The mechanisms underlying the GPCR-mediated chemotaxis in D. discoideum

D. discoideum GPCR 介导的趋化机制

• 批准号: 8745398
• 负责人: Tian Jin
• 金额: $ 62.32万
• 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/8745398
• 关键词: Actins; Affinity; Arrestins; Binding; Biochemical; Biological Models; Biological Phenomena; Cell membrane; Cells; Chemicals; Chemotactic Factors; Chemotaxis; Collaborations; Computer Assisted; Coupled; Cyclic AMP; Cyclic AMP Receptors; Cytoskeleton; Development; Dictyostelium discoideum; Dissociation; Eukaryota; Eukaryotic Cell; Face; Fluorescent Probes; Frequencies; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Guanine; Guanine Nucleotide Exchange Factors; Heterotrimeric GTP-Binding Proteins; Hormones; Life; Ligands; MAPK1 gene; Mammalian Cell; Mediating; Membrane; Modeling; Molecular; Monitor; Movement; Neurotransmitters; Organism; Phospholipids; Physiological; Play; Process; Proteins; Recruitment Activity; Regulation; Reporting; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Source; Staging; Stimulus; base; cell motility; chemokine; directional cell; extracellular; fluorescence imaging; imaging modality; inhibitor/antagonist; intercellular communication; response; spatiotemporal

1: cAR1-mediated spatiotemporal dynamics of Ras signaling. To reveal the mechanism, we monitored spatiotemporal activation of Ras by monitoring the membrane translocation of a fluorescent probe, active Ras Binding Domain fused to GFP (RBD-GFP) in response to various stimuli. The Ras-activation dynamics we observed indicate that a negative regulator, possibly a RasGAP, is gradually recruited to the membrane and promotes Ras inactivation. In mammalian cells, a RasGAP containing a phospholipid-binding domain has been shown to translocate to the plasma membrane's inner face, deactivate Ras, and thereby inhibit PI3K. We proposed that a similar phospholipid-bound RasGAP is likely to be an important inhibitor in cAR1-mediated chemosensing in D. discoideum. We have revealed the roles of RasGAP in chemosensing in D. discoideum (Xu et al., in prep). 2: We have discovered that arrestins are key components of the signaling circuit involved in the oscillatory cell-cell signaling in eukaryotes. We have found that cAR1 GPCR-mediated arrestin function regulates the period of transient ERK2 activation that controls the frequency of cAMP oscillations in D. discoideum. Oscillation of chemical signals is a common biological phenomenon but its regulation is poorly understood. At the aggregation stage of Dictyostelium discoideum development, the chemoattractant cAMP is synthesized and released at 6 min intervals, directing cell migration. Although the cAMP receptor cAR1 GPCR and ERK2 are both implicated in regulating the oscillation, the signaling circuit remains unknown. Here, we report that D. discoideum arrestins (AdcB and AdcC) play a critical role in regulating the frequency of cAMP oscillation. Activation of cAR1 promotes membrane recruitment of AdcC, and AdcC associates with ERK2. Cells lacking arrestins (adcB-C-) have shorter periods (3 min) of both cAR1-triggered transient ERK2 activation and cAMP oscillations, suggesting that cAR1-controlled arrestin function regulates the period of transient ERK2 activation that mediates the frequency of cAMP oscillations at the aggregation stage of D. discoideum development. In addition, D. discoideum utilizes arrestins for ligand-induced cAR1 internalization to achieve the switch from high-affinity to low-affinity cAMP receptors for its multicellular development (Yan et al., PNAS, in revision). In collaboration with Dr. Kortholt, we reported that Ric8 protein is a nonreceptor guanine exchange factor for heterotrimeric G proteins and is important for development and chemotaxis in D.d sicoideum (Kataria, et al PNAS, 2013).

1：cAR 1介导的Ras信号传导的时空动力学。为了揭示其机制，我们通过监测荧光探针的膜易位来监测Ras的时空激活，活性Ras结合结构域融合到GFP（RBD-GFP）以响应各种刺激。我们观察到的Ras激活动力学表明，一个负调节，可能是一个RasGAP，逐渐招募到膜，促进Ras失活。在哺乳动物细胞中，含有磷脂结合结构域的RasGAP已被证明可以移位到质膜的内表面，使Ras失活，从而抑制PI 3 K。我们提出，一个类似的磷脂结合的RasGAP可能是一个重要的抑制剂，在cAR 1介导的化学传感在D。盘状突我们揭示了RasGAP在D.盘状体（Xu等人，准备中）。 2：我们发现抑制蛋白是真核生物中参与振荡细胞-细胞信号传导的信号通路的关键组分。 我们已经发现，cAR 1 GPCR介导的抑制蛋白功能调节ERK 2瞬时激活的时间，从而控制D.盘状突。 化学信号的振荡是一种常见的生物学现象，但其调控机制却知之甚少。 在盘基网柄藻发育的聚集阶段，化学引诱物cAMP以6分钟的间隔合成和释放，指导细胞迁移。 虽然cAMP受体cAR 1 GPCR和ERK 2都参与调节振荡，但信号通路仍不清楚。 在这里，我们报告说，D。discoideum arrestins（AdcB和AdcC）在调节cAMP振荡频率中起关键作用。 cAR 1的激活促进AdcC的膜募集，并且AdcC与ERK 2相关联。 缺乏arrestins（adcB-C-）的细胞具有更短的cAR 1触发的瞬时ERK 2激活和cAMP振荡的周期（3 min），这表明cAR 1控制的arrestin功能调节瞬时ERK 2激活的周期，介导D.盘状突发育 此外，D. discoideum利用抑制蛋白进行配体诱导的cAR 1内化，以实现从高亲和力到低亲和力cAMP受体的转换，用于其多细胞发育（Yan等人，PNAS，修订版）。 与Dr. Kortholt合作，我们报道了Ric 8蛋白是异源三聚体G蛋白的非受体鸟嘌呤交换因子，并且对D.d sicoideum的发育和趋化性很重要（Kataria，et al PNAS，2013）。