Friday, April 26, 2024
4/26/2024
Project Summary My thesis project aims to clarify the signaling mechanism of the most abundant Ga protein subunit in the brain, Gao. Most neurotransmitters can bind to and activate G Protein Coupled Receptors (GPCRs) that signal through Gao, and alterations in Gao signaling have been implicated in a number of neurological disorders. GPCRs activate Gao by promoting exchange of a bound GDP for GTP. This causes the dissociation of the Gß¿ subunits from Gao and potentially allows both Gao and Gß¿ to bind and modulate the behavior different target molecules, known as effectors. Genetic studies show that Gao functions to prevent the release of neurotransmitters, but the molecular details of how this occurs remains unclear, largely because the effector(s) that Gao binds to and regulates remain unknown. While some field have speculated that Gao may simply serve to release the Gß¿ dimer to carry out signaling, studies in C. elegans refute this idea and suggest that Gao must directly signal through its own effectors. I hypothesize that Gao signals by directly binding effector protein(s) and that identifying and analyzing these effectors will be the key to understanding signaling by the major G protein of the brain. I have employed immunopurification of activated and inactive Gao protein complexes from mouse brain followed by mass spectrometry to identify candidate Gao effector molecules. I have already generated a large set of mass spectrometry data and have identified the relatively unstudied Ras GTPase activators Rasa2/3 as strong candidates to be the long-sought Gao effectors. In this proposal I will use in vitro and in vivo experimental approaches to characterize the interaction between Gao and Rasa2/3. My first is aim is to characterize the biochemical interactions between G¿o and Rasa2/3 using purified proteins. I will purify Gao and Rasa2/3 as well as a control Rasa-binding protein and a control Gao-GTP binding protein. I will measure the binding affinities of active and inactive Gao for Rasa2/3 and determine if the small- molecule ligands of Rasa2/3, Ca2+ and IP3, alter this binding. I will map the binding interface of Rasa2/3 for Gao. My second aim is to use C. elegans genetics to analyze the functions of GAP-1, the close C. elegans ortholog of mammalian Rasa proteins, to determine if and how it functions in Gao signaling in vivo. I have obtained a null mutant gap-1 and will analyze to determine if it phenocopies aspects of the already extensively-characterized effects of Gao mutations on specific behaviors in C. elegans. I will also determine which neurons express gap-1 and direct my analysis to functions of those neurons. I will use double-mutant studies to understand the in vivo functional relationship between Gao, GAP-1, and Ras.
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