Summary Heterotrimeric G protein signaling pathways are of tremendous importance to human health. Mutation of G protein subunits causes genetic disease, developmental abnormalities, and altered infectious disease susceptibility. Indeed, G protein pathways are the targets of approximately a third of all drugs under clinical use. The Assmann laboratory has furthered fundamental understanding of heterotrimeric G protein signaling through elucidation of G protein-mediated signaling cascades and phenotypes. The emphasis of the parent award is on mechanisms of phospho-regulation of G protein signaling that are evolutionarily conserved but have been oft overlooked in mammalian systems. In this research, the model plant Arabidopsis is used as a facile system to investigate kinase-mediated phosphorylation of the canonical G protein α (Gα) subunit, GPA1, and to explicate the downstream signaling impacts of this phosphorylation; in particular, how phosphorylation biases interactions with downstream effector proteins. In parallel, relevance to human health is demonstrated through assessment of the impacts of analogous phosphorylation events on human Gα subunits in vitro, using BODIPY-GTP binding and hydrolysis assays on the corresponding human phosphomimic mutants. This supplement requests funding for the purchase of a BioTek Synergy Neo2 plate reader to increase the throughput and reliability of the BODIPY- GTP activity assays and to allow the implementation of orthogonal methods, particularly transcreener assays, that will allow independent validation of these biochemical data. The dual monochromators of the Synergy Neo2 plate reader will allow for assay versatility with a multitude of fluorophores. The speed and sensitivity of the Synergy Neo2 far outpaces that of the extant obsolete Flx800 plate reader, allowing for finer timescale measurements of a greater number of Gα variants. In addition, the capability of the Synergy Neo2 to assay luciferase activity will facilitate assessment of biased signaling arising from phosphorylation-dependent protein- protein interactions, as the Synergy Neo2 has the capability for high-throughput measurements of protein-protein interaction using the split-luciferase method. In summary, the proposed state-of-the-art plate reader will provide reliable, sensitive, and rapid data acquisition as well as entirely new capabilities to probe the molecular effects of G protein phosphorylation. Finally, the instrumentation will be available to other NIH-funded researchers at Penn State in the Biology and Chemistry Departments.