DISSECTING G PROTEIN PATHWAYS

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Although heterotrimeric G proteins account for a large fraction of the signals in the mammalian brain, many of the second messengers and ion channels regulated by these pathways have not been identified, little is known about cross-talk between G protein pathways, and about how these signals determine changes in behavior. We have undertaken a comprehensive analysis of signaling by the G protein G/o in the nervous system of the nematode C. elegans. We showed that GOA-1 (which encodes the alpha subunit of G/o) plays an important role in signaling by serotonin (5-HT). We devised behavioral assays for signaling by GOA-1 and by Gs. Using these assays, we identified 13 mod (neuromodulation defective) genes that genetically act downstream of GOA-1 and one downstream of Gs. We have cloned three of these downstream genes. dgk-1 and egl-36 are downstream of GOA-1 and encode a diacylglycerol kinase and a Shaw type potassium channel subunit, respectively. acy-1 is downstream of Gs and encodes an adenylate cyclase. Interestingly, acy-1 mutations also reduce signaling by GOA- 1, implying that these pathways interact in some manner. We propose behavioral, genetic, biochemical, and biophysical experiments to determine what role DGK-1, EGL-36, and ACY-1 play in GOA-1 signaling. First, we will determine where GOA-1 acts to control several behaviors. If EGL-36, DGK-1, and ACY-1 biochemically interact with GOA-1, they must be expressed in these same cells. Second, we will determine whether GOA-1 can regulate the activities of DGK-1, and ACY- 1 in transfected cell lines. We will also test if dependence on DGK-1 reflects GOA-1 regulation of phospholipase C (PLC) by determining whether any of the five C. elegans isoforms of PLC are co-expressed with GOA-1, and if GOA-1 can regulate co-expressed PLCs in transfected cells. Third, we will characterize EGL-36 channel activity, its interactions with two other C. elegans Shaw subunits, and whether it is regulated by GOA-1 in transfected tissue culture cells. And fourth, to identify another potential GOA-1 target, we will clone one more downstream gene, mod(nu62). In summary, 5-HT has been implicated in depression, alcoholism, obesity, and suicidality. Given the strong conservation of these pathways across phylogeny, it is likely that our experiments will identify new targets for pharmaceuticals to treat these clinically important human disorders.