We seek to understand how neurotransmitters signal through heterotrimeric G proteins to modulate the activities of neurons. Addictive drugs activate G protein-coupled receptors, and a number of mental diseases are due to alterations in neurotransmission through G proteins, so it is critical to understand the fundamental mechanims of G protein signaling in neurons. Gao is the major brain G protein activated by G protein- coupled neurotransmitter receptors, but little is known about its mechanism of signaling. A recent discovery is that Gao acts in dividing cells to regulate force on microtubules, raising the hypothesis that Gao may also act in neurons via microtubule force to modify cell structure. Gao signaling inhibits egg-laying behavior in C. elegans, and our first aim is to use this as a model to study the Gao signaling mechanism. Thus we will identify the cells of the egg-laying system that generate and receive Gao-mediated signal(s). We will analyze three new genes we have genetically identified that are required for this signaling, one of which we have already cloned and shown to encode a TRP ion channel. We will test the hypothesis that Gao acts via the cytoskeleton to alter the structure of neurons by fluorescently labeling the neural processes and synapses of the egg-laying system in wild-type and Gao-mutant animals.
Our second aim exploits a second model for Gao signaling. Thus we will study the mechanism of Gao-mediated signaling by serotonin, a neurotransmitter involved in depression in humans. We are screening for mutants of C. elegans that fail to respond to serotonin. We will complete this screen and clone and analyze a new serotonin signaling gene that we have already identified by this approach. We will also analyze the expression patterns and knockout phenotypes for a set of C. elegans serotonin receptor homologs.
Our third aim i s to identify and analyze the molecules that act downstream of Gao to mediate its effects. We will complete a genetic screen for mutants that disrupt neurotransmitter signaling downstream of Gao.Using this screen and the serotonin screen, we have already isolated five mutations that appear to disrupt both Gao-mediated neurotransmission as well as microtubule force generation in dividing cells. This strongly supports the hypothesis that Gao acts by the same mechanism for both functions. Three of the mutations identify a single gene that, by epistasis analysis, appears to function downstream of Gao. We will clone and analyse the gene(s) identified by these mutations.
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