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

使用 FRET 探测膜上 CD4、CXCR4 和 CCR5 的空间分布

• 批准号: 7592286
• 负责人: Tian Jin
• 金额: $ 50.24万
• 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/7592286
• 关键词: Address; Affect; Binding; CCR5 gene; CD4 Antigens; CXCR4 gene; Caliber; Cell Surface Proteins; Cell membrane; Cells; Chimera organism; Cholesterol; Complex; Cyclophosphamide/Fluorouracil/Prednisone; Diffusion; Fluorescence Recovery After Photobleaching; Fluorescence Resonance Energy Transfer; Foundations; Glycoproteins; Glycosphingolipids; Goals; HIV; HIV Envelope Protein gp120; HIV Infections; HIV envelope protein; Image; Immunoelectron Microscopy; Lateral; Life; Ligands; Ligation; Lipids; Measurement; Measures; Membrane; Membrane Microdomains; Modeling; Molecular; Monitor; Personal Satisfaction; Physiology; Play; Preclinical Drug Evaluation; Process; Proteins; Receptor Signaling; Resolution; Role; Signal Transduction; Spatial Distribution; Sphingomyelins; Staining method; Stains; Structure; Surface; System; Viral; base; chemokine; chemokine receptor; design; insight; particle; protein protein interaction; receptor; research study; single-molecule FRET

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.

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异源二聚体以三聚体形式结合，在病毒表面形成尖刺。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gp120与趋化因子受体相互作用的尖峰的构象变化。Gp120与CD4和趋化因子受体的连接触发了进一步的结构变化，使gp41插入到靶细胞膜中。在目前的模型中，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，在接触HIV之前，CD4、CXCR4和CCR5各自形成独立的微团簇，相互之间的距离约为10 nm。基于结构和免疫-EM研究，我们估计当HIV进入复合体形成时，CD4和趋化因子受体之间的距离在10 nm以内。我们支持的假设是，当gp120三聚体与CD4分子结合时，gp120-CD4复合体诱导趋化因子受体与三聚体结合形成有效的HIV进入复合体。免疫组织化学染色显示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。 我们利用荧光共振能量转移(FRET)技术，在与CFP或YFP融合的CD4、CXCR4和CCR5嵌合体之间探索它们的膜分布，并观察活细胞中CD4和趋化因子受体的分子相互作用。我们还使用荧光漂白后恢复(FRAP)来测量活细胞膜中分别与CFP或YFP融合的CD4和CCR5的横向扩散，以探索它们的空间分布。我们发现gp120诱导了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之间的结合。质膜由各种脂类和蛋白质组成的复杂集合，分布在不同的脂微环境区域，称为脂筏或非脂筏微域。脂筏被定义为富含胆固醇、鞘糖脂和鞘磷脂的微域。脂类和非脂类RAFT微域都含有多种蛋白质，它们通过配体、受体和信号元件之间复杂的蛋白质相互作用在信号转导中发挥关键作用。为了检验脂筏微环境是否对gp120诱导的CD4和CCR5之间的联系是必不可少的，我们通过用MRCD耗尽胆固醇来破坏质膜上的这些微环境，并测量了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，我们发现在质膜上，CD4比CCR5具有更强的流动性。然而，当膜上同时表达CD4和CCR5时，它们的迁移率变得相似，这表明即使在没有gp120的情况下，这两种受体在膜上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。