G Protein Signaling in the C. elegans Nervous System

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DESCRIPTION (provided by applicant): 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 mechanisms of G protein signaling in neurons. Galpha-o 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 Galpha-o acts in dividing cells to regulate force on microtubules, raising the hypothesis that Galpha-o may also act in neurons via microtubule force to modify cell structure. Galpha-o signaling inhibits egg-laying behavior in C. elegans, and our first aim is to use this as a model to study the Galpha-o signaling mechanism. Thus we will identify the cells of the egg-laying system that generate and receive Galpha-o-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 Galpha-o 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 Galpha-o-mutant animals. Our second aim exploits a second model for Galpha-o signaling. Thus we will study the mechanism of Galpha-o-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 is to identify and analyze the molecules that act downstream of Galpha-o to mediate its effects. We will complete a genetic screen for mutants that disrupt neurotransmitter signaling downstream of Galpha-o. Using this screen and the serotonin screen, we have already isolated five mutations that appear to disrupt both Galpha-o-mediated neurotransmission as well as microtubule force generation in dividing cells. This strongly supports the hypothesis that Galpha-o 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 Galpha-o. We will clone and analyze the gene(s) identified by these mutations.