Heterotrimeric G-protein regulation of neurotransmission in C. elegans

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Signaling through heterotrimeric GTPases (G-proteins) activates cellular effector enzymes and ion channels to regulate cell growth, mitotic division, and sensory perception. G-protein coupled receptors are among the most commonly used signal transducers in eukaryotes and are the targets of about half of prescribed Pharmaceuticals. The soil nematode, Caenorhabditis elegans, offers many experimental advantages to investigate the conserved features of heterotrimeric G-protein signaling. In embryos, G- proteins regulate microtubule forces that control mitotic spindle positioning during asymmetric cell division. In adults, G-proteins coordinate animal feeding, motility, and other behaviors by altering synaptic activity and the frequency of muscle contractions. These behaviors offer convenient and quantitative assays to study intracellular and intercellular signaling genetically. Through three independent lines of experimentation, I will study the molecular and cellular consequences of signaling through two G-proteins, EGL-30 (Gctq) and GOA-1 (Ga0). Activated EGL-30 interacts with EGL- 8, the phosphatidylinositol (4,5) bisphosphate (PlPz)-specific Phospholipase C (p) family member. Hydrolysis of PIP? by EGL-8 releases the second messengers inositol 1,4,5-triphosphate (IP3) and 1,2- diacylglycerol (DAG). In contrast, direct effectors of activated GOA-1 remain unknown. To find these, I will isolate mutants that suppress hyperactivated GOA-1 signaling phenotypes. One suppressor has already been mapped by members of the Koelle lab, and I will map additional suppressor mutations, determine how the encoded factors regulate signaling, and test whether they act as direct effectors for GOA-1. Second, to study how GOA-1 antagonizes EGL-30 signaling to modulate synaptic activity, I will visualize EGL-8 activity in vivo using established fluorescent sensors of Ca+2 and specific phospholipids. I will test how mutations that impair or stimulate inhibitory GOA-1 signaling affect the behavior and distribution of these indicators. These in situ biochemical experiments will reveal the cell biological consequences of the signaling pathways defined genetically. Finally, to find genes whose expression is regulated by signaling, I will compare gene expression profiles in goa-1 and egl-30 mutants by microarrayanalysis. This research will shed light on how the nervous system controls the frequency of muscle contractions. As these functions are perturbed during human brain seizures, muscle tremors, and neurodegenerative diseases such as Parkinson's, these studies should inform a rational basis for targeted therapies.