FRET Probe of Spatial Distributions of CD4/CXCR/CCR5

CD4/CXCR/CCR5空间分布的FRET探针

• 批准号: 7196712
• 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/7196712
• 关键词: CD4 molecule; HIV envelope protein gp120; biological signal transduction; cell line; cell surface receptors; chemokine receptor; confocal scanning microscopy; fluorescence recovery after photobleaching; fluorescence resonance energy transfer; green fluorescent proteins; host organism interaction; human immunodeficiency virus 1; membrane proteins; receptor binding; single cell analysis; tissue /cell culture; virus infection mechanism

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的顺序相互作用进入细胞。病毒包膜GP120-GP41异二聚体在三聚体中辅助在病毒表面形成尖峰。结构研究表明，成熟的艾滋病毒颗粒包含72个尖峰，两个尖峰之间的距离为21-22 nm，尖峰形成直径为14 nm的旋钮。 CD4与GP120的结合导致尖峰的构象变化，该尖峰使GP120与趋化因子受体相互作用。 GP120与CD4的连接和趋化因子受体触发了进一步的结构变化，从而使GP41插入靶细胞膜。在当前模型中，一个GP120与一个CD4和一个趋化因子受体相互作用。免疫电子显微镜的证据表明，在与HIV，CD4，CXCR4和CCR5接触之前，每个距离分开的距离约为10 nm。基于结构和免疫EM研究，我们估计形成HIV进入复合物时CD4和趋化因子受体之间的距离在10nm之内。我们最喜欢的假设是，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的缔合形成生产性HIV进入配合物。使用免疫染色显示了由GP120诱导的CD4和CXCR4或CCR5的共定位。但是，由于免疫染色的分辨率限制，CD4和趋化因子受体之间的时间和空间布置以及物理相互作用仍不清楚。 我们已经在与CFP或YFP融合的CD4，CXCR4和CCR5之间使用荧光共振能量转移（FRET），以探测其膜分布，并可视化CD4的分子相互作用和活细胞中的趋化因子受体。我们还使用光漂白后的荧光回收率（FRAP）来测量CD4和CCR5的横向扩散，每个CCR5都在活细胞膜中大惊小怪或YFP，以探测其空间分布。我们发现GP120诱导CD4-YFP和CCR5-CFP之间的FRET增加，这表明HIV包膜蛋白促进了细胞膜上CD4和CCR5之间的关联。质膜由各种脂质和蛋白质的复杂组装组成，这些组合分布在不同的脂质微环境（称为脂质筏或非生育微区）的区域。脂质筏被定义为富含胆固醇，糖磷脂脂和鞘磷脂的微域。脂质和非脂筏微区都包含多种蛋白质，这些蛋白质通过配体，受体和信号成分之间的复杂蛋白质 - 蛋白质相互作用在信号转导中起关键作用。为了检查脂质筏微环境是否对于CD4和CCR5之间的GP120诱导的关联至关重要，我们通过用M？OCD耗尽胆固醇并测量CD4-YFP和CCR5 CFP之间的胆固醇来破坏质膜上的这些微环境。我们有趣的是，耗尽的胆固醇对CD4和CCR5之间的GP120促进的关联没有影响。使用FRAP测量，我们表明CD4在质膜上比CCR5更可移动。但是，当CD4和CCR5都在膜上表达时，它们的迁移率变得相似，即使在没有GP120的情况下，这两个受体之间的迁移率也相似。我们正在进行完成FRET和FRAP实验的过程，这可能会深入了解CD4和趋化因子受体之间的分子相互作用。