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).
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