G-protein Coupled Receptor Mediated Chemoattractant Sensing and Phagocytosis

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We are investigating the following questions: 1. how a GPCR chemosensing network regulates the polarized reorganization of the actin cytoskeleton required for protrusion of the cell's front and retraction of its back during chemotaxis. 2. Coupling mechanism of a GPCR and heterotrimeric G proteins during chemoattractant gradient sensing. 3. What are the molecular mechanisms underlying phagosome maturation process during phagocytosis? 1. Chemoattractant GPCRs control the actin cytoskeleton during cell migration. GPCR activation induces dissociation of the heterotrimeric G-protein into G and G, which promote formation of new actin filaments via the Arp2/3 complex in migrating cells. The Arp2/3 complex is thought to be activated by Rac, and Elmo/Dock180 serves as a GEF for Rac activation. However, it has been unclear how a GPCR signals to Elmo/Dock180. Here, we have characterized a new Elmo protein, ElmoE in Dictyostelium, and demonstrated that it is required for cAR1 GPCR-mediated chemotaxis. Remarkably, ElmoE physically associates with G to activate Rac and this association is mediated by cAR1. These results have uncovered a new pathway of the GPCR-mediated regulation on the actin cytoskeleton, which involves a GPCR, G, Elmo, Rac, Arp2/3, and actin filaments. The pathway may spatially direct the growth of a dendritic actin network in pseudopod of eukaryotic cells during chemotaxis (Yan et al, submitted). 2. The coupling of heterotrimeric guanine nucleotideVbinding protein (G protein)-coupled receptors (GPCRs) with G proteins is fundamental for GPCR signaling; however, the mechanism of coupling is still debated. Moreover, it remains unclear how the proposed mechanisms affect the dynamics of downstream signaling. Here, through experiments involving fluorescence recovery after photobleaching and single-molecule imaging, we directly measured the mobilities of cAR1, a chemoattractant receptor, and a G protein subunit in live cells. We found that cAR1 diffused more slowly in the plasma membrane than did G. Upon binding of ligand to the receptor, the mobility of cAR1 was unchanged, whereas the speed of a fraction of the faster-moving G subunits decreased. Our measurements showed that cAR1 was relatively immobile and G diffused freely, suggesting that chemoattractant-bound cAR1 transiently interacted with G proteins. Through the use of models that describe possible coupling mechanisms, we computed the temporal kinetics of G protein activation. Our fluorescence resonance energy transfer imaging data showed that fully activated cAR1 induced the sustained dissociation of G protein - and -subunits, which indicated that ligand-bound cAR1 activated G proteins continuously. Finally, our simulations indicated that a high-affinity coupling of ligand-bound receptors and G proteins was essential for cAR1 to translate extracellular gradient signals into directional cellular responses. We suggest that chemoattractant receptors use a ligand-induced coupling, rather than a pre-coupled, mechanism to control the activation of G proteins during chemotaxis (Xu et al., Science Signaling, in press). 3. Phagocytosis is crucial for host defense against microbial pathogens and for obtaining nutrients in Dictyostelium discoideum. Phagocytosed particles are delivered from phagosomes to lysosomes for degradation, but the molecular mechanism regulating phagosome maturation remains unclear. Using D. discoideum as a model system, we plan to reveal important components involved in phagosome maturation. We have identified 3 novel vesicle-associated receptor tyrosine kinases, VSK1-3, in D. discoideum. Our previous study suggests that localized VSK3 tyrosine kinase signaling on the surface of endosome/lysosomes represents a new control mechanism for phagosome maturation. We are identifying targets of VSK 2 and 3. This study will provide a foundation for understanding the molecular mechanism of VSK signaling that regulate phagosome maturation.