1: A GPCR-mediated signaling network enables a chemotactic cell to generate adaptation responses to a large range of concentrations of a chemoattractant and polarized responses to a chemoattractant gradient. However, it is unclear how the signaling network mechanistically organizes at the molecular level, dynamically encodes information at each signaling step, and systematically produces outputs during chemotaxis. Here, we developed a systems biology approach to investigate GPCR cAR1-mediated chemotactic responses and to reveal missing regulatory mechanisms in the signaling network. Chemoattractant cAMP binding to cAR1 activates heterotrimeric G-proteins, Ras signaling and PIP3 production, which regulate chemotaxis of D. discoideum cells. Using live cell imaging, we found that each signaling event has distinct kinetic patterns in response to stimuli. Specifically, the cell produces the persistent G-protein activation, the imperfectly adapted Ras activation, and the perfectly adapted PIP3 production in response to cAMP stimulation at various concentrations (10-9 to 10-5M). Using these experimentally measured dynamics as the foundation, we constructed mechanistic diffusion models of cAR1-mediated Ras signaling network by incorporating potential regulatory mechanisms of Ras signaling, which are an activator (RasGEF) and an inhibitor (RasGAP), and simulated spatiotemporal dynamics of signaling events, including persistent G-protein activation and imperfectly adapted Ras activation. Using our computational models, we explored the roles of RasGEF and RasGAP in generating Ras adaptation in response to various cAMP stimuli. Furthermore, our computer simulations revealed that a cAMP gradient induces imperfect Ras adaption around the cell surface, and that the signaling network of an adaptation model is unable to produce significant spatial amplification at the step of either G-protein activation or Ras activation. Together, our study showed that adaption models require additional mechanisms to explain the amplification of directional cues at the steps of G-protein activation and Ras activation into highly polarized responses of PIP3 production and actin polymerization for chemotaxis. Our model construction and computer simulation approach can help to allow a quantitative and predictive understanding of GPCR-mediated signaling network for adaptation and amplification responses and to identify missing components that are required for eukaryotic chemotaxis. (Xu et al., in revision). 2: Eukaryotic cells chemotax in a wide range of chemoattractant concentration gradients, and thus need inhibitory processes that terminate cell responses to reach adaptation while maintaining sensitivity to higher-concentration stimuli. However, the molecular mechanisms underlying inhibitory processes are still poorly understood. Here, we reveal a locally controlled inhibitory process in a GPCR-mediated signaling network for chemotaxis in Dictyostelium discoideum. We discover a novel negative regulator of Ras signaling, C2GAP1, which localizes at the leading edge of chemotaxing cells, is activated by and is essential for GPCR-mediated Ras adaptation. We show that both C2 and GAP domains are required for the membrane targeting of C2GAP1, and that GPCR-triggered Ras activation recruits C2GAP1 from cytosol and retains it on the membrane to locally inhibit Ras signaling. The altered Ras activation results in impaired gradient sensing and excessive polymerization of F-actin in c2gap1 knockout (c2gap1-) cells, leading to chemotaxis defects. Remarkably, c2gap1- cells display altered cell response, impaired directional sensing, and chemotaxis defects in a chemoattractant concentration-dependent fashion. Thus, we have uncovered a novel inhibitory mechanism required for the adaptation and long-range chemotax.(Xu, Quan et al., in submission). 3: Eukaryotic phagocytes search and destroy invading microorganisms via chemotaxis and phagocytosis. Social ameba Dictyostelium discoideum are professional phagocytes that chase bacteria through chemotaxis and engulf them as food via phagocytosis. G-proteincoupled-receptors (GPCRs) are known for detecting chemoattractants and directing cell migration, but their roles in phagocytosis are not clear. Here we uncovered an orphan GPCR as the long-sought-after receptor for folic acid, a chemoattractant for D. discoideum by using a quantitative phosphoproteomic approach. Significantly, we discovered that this receptor is essential for both chemotaxis and phagocytosis of bacteria, thereby representing the first identified chemoattractant GPCR that is required for not only chasing but also catching and ingesting bacteria. (Pan et al. Developmental Cell, 2016)
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