GPCR Mediated Directional Sensing And Cell Movement

GPCR 介导的定向传感和细胞运动

• 批准号: 7196703
• 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/7196703
• 关键词: G protein; biological signal transduction; calcium flux; calcium indicator; cell migration; cell surface receptors; chemokine receptor; chemotaxis; confocal scanning microscopy; cyclic AMP; fluorescence recovery after photobleaching; fluorescence resonance energy transfer; green fluorescent proteins; intermolecular interaction; membrane structure; protein localization; receptor binding; receptor expression; single cell analysis; tissue /cell culture

How a eukaryotic cell translates the small concentration difference of a chemoattractant on its surface into highly polarized intracellular responses is a fundamental question in chemotaxis. Chemoattractants are detected by G-protein coupled receptors (GPCRs). Receptor activation upon ligand binding induces the dissociation of heterotrimeric G-protein into Galpha; and Gbetagamma subunits. These subunits, in turn, modulate the function of other intracellular signaling components to generate biochemical responses that exhibit a high degree of spatial polarization if there are asymmetries in the activation of the surface receptors. The ability to translate small asymmetry in stimulation into substantial intracellular signaling polarity provides chemotaxing cells with an acute sense of direction. They are capable of orientation in very shallow gradients with as little as a 2% difference in chemoattractant concentration between the front and back of the migrating cell. Activation of G-protein-coupled chemoattractant receptors triggers dissociation of Galpha and Gbetagamma subunits. These subunits induce intracellular responses that can be highly polarized when a cell experiences a gradient of chemoattractant. Exactly how a cell achieves this amplified signal polarization is still not well understood. Here, we quantitatively measure temporal and spatial changes of receptor occupancy, G-protein activation by FRET imaging, and PIP3 levels by monitoring the dynamics of PHCrac-GFP translocation in single living cells in response to different chemoattractant fields. Our results provided the first direct evidence that G-proteins are activated to different extents on the cell surface in response to asymmetrical stimulations. A stronger, uniformly applied stimulation triggers not only a stronger G-protein activation but also a faster adaptation of downstream responses. When naive cells (which have not experienced chemoattractant) were abruptly exposed to stable cAMP gradients, G-proteins were persistently activated throughout the entire cell surface, whereas the response of PHCrac-GFP translocation surprisingly consisted of two phases, an initial transient and asymmetrical translocation around the cell membrane, followed by a second phase producing a highly polarized distribution of PHCrac-GFP. We propose a revised model of gradient sensing, suggesting an important role for locally controlled components that inhibit PI3Kinase activity (Xu et al., 2005). Chemotactic cytokines (chemokines) bind to cell surface receptors that are linked to heterotrimeric G-proteins. Upon binding to ligands, chemokine GPCR receptors activate G-proteins and modulate intracellular signaling events. Chemokines and their receptors play critical roles in many physiological processes including inflammation, allergy, tumor progression and HIV infection. Several chemokine receptors have been found in lipid rafts of membranes. Regulation of the localization of chemokine receptors may modulate the function of some, if not all, chemokine receptors (Manes et al., 2001). The goal of this project is to determine if chemokine receptor function depends on the integrity of lipid rafts and if the membrane distribution of the receptors is altered upon binding to ligands. Ligand binding to a chemokine receptor triggers signaling events through heterotrimeric G-proteins. The mechanisms underlying receptor-mediated G-protein activation in the heterogeneous microenvironments of the plasma membrane are unclear. Here, using live cell FRET imaging to detect the proximity between CXCR1-CFP and fluorescence probes that label lipid raft or non-raft microdomains, and FRAP analysis to measure the lateral diffusion of CXCR1-CFP, we found that IL-8 induces association between the receptors and lipid-raft microenvironments. Disruption of lipid rafts impaired G-protein-dependent signaling such as Ca2+ responses and PI3K activation, but had no effect on ligand-binding function and did not completely abolish ligand-induced receptor phosphorylation. Our results suggest a novel mechanism by which ligand binding to CXCR1 promotes lipid raft partitioning of receptors and facilitates activation of heterotrimeric G-proteins (Jiao et al., 2005).

真核细胞如何将其表面趋化剂的微小浓度差转化为高度极化的细胞内反应是趋化性的一个基本问题。化学引诱剂由 G 蛋白偶联受体 (GPCR) 检测。配体结合后受体激活诱导异源三聚体 G 蛋白解离为 Galpha；和 Gbetagamma 亚基。这些亚基反过来调节其他细胞内信号传导成分的功能，以产生生化反应，如果表面受体的激活存在不对称性，则表现出高度的空间极化。将刺激中的微小不对称性转化为实质的细胞内信号极性的能力为趋化细胞提供了敏锐的方向感。它们能够以非常浅的梯度进行定向，迁移细胞前后的趋化剂浓度差异只有 2%。 G 蛋白偶联趋化受体的激活触发 Galpha 和 Gbetagamma 亚基的解离。当细胞经历化学引诱剂梯度时，这些亚基诱导细胞内反应，这些反应可以高度极化。细胞究竟如何实现这种放大的信号极化仍不清楚。在这里，我们通过监测单个活细胞中 PHCrac-GFP 易位对不同趋化场的反应动态，定量测量受体占用、FRET 成像 G 蛋白激活和 PIP3 水平的时间和空间变化。我们的结果提供了第一个直接证据，证明细胞表面的 G 蛋白响应不对称刺激而不同程度地被激活。更强、均匀的刺激不仅会触发更强的 G 蛋白激活，还会更快地适应下游反应。当幼稚细胞（未经历化学引诱剂）突然暴露于稳定的 cAMP 梯度时，G 蛋白在整个细胞表面持续激活，而 PHCrac-GFP 易位的反应令人惊讶地由两个阶段组成，即初始瞬时易位和不对称易位细胞膜周围，然后是产生高度极化分布的 PHCrac-GFP 的第二阶段。我们提出了一种修正的梯度传感模型，表明抑制 PI3Kinase 活性的局部控制成分发挥着重要作用（Xu 等人，2005）。 趋化细胞因子（趋化因子）与与异三聚体 G 蛋白相连的细胞表面受体结合。与配体结合后，趋化因子 GPCR 受体激活 G 蛋白并调节细胞内信号传导事件。趋化因子及其受体在许多生理过程中发挥着关键作用，包括炎症、过敏、肿瘤进展和艾滋病毒感染。在膜的脂筏中发现了几种趋化因子受体。趋化因子受体定位的调节可能会调节一些（如果不是全部）趋化因子受体的功能（Manes 等，2001）。该项目的目标是确定趋化因子受体的功能是否取决于脂筏的完整性，以及受体的膜分布是否在与配体结合后发生改变。配体与趋化因子受体的结合通过异三聚体 G 蛋白触发信号事件。质膜异质微环境中受体介导的 G 蛋白激活机制尚不清楚。在这里，使用活细胞 FRET 成像来检测 CXCR1-CFP 和标记脂筏或非筏微域的荧光探针之间的接近度，并使用 FRAP 分析来测量 CXCR1-CFP 的横向扩散，我们发现 IL-8 诱导受体和脂筏微环境。脂筏的破坏损害了 G 蛋白依赖性信号传导，例如 Ca2+ 反应和 PI3K 激活，但对配体结合功能没有影响，也没有完全消除配体诱导的受体磷酸化。我们的结果提出了一种新的机制，通过该机制配体与 CXCR1 的结合促进受体的脂筏分配并促进异源三聚体 G 蛋白的激活（Jiao 等人，2005）。