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

1：cAR1介导的RAS信号的时空动力学。为了揭示这一机制，我们通过监测荧光探针活性RAS结合结构域与GFP融合(RBD-GFP)对各种刺激的反应来监测RAS的时空激活。我们观察到的RAS激活动力学表明，一个负调控因子，可能是RasGAP，逐渐被招募到膜上，促进RAS失活。在哺乳动物细胞中，含有磷脂结合域的RasGAP被证明移位到质膜内表面，使RAS失活，从而抑制PI3K。我们认为，一个类似的磷脂结合的RasGAP很可能是盘状螺旋藻中cAR1介导的化学传感的重要抑制物。我们已经揭示了RasGAP在盘状芽孢杆菌化学传感中的作用(Xu等人，在PREP中)。 2：我们发现，在真核生物中，阻滞素是参与细胞-细胞振荡信号传递的信号通路的关键组成部分。我们发现，cAR1GPCRi介导的arrestin功能调节ERK2的瞬时激活周期，从而控制盘状芽孢杆菌cAMP振荡的频率。化学信号的振荡是一种常见的生物现象，但人们对其调控知之甚少。在盘基网柄菌发育的聚集期，趋化剂cAMP每隔6分钟合成并释放一次，指导细胞迁移。虽然cAMP受体cAR1、gpr和ERK2都参与了对这种振荡的调节，但信号通路仍不清楚。在此，我们报道了盘状假丝酵母菌拦阻蛋白(ADCB和ADCC)在调节cAMP振荡频率中起关键作用。激活cAR1促进ADCC的膜募集，ADCC与ERK2结合。缺乏阻滞素的细胞(ADCB-C-)具有较短的cAR1触发的瞬时ERK2激活和cAMP振荡周期(3min)，这表明cAR1控制的arrestin功能调节瞬时ERK2激活的周期，该周期调节盘状芽孢杆菌聚集期cAMP振荡的频率。此外，盘状芽孢杆菌利用阻滞素进行配体诱导的cAR1内化，以实现从高亲和力到低亲和力的cAMP受体的切换，从而实现其多细胞发育(Yan等人，PNAS，修订中)。 在与Koreolt博士的合作中，我们报道了Ric8蛋白是异三聚体G蛋白的非受体鸟嘌呤交换因子，并且对D.dsicoideum的发育和趋化作用非常重要(Kataria，et al PNAS，2013)。

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).