Neuronal circuitry underlying attraction to BZ, BU, and IAA includes AWC sensory neurons and ∼20 interneurons and motor neurons. Thus, expression of RGEF-1b-GFP in a few neurons might restore chemotaxis in rgef-1−/− animals. Three promoters were used to drive RGEF-1b-GFP expression.

The odr-1 promoter is see more active in AWC, AWB, and I1 neurons; odr-3 drives transcription in AWA, AWB, AWC, and four other neurons, and the gpa-3 promoter is silent in chemosensory neurons, but active in a dozen distinct neurons ( L’Etoile and Bargmann, 2000 and Lans et al., 2004). An odr-1:: RGEF-1b-GFP transgene restored CI values to near-WT levels in rgef-1−/− animals ( Figures 3E and 3F). Substantial, but incomplete rescue of chemotaxis ABT-199 was achieved with an odr-3::REGF-1b-GFP transgene. Partial restoration of chemotaxis may be due to lower transcriptional activity of odr-3 relative

to odr-1 and rgef-1 promoters. Although the gpa-3 promoter enables RGEF-1b-GFP accumulation in multiple neurons, the rgef-1−/− phenotype persisted ( Figures 3E and 3F). I1, AWB and AWA neurons are not involved in BZ- or BU-induced chemotaxis. Thus, the results suggest that RGEF-1b GTP exchange activity in AWC sensory neurons is indispensable for activating neuronal circuitry underlying chemotaxis behavior. The observations establish a neuron-specific physiological role for a RasGRP. Conserved Arg290 in the RGEF-1b catalytic domain was mutated to Ala (Park et al., 1994). RGEF-1bR290A protein accumulated in transfected cells, but did not promote loading of GTP onto LET-60 (Figure 4A, lanes 3 and 4). Cells expressing bombesin receptor were cotransfected with transgenes encoding WT RGEF-1b (0.2 μg DNA), Flag-LET-60

(1 μg DNA), and RGEF-1bR290A (plasmid DNA varied from 0–1 μg). In both bombesin-treated and unstimulated cells, LET-60-GTP content decreased as the ratio Etomidate of RGEF-1bR290A cDNA:WT RGEF-1b cDNA increased (Figure 4B, lanes 1–8). A 4-fold excess of RGEF-1bR290A transgene eliminated bombesin-induced GTP exchange activity of WT RGEF-1b (Figure 4B, lanes 7 and 8). Thus, RGEF-1bR290A is a dominant-negative RasGRP. Expression of RGEF-1bR290A-GFP throughout the nervous system did not rescue chemotaxis in rgef-1−/− animals ( Figure 4C). Thus, RGEF-1b does not function as a scaffold in vivo; its GTP exchange activity is required for chemotaxis. Moreover, Arg290 was identified as an essential amino acid in the catalytic domain. Expression of RGEF-1bR290A-GFP in the WT background suppressed chemotaxis to odorants detected by AWC neurons ( Figure 4D). The data show that RGEF-1b is essential for odorant-activated chemotaxis in a WT context. By analogy with mammalian Ras and Rap1 mutants (Campbell et al., 2006), substitution of Gly12 with Val should ablate GTPase activity, thereby locking LET-60 or RAP-1 in an active, GTP-bound state (Figure 5A). Replacement of Ser17 with Asn should generate dominant-negative mutants of LET-60 and RAP-1.