没有影响。通过 FRAP 测量，我们发现 CD4 在质膜上比 CCR5 更具移动性。然而，当 CD4 和 CCR5 都在膜上表达时，它们的迁移率变得相似，这表明即使在不存在 gp120 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 CD4 和趋化因子受体之间的分子相互作用。