1: cAR1-mediated spatiotemporal dynamics of Ras signaling. GPCR-mediated Ras activation is a key signaling step for eukaryotic cells to generate temporal adaptation or to cause spatial amplification in response to spatially uniform stimuli or a chemoattractant gradient. It is not clear how a GPCR/G-protein machinery regulates spatiotemporal dynamics of Ras activation to achieve these cellular responses. Here, using quantitative live cell imaging methods, we measured spatiotemporal dynamics of Ras activation in Dictyostelium discoideum cells in response to various cAMP stimuli. While regulators of Ras and other components have not been identified, our quantitative measurements demonstrate that different signaling events downstream of GPCR have distinct kinetic patterns, and provide a foundation for understanding how these events are linked to each other to produce chemotactic responses. We then constructed a spatially resolved model of cAR1-mediated Ras signaling network based on detailed molecular interactions, and selected a set of parameters of defined molecules for the model to generate dynamics matching those determined experimentally. This was done by using the computer interfaces of SIMMUNE, a software package that allows biologists to construct a spatially resolved computational models of a signaling network based on molecular interactions, to carry out computer simulations that test performance of a model in response to various stimuli. These analyses allowed us to propose molecular mechanisms of Ras regulators, RasGEF and RasGAP, and also to develop computational models by incorporating detailed molecular mechanisms. We then examined dynamic behaviors of potential RasGAP regulatory mechanisms in a GPCR-mediated Ras signaling network by incorporating different molecular interactions in our models. Our study proposes detailed spatially resolved computational models that allow us to examine how a GPCR-mediated signaling network organizes at a molecular level, dynamically encodes information at each signaling steps, and systematically produces outputs to achieve chemoattractant gradient sensing. In addition, we showed a novel approach to generate specific mutations in a signaling network, in silico, and to study molecular mechanisms at a systems level using the SIMMUNE software. (Xu et al., to be submitted). 2: Based on our proposed molecular mechanisms of Ras regulators from computational models, we identified a novel negative regulator of Ras, C2RasGAP1, and revealed how the Ras signaling guides directional responses in chemotaxis. A signaling network controlling chemotaxis require an inhibition process for cells to instantly respond to a chemoattractant at various concentrations, and also to quickly reset themselves to any given existing stimulus, so these cells can migrate in a chemoatrractant gradient. However, molecular mechanisms underlying the inhibition process are still poorly understood. In this study, we revealed a locally controlled inhibitory process in a GPCR-mediated signaling network for chemotaxis of Dictyostelium cells. We discovered a novel negative regulator of Ras, C2RasGAP that is essential for a chemoattractant cAMP-mediated Ras adaptation. We found that cells lacking C2RasGAP failed to generate proper spatiotemporal responses of Ras and PIP3 upon either a uniform or a gradient of chemoattractant stimulations. The c2rasgap knockout cells displayed impaired directional sensing and chemotaxis, especially in high concentrations of cAMP, suggesting that defects are chemoattractant concentration dependent. Taken together, our results uncover a novel inhibitory mechanism by C2RasGAP to suppress Ras signaling. We found the level of inhibition is controlled by a chemoattractant GPCR in a concentration-independent manner for cells to carry out chemotaxis in a large range of chemoattractant concentrations (Xu, Quan et al., in submission). 3: We developed a quantitative phosphoproteomic approach to monitor the global phosphorylation changes upon activation of a chemoattractant GPCR. Our phosphoproteomic approach combines tandem mass tag (TMT) chemical labeling for quantification, basic reverse-phase high-performance liquid chromatography (HPLC) and immobilized metal affinity chromatography (Fe-NTA) for phosphopeptide enrichment, and multi-stage MS for identification of phosphopeptides and quantification of phosphorylation sites. Over 4,000 phosphorylation sites with a dynamic change (phosphorylation level increase or decrease) upon stimulations of a chemoattractant (cAMP or folic acid) were determined. We discovered many new phosphorylation sites on known components in the signaling network. In addition, we also found a variety of previously uncharacterized proteins exhibiting chemoattractant-dependent phosphorylation level change, which are currently under investigation (Pan et al). 4: Using the quantitative phosphoproteomic approach, we identified a novel chemoattractant GPCR that controls both chemotaxis and phagocytosis. Eukaryotic phagocytes search and destroy invading microorganisms via chemotaxis and phagocytosis. Social ameba D. discoideum are professional phagocytes that chase bacteria through chemotaxis and engulf them as food via phagocytosis. G-protein-coupled-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 (FR), a chemoattractant for D. discoideum, by using above-mentioned quantitative phosphoproteomic approach. We found that FR is localized on the cell surface and binds to folic acid directly. Using the FR knock out cell lines, we demonstrated that FR is required for folic acid-induced ERK signaling and PIP3 signaling. Furthermore, folic acid-mediated chemotaxis was impaired in FR null cells, and chemotaxis phenotypes were rescued by expressing FR in FR knock cell lines. 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. in submission)
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