Negative and positive feedback in cell signaling

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PROJECT SUMMARY Over the past half century scientists have learned much about how cells detect hormones, nutrients and other chemical cues. Typically, these signals are detected by a G protein-coupled receptor and are transduced by a chemical second messenger, such as cAMP or calcium. Our lab has long focused on the identification and characterization of feedback regulators, most notably RGS proteins. Whereas receptors activate G proteins, RGS proteins inactivate G proteins through accelerated GTP hydrolysis. In the next five years our lab will focus on the identification and characterization of novel second messengers and their roles in two aspects of signal coordination. The first is focused on signal coordination by a pheromone GPCR and by another GPCR that detects nutrients. The second is how the pheromone GPCR regulates nutrient availability, through autophagy and the recycling of molecular building blocks. This new initiative builds on our discovery that G proteins regulate, and are regulated by, physiological changes in intracellular pH and of 2-hydroxy branched chain amino acids. These metabolites are second messengers of glucose limitation and osmotic stress, respectively, and share the ability to trigger G protein phosphorylation and inactivation. Our overall approach is to systematically determine the effects of stimulating (pharmacologically) the cell, deleting (genetically) signaling pathway components, measuring (NMR and mass spectrometry) changes in cellular proteins and metabolites, reconstituting (biochemically) the activity of G proteins and effector kinases, and solving (x-ray and NMR) the structures of second messengers bound to their physiological targets. If successful, our research program will lead to the identification of novel second messengers, their mechanism of action, and their functional consequences in the cell. Our investigations will require the implementation of multiple research platforms and strategies, and thus benefit greatly from the flexibility and stability provided by the MIRA program.