Characterization of odr-3, tax-2, and tax-4 mutants indicated that odorant-induced AWC hyperpolarization

is a prerequisite for MPK-1 activation Ulixertinib cell line by IAA ( Hirotsu et al., 2000). Thus, the LET-60-MPK-1 pathway functions downstream from TAX-2/TAX-4 channels. The inability of odorants to activate MPK-1 in tax-2 or tax-4 mutants excludes the possibility that odorant receptor controls LET-60 activation via ODR-3. Phosphorylated MPK-1 accumulated principally in the AWC cell body of IAA- and BZ-treated WT animals. Output from the LET-60-MPK-1 cascade evidently modulates an odorant-induced, ion-based signal at a site segregated from ciliary odor sensing machinery. A modulatory role explains why a combination of odorant and AWC-targeted expression of constitutively active LET-60 (or MEK-2) restores chemotaxis in rgef-1−/− animals. PMA and DAG elicited

translocation of RGEF-1b from cytoplasm to ER in HEK293 cells. Diminished DAG binding affinity of the RGEF-1bP503G C1 domain markedly decreased translocation, thereby NVP-BKM120 in vitro segregating the GTP exchanger from LET-60. When RGEF-1b was anchored to ER by a Tb5 domain, only basal catalytic activity was observed. PMA (50 nM) robustly activated ER-tethered RGEF-1b, but only minimally stimulated ER-bound RGEF-1bP503G. Thus, avid PMA/DAG binding by the C1 domain is crucial for (1) colocalizing RGEF-1b with LET-60 and (2) inducing or stabilizing a conformation of RGEF-1b that expresses high level catalytic activity. RGEF-1P503G did not restore chemotaxis or MPK-1 phosphorylation to rgef-1−/− animals. Thus, C1-mediated targeting of RasGRP to membranes is a critical step in switching on the Ras/ERK pathway in vivo. LET-60 is maximally homologous with K-Ras, which is farnesylated and often activated at the ER. Subsequently, K-Ras is routed to effector

locations without passage through Golgi membranes (Karnoub and Weinberg, 2008). RasGRP-mediated activation of K-Ras (LET-60) at the ER may be a conserved mechanism for routing regulatory signals. LET-60-GTP could be guided to various ER-proximal locations by its membrane binding properties, affinities for effectors, and association with specific transport vesicles. Concentrating RGEF-1b (presumably at ER) in the AWC axon through and cell body enabled MPK-1 activation and chemotaxis. Sequestration in nonaxonal compartments evidently separated RGEF-1b from its substrate, thereby disrupting its function. RGEF-1b apparently exerts physiological effects at sites far removed from cilia. These observations and evidence for unimpaired odorant detection in rgef-1−/− animals, suggest the RGEF-1b-LET-60-MPK-1 pathway modulates olfactory signal transduction within AWC and/or synaptic transmission to interneurons. Distinct DAG effectors modulate synaptic transmission in AWC and motor neurons.