The long-term goal of this proposal is to understand how neurotransmitters signal through G protein coupled receptors to modulate the activities of neurons. Egg-laying behavior in C. elegans is regulated by neurotransmitter signaling through the G proteins Ga0 and Ga. To identify novel G protein signaling components and understand their mechanisms of action, we: isolate and analyze mutations that disrupt C. elegans egg-laying behavior; 2) clone the genes identified by these mutations; 3) purify the signaling proteins encoded by the cloned genes; and 4) study the properties and interactions of the purified proteins. The signaling proteins analyzed so far have close homologs expressed in the mammalian brain, suggesting that C. elegans is a useful model for understanding neurotransmission through G proteins in humans. The potential of our approach is illustrated by the fact that we have already used it to discover a novel class of regulators of G protein signaling (RGS proteins) that inhibit signaling by acting as G protein GTPase activators. The first major aim of this proposal is to exploit C. elegans genetics to identify and analyze more G protein signaling pathway components. We recently isolated mutations in at least three novel signaling genes. We will clone and molecularly analyze these genes. We also recently cloned an additional genetically identified signaling gene, egl-47, that encodes two putative G protein-coupled receptors, and propose further analysis of their functions. As an additional genetic project, we will knock out the genes encoding most or all of the 13 RGS proteins of C. elegans and use these mutants to define the in vivo functions of RGS proteins. The second major aim of this proposal is to carry out biochemical studies of the signaling proteins already identified. First, we will carry out enzymological studies of diacylglycerol kinase-1, which genetic and biochemical experiments show is a direct effector of Ga0. We will characterize the activities of the purified enzyme and its activation by purified Galphao. Second, we will study the in vitro activities of the RGS proteins EGL-lO and EAT-16. They contain a G gamma-like domain via which they complex with the GB5-like subunit GPB-2 to regulate Galphao and Galphaq. These proteins may form a novel type of G protein heterotrimer containing a Ga subunit, the GB protein GPB-2, and an RGS protein rather than a conventional gamma subunit. We will examine formation of complexes between the RGS, GB and Ga subunits in vitro and determine which regions of the RGS proteins direct them to act specifically on their genetically identified Ga targets.
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