Michael R. Koelle, Dept. of Molecular Biophysics & Biochemistry, Yale University
Dysregulation of serotonin signaling is associated with drug addiction, but to understand this association in detail it is first necessary to obtain a proper basic science understanding of the mechanisms that regulate serotonin signaling. The nematode C. elegans uses serotonin as a neurotransmitter and has close homologs of human serotonin signaling proteins including receptors, reuptake transporters, and G proteins. Using the unique advantages of this experimental system, we have carried out a genetic screen for mutants of C. elegans that fail to respond to serotonin. We identified mutations in genes encoding four proteins required for serotonin response: two serotonin receptors, a G protein, and a cytoplasmic enzyme complex called elongator that modifies other proteins through acetylation of lysine residues. Elongator is highly-conserved from worms to humans and is expressed throughout the nervous system. Our work is the first implicating elongator in serotonin signaling. We hypothesize that elongator affects serotonin signaling by acetylating lysine residues on one of the serotonin receptors or the G protein identified by our genetic screen. Currently the best candidate for the acetylated protein is the Gαo, the major G protein of the nervous system. We found that C. elegans Gαo exists as a series of species separable by isoelectric focusing, demonstrating that it is heterogeneously post-translationally modified in a manner that alters its charge. Our specific aims are 1) To use mass spectrometry of immunoprecipitated Gαo from C. elegans lysates to identify acetylated lysine residues; and 2) To similarly analyze Gαo immunoprecipitated from mouse brain extracts to identify acetylated lysine residues in mouse Gαo. Results from these studies will allow us to apply the full power of the C. elegans system to determine the precise role of lysine acetylation in serotonin signaling. Comparative studies of Gαo modification in the mouse brain we will determine the extent to which our results in C. elegans apply to the mammalian brain and may be relevant to drug addiction in humans.