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).

真核细胞如何将趋化因子在其表面上的小浓度差异转化为高极化的细胞内反应是趋化性的基本问题。通过G蛋白偶联受体（GPCR）检测到化学吸引剂。配体结合后的受体激活诱导异三聚体G蛋白分离为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中存在不对称，则表现出高度的空间极化。将刺激中小的不对称性转化为实质细胞内信号传导极性的能力为趋化细胞具有急性的方向感。它们能够在非常浅的梯度中取向，在迁移细胞的正面和背面之间的趋化剂浓度差异只有2％。 G蛋白偶联的趋化受体的激活触发Galpha和Gbetagamma亚基的解离。这些亚基诱导细胞内反应，当细胞经历趋化剂梯度时，这些反应可能会高度极化。确切的细胞如何实现这种扩增的信号极化仍未得到充分了解。在这里，我们通过监测单个活细胞中PHCRAC-GFP易位的动力学来定量测量受体占用，G蛋白激活以及PIP3水平的时间和空间变化，以响应不同的趋化剂领域。我们的结果提供了第一个直接证据，表明G蛋白因不对称刺激而激活了细胞表面的不同量。更强，均匀应用的刺激不仅触发了更强的G蛋白激活，而且还可以更快地适应下游反应。当幼稚细胞（尚未经历过介摄取剂）突然暴露于稳定的营地梯度时，G蛋白在整个细胞表面持续激活，而PHCRAC-GFP的响应令人惊讶地由两个相组成，由两个初始的短暂性和不对称的细胞易位围绕细胞膜周围产生了phcrAc-gfp phcraC-gfp，则是细胞膜周围的不对称转移。我们提出了一个修订后的梯度传感模型，这表明抑制PI3Kinase活性的局部控制成分的重要作用（Xu等，2005）。 趋化性细胞因子（趋化因子）与与异三聚体G蛋白相关的细胞表面受体结合。与配体结合后，趋化因子GPCR受体会激活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起关键作用，包括炎症，过敏，肿瘤进展和HIV感染。在膜的脂质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某些（如果不是全部）趋化因子受体的功能（Manes等，2001）。该项目的目的是确定趋化因子受体功能是否取决于脂质筏的完整性，以及在与配体结合时是否改变了受体的膜分布。配体与趋化因子受体结合通过异三聚体G蛋白触发信号事件。质膜异质微环境中的受体介导的G蛋白激活的基础机制尚不清楚。在这里，使用活细胞FRET成像来检测CXCR1-CFP和标记脂质筏或非系列微域的荧光探针之间的接近度，以及测量CXCR1-CFP的FRAP分析，我们发现IL-8诱导受体和脂质 - 激流之间的均匀诱导。脂质筏的破坏会损害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，也没有完全废除配体诱导的受体磷酸化。我们的结果表明了一种新的机制，通过该机制，配体与CXCR1的结合促进受体的脂质筏分配，并促进异三聚体G蛋白的激活（Jiao等，2005）。