The

data suggest PMA and DAG enhance GTP exchange activity by recruiting RGEF-1b to internal membranes that contain transiently or persistently colocalized LET-60. C1 domains are composed of ∼50 amino acids and contain two His and six Cys residues that are conserved (Figure 7B). Collectively, the Cys and His side chains ligate two zinc ions, thereby creating a rigid structure that supports a binding pocket. Three β strands and two hydrophobic loops (A and B) (Figure 7B) generate surfaces that bind DAG/PMA and facilitate partial immersion of C1 domains into a lipid bilayer. A conserved Pro in loop A is critical for high-affinity PMA binding (Hurley and Misra, 2000). Consequently, Pro503 in RGEF-1b was mutated to Gly. The mutation extinguished basal GTP exchange (Figure 7C, lanes 1 and 3) MG-132 manufacturer and sharply suppressed GTP loading activity at a concentration of PCI32765 PMA (50 nM) that maximally activated WT RGEF-1b (Figure 7C, lanes 2 and 4; Figure 7D lane 1, upper and lower panels). However, incubation of cells with high concentrations of PMA (200–400 nM) elicited similar, maximal catalytic activities for RGEF-1bP503G and RGEF-1b (Figure 7D). The results indicate that the mutant exchanger folds normally, but Pro503 is essential for high-affinity PMA binding by the C1 domain. RGEF-1bP503G failed to activate LET-60 when bombesin triggered DAG synthesis (Figure 7C, lane 8). Bombesin peptide concentration (200 nM) was

20-fold higher than necessary to saturate bombesin receptors and optimally activate PLCβ (Feng et al., 2007). Thus, Pro503 is indispensable for coupling increased DAG content to RGEF-1b activation. Maximal, bombesin-induced production of DAG did not compensate for the diminished C1 domain function of RGEF-1bP503G. RGEF-1bP503G was

not stably recruited to membranes by 50 nM PMA or 200 nM bombesin (Figure 7Af and 7Ah). Evidently, transient association of RGEF-1bP503G with membrane-bound, nonmetabolized PMA was sufficient to modestly activate LET-60 (Figure 7C, lane 4). Reduced binding affinity of RGEF-1bP503G from for transiently synthesized, rapidly metabolized DAG prevented exchanger activation by endogenous second messenger (Figure 7C, lane 8). Thus, high-affinity DAG binding activity of the C1 domain is essential for intracellular targeting and maximal catalytic activity of RGEF-1b. In vivo consequences of RasGRP C1 domain dysfunction are unknown. Therefore, animals expressing rgef-1::RGEF-1b or rgef-1::RGEF-1bP503G transgenes (rgef-1−/− background) were incubated with odorants detected by AWC or AWA neurons. RGEF-1b restored chemotaxis to both odorants ( Figure 7E). In contrast, panneuronal expression of RGEF-1bP503G did not alter low CI values observed in RGEF-1b-deficient C. elegans ( Figure 7E). Thus, C1 domain-mediated targeting to membranes is a key determinant of a RasGRP function (chemotaxis) in vivo. MPK-1 phosphorylation was assayed to determine if the RGEF-1bP503G mutation affected signaling in AWC neurons.