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

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1: cAR1-mediated spatiotemporal dynamics of Ras signaling. To reveal the inhibitory mechanism, we monitored spatiotemporal activation of Ras by observing the membrane translocation of a fluorescent probe, active Ras Binding Domain fused to GFP (RBD-GFP) in response to a cAMP gradient. 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. To test this hypothesis, we are studying the roles of RasGAP in chemosensing in D. discoideum. 2: A novel Gbg effector, ElmoE, transduces GPCR signaling to the actin network during chemotaxis. Activation of G-protein-coupled receptors (GPCRs) leads to the dissociation of heterotrimeric G-proteins into Ga and Gbg subunits, which go on to regulate various effectors involved in a panoply of cellular responses. During chemotaxis, Gbg subunits regulate actin assembly and migration, but the protein(s) linking Gbg to the actin cytoskeleton remains unknown. Here, we identified a new Gbg effector, ElmoE in Dictyostelium, and demonstrated that it is required for GPCR-mediated chemotaxis. Remarkably, ElmoE interacts with Gbg and Dock-like proteins to activate the small GTPase Rac, in a GPCR-dependent manner, and also associates with Arp2/3 complex and F-actin. Thus, ElmoE serves as the first direct link between chemoattractant GPCRs, G-proteins and the actin cytoskeleton. The pathway, consisting of GPCR, Gbg, Elmo/Dock, Rac, and Arp2/3, spatially guides the growth of dendritic actin networks in pseudopods of eukaryotic cells during chemotaxis (Yan et al Dev. Cell 2012).