Spatial and temporal regulation of G-protein signaling elucidated by computational modeling and live cell FRET imaging

Regulation of G-protein signaling occurs spatially between microdomains of cellular membranes, and temporally due largely to ligand-bound G-protein couple receptors (GPCRs) that speed up G-protein activation and regulator of G-protein signaling (RGS) proteins that speed up deactivation. This dissertation investigates spatial and temporal regulation of G-protein activity primarily through computational modeling and live cell FRET imaging. Computational modeling was used to study how variation in local concentrations of active GPCR, G-protein, and RGS protein affect local G-protein activity and turnover; results predict 16 distinct signaling regimes and numerous intermediate signaling phenomena. Computational methods were also developed to simplify complex biochemical networks; results indicate that a GPCR-Galpha-RGS complex is required in muscarinic receptor signaling to Galphaq, and predict that RGS protein can promote biphasic (bell-shaped) dose-response to ligand. To study spatial and temporal regulation of G-protein activity experimentally, two FRET probes of YFP-tagged Galphai were developed: GTP-BODIPY TR (for membrane-based high throughput assays) and RGS-CFP (for live-cell imaging). RGS-CFP, unlike previous FRET probes for G-protein activity, is untargeted thereby functioning throughout the cell. RGS-CFP was used to investigate spatial regulation of G-protein activity during cell migration, in which GPCRs and G-proteins are thought, based primarily on chemotaxis of the slime mold Dictyostelium discoideum towards GPCR-ligand, to be distributed roughly homogeneously around the plasma membrane with subtly enhanced G-protein signaling at the front of the cell (a.k.a. leading edge). We studied migration in an alternative system: HeLa cells migrating to close a wound. Here, migration required Gi3 (together with a protein called GIV/Girdin) as did associated processes like actin remodeling and Akt activity; spatially, Gi3-YFP translocated from the Golgi to the leading edge where it accumulated and RGS-CFP, which is primarily cytosolic in resting cells, colocalized with Gi3-YFP at the leading edge where high FRET was observed indicating Gi3-YFP activity. These data demonstrate for the first time specific local regulation of Galphai activity at the leading edge of cell migrating to close a wound. Taken together, this dissertation combining computational modeling and live cell FRET imaging sets the stage for quantitative research into G-protein signal transduction at the subcellular level.