G-protein Coupled Receptor Mediated Chemoattractant Sensing and Phagocytosis

G 蛋白偶联受体介导的趋化剂感应和吞噬作用

• 批准号: 8156943
• 负责人: Tian Jin
• 金额: $ 63.55万
• 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/8156943
• 关键词: Chemotactic Factors; G-Protein-Coupled Receptors; Mediating; Phagocytosis

We are investigating the following questions: 1. how a GPCR chemosensing network regulates the polarized reorganization of the actin cytoskeleton required for protrusion of the cell's front and retraction of its back during chemotaxis. 2. Coupling mechanism of a GPCR and heterotrimeric G proteins during chemoattractant gradient sensing. 3. What are the molecular mechanisms underlying phagosome maturation process during phagocytosis? 1. Chemoattractant GPCRs control the actin cytoskeleton during cell migration. GPCR activation induces dissociation of the heterotrimeric G-protein into G and G, which promote formation of new actin filaments via the Arp2/3 complex in migrating cells. The Arp2/3 complex is thought to be activated by Rac, and Elmo/Dock180 serves as a GEF for Rac activation. However, it has been unclear how a GPCR signals to Elmo/Dock180. Here, we have characterized a new Elmo protein, ElmoE in Dictyostelium, and demonstrated that it is required for cAR1 GPCR-mediated chemotaxis. Remarkably, ElmoE physically associates with G to activate Rac and this association is mediated by cAR1. These results have uncovered a new pathway of the GPCR-mediated regulation on the actin cytoskeleton, which involves a GPCR, G, Elmo, Rac, Arp2/3, and actin filaments. The pathway may spatially direct the growth of a dendritic actin network in pseudopod of eukaryotic cells during chemotaxis (Yan et al, submitted). 2. The coupling of heterotrimeric guanine nucleotideVbinding protein (G protein)-coupled receptors (GPCRs) with G proteins is fundamental for GPCR signaling; however, the mechanism of coupling is still debated. Moreover, it remains unclear how the proposed mechanisms affect the dynamics of downstream signaling. Here, through experiments involving fluorescence recovery after photobleaching and single-molecule imaging, we directly measured the mobilities of cAR1, a chemoattractant receptor, and a G protein subunit in live cells. We found that cAR1 diffused more slowly in the plasma membrane than did G. Upon binding of ligand to the receptor, the mobility of cAR1 was unchanged, whereas the speed of a fraction of the faster-moving G subunits decreased. Our measurements showed that cAR1 was relatively immobile and G diffused freely, suggesting that chemoattractant-bound cAR1 transiently interacted with G proteins. Through the use of models that describe possible coupling mechanisms, we computed the temporal kinetics of G protein activation. Our fluorescence resonance energy transfer imaging data showed that fully activated cAR1 induced the sustained dissociation of G protein - and -subunits, which indicated that ligand-bound cAR1 activated G proteins continuously. Finally, our simulations indicated that a high-affinity coupling of ligand-bound receptors and G proteins was essential for cAR1 to translate extracellular gradient signals into directional cellular responses. We suggest that chemoattractant receptors use a ligand-induced coupling, rather than a pre-coupled, mechanism to control the activation of G proteins during chemotaxis (Xu et al., Science Signaling, in press). 3. Phagocytosis is crucial for host defense against microbial pathogens and for obtaining nutrients in Dictyostelium discoideum. Phagocytosed particles are delivered from phagosomes to lysosomes for degradation, but the molecular mechanism regulating phagosome maturation remains unclear. Using D. discoideum as a model system, we plan to reveal important components involved in phagosome maturation. We have identified 3 novel vesicle-associated receptor tyrosine kinases, VSK1-3, in D. discoideum. Our previous study suggests that localized VSK3 tyrosine kinase signaling on the surface of endosome/lysosomes represents a new control mechanism for phagosome maturation. We are identifying targets of VSK 2 and 3. This study will provide a foundation for understanding the molecular mechanism of VSK signaling that regulate phagosome maturation.

我们正在调查以下问题：1。GPCR化学传感网络如何调节细胞前部突出所需的肌动蛋白细胞骨架的两极分化重组和在趋上期间其背部的缩回。 2。在趋化剂梯度传感期间，GPCR和异三聚体G蛋白的耦合机制。 3。吞噬作用期间吞噬体成熟过程的分子机制是什么？ 1。趋化剂GPCR控制细胞迁移过程中肌动蛋白细胞骨架。 GPCR激活诱导异三聚体G蛋白在G和G中解离，从而促进通过迁移细胞中ARP2/3复合物促进新肌动蛋白丝的形成。 ARP2/3复合物被认为是由RAC激活的，Elmo/Dock180是RAC激活的GEF。但是，目前尚不清楚GPCR信号是如何向Elmo/Dock180发出的。 在这里，我们表征了一种新的Elmo蛋白，即Dictyostelium中的Elmoe，并证明了CAR1 GPCR介导的趋化性所必需的。 值得注意的是，Elmoe将与G的物理联系起来激活RAC，并且该关联是由CAR1介导的。这些结果发现了肌动蛋白细胞骨架上GPCR介导的调节的新途径，该调节涉及GPCR，G，Elmo，RAC，RAC，ARP2/3和肌动蛋白丝。 该途径可以在趋化性期间在真核细胞伪脚下的树突状肌动蛋白网络的空间引导（Yan等人，提交）。 2。异三元素鸟嘌呤核苷酸蛋白（G蛋白）偶联受体（GPCR）与G蛋白的偶联是GPCR信号传导的基础。但是，耦合机制仍在争论中。此外，尚不清楚提出的机制如何影响下游信号的动力学。在这里，通过涉及光漂白和单分子成像后荧光恢复的实验，我们直接测量了Live细胞中CAR1的迁移率，趋化受体和G蛋白亚基。我们发现，CAR1在质膜中的扩散较慢，而G。在配体与受体的结合后，CAR1的迁移率不变，而更快的G亚基的速度降低了。我们的测量结果表明，CAR1是相对不动的，并且G自由扩散，这表明趋化剂结合的CAR1与G蛋白瞬时相互作用。通过使用描述可能的耦合机制的模型，我们计算了G蛋白激活的时间动力学。我们的荧光共振能量传递成像数据表明，完全活化的CAR1诱导了G蛋白 - 和 - 亚基的持续解离，这表明配体结合的CAR1激活的G蛋白不断激活G蛋白。最后，我们的模拟表明，配体结合的受体和G蛋白的高亲和力偶联对于CAR1将细胞外梯度信号转化为方向性细胞反应至关重要。我们建议趋化受体使用配体诱导的耦合，而不是预先耦合的机制来控制趋化过程中G蛋白的激活（Xu等，Science Signal传导，印刷中）。 3。吞噬作用对于针对微生物病原体的宿主防御和在迪斯特尔迪斯特尔氏菌中获得营养至关重要。吞噬细胞颗粒从吞噬体传递到溶酶体以降解，但是调节吞噬体成熟的分子机制尚不清楚。使用D. Discoideum作为模型系统，我们计划揭示涉及吞噬体成熟的重要组成部分。我们已经在D. discoideum中确定了3个新型囊泡相关的受体酪氨酸激酶VSK1-3。我们先前的研究表明，内体/溶酶体表面上的局部VSK3酪氨酸激酶信号传导代表了吞噬体成熟的新控制机制。我们正在确定VSK 2和3的靶标。这项研究将为了解调节吞噬体成熟的VSK信号传导的分子机制提供基础。