A RasGRP, C. elegans RGEF-1b, couples external stimuli to behavior by activating LET-60 (Ras) in sensory neurons

Abstract

RasGRPs, which load GTP onto Ras and Rap1, are expressed in vertebrate and invertebrate neurons. The functions, regulation, and mechanisms of action of neuronal RasGRPs are unknown. Here, we show how C. elegans RGEF-1b, a prototypical neuronal RasGRP, regulates a critical behavior. Chemotaxis to volatile odorants was disrupted in RGEF-1b-deficient (rgef-1⁻/⁻) animals and wild-type animals expressing dominant-negative RGEF-1b in AWC sensory neurons. AWC-specific expression of RGEF-1b-GFP restored chemotaxis in rgef-1⁻/⁻ mutants. Signals disseminated by RGEF-1b in AWC neurons activated a LET-60 (Ras)-MPK-1 (ERK) signaling cascade. Other RGEF-1b and LET-60 effectors were dispensable for chemotaxis. A bifunctional C1 domain controlled intracellular targeting and catalytic activity of RGEF-1b and was essential for sensory signaling in vivo. Chemotaxis was unaffected when Ca²+-binding EF hands and a conserved phosphorylation site of RGEF-1b were inactivated. Diacylglycerol-activated RGEF-1b links external stimuli (odorants) to behavior (chemotaxis) by activating the LET-60-MPK-1 pathway in specific neurons.

Figure 1. RGEF-1b loads GTP onto LET-60 and RAP-1

A,HEK293 cells were transfected with a FLAG-LET-60 transgene and either control plasmid (lanes 1 and 2) or plasmid encoding RGEF-1b (lanes 3 and 4). After incubation with vehicle or 50 nM PMA for 15 min, cells were lysed and LET-60-GTP was precipitated from samples containing 300 μg of protein (Experimental Procedures). Precipitated proteins and samples of total detergent soluble protein (30 μg/lane) were assayed by Western immunoblot analysis. LET-60-GTP and total LET-60 were detected by probing blots with anti-Flag IgGs. RGEF-1b was detected with anti-RGEF-1b IgGs. Incubation with secondary IgGs, and recording and quantification of chemiluminescence signals were performed as described in Experimental Procedures. Chemiluminescence signals, recorded on x-ray film, are shown. B, cells were co-transfected with FLAG-LET-60 and bombesin receptor transgenes and either control vector or expression plasmid encoding RGEF-1b. Cells were incubated with vehicle or 200 nM bombesin (10 min) before lysis. Samples were processed as described in A. C, cells were co-transfected a FLAG-RAP-1 transgene and either empty vector or RGEF-1b transgene. Assays were performed as described in A. Similar results were obtained in 3 experiments.

Figure 2. The rgef-1 promoter is active in neurons

An rgef-1::GFP transgene was expressed in C. elegans. Accumulation of GFP was monitored by fluorescence microscopy. A, C and E show GFP fluorescence; B, D and F are corresponding phase micrographs. A and C show the rgef-1 promoter is active throughout the nervous system. GFP is evident in head neurons (H), nerve ring (NR), tail neurons (T) and nerve cord (NC). E, rgef-1 promoter activity is initially observed in late embryos.

Figure 3. RGEF-1b is essential for chemotaxis to volatile odorants

A,WT, rgef-1(−/−) animals and animals expressing an rgef-1::RGEF-1b-GFP transgene (rgef-1(−/−) background) were assayed for chemotaxis. Chemotaxis index (CI) values are plotted; error bars are SEM. *P< 0.0005 compared with WT animals, Dunnett’s t test. B, WT or rgef-1(−/−) animals were assayed for locomotion on a bacterial lawn. The number of body bends in a 30 s time interval was determined for 20 WT or rgef-1(−/−) animals. Mean values and SEM are shown. C, HEK293 cells were transfected with transgenes encoding FLAG-LET-60 and either RGEF-1b (lanes 1, 2) or RGEF-1b-GFP (lanes 3, 4). Cells were incubated with vehicle or PMA and assayed for LET-60-GTP, LET-60 and RGEF-1b content by immunoblotting. D, an hsp-16.2::RGEF-1b transgene was inserted into rgef-1(−/−) animals. WT, rgef-1(−/−) and transgenic C. elegans were grown to the young adult stage at 20ºC. Control transgenic animals were maintained at 20ºC. Others were incubated at 32ºC for 60 min and returned to 20ºC for 2 h to enable recovery from heat stress. Subsequently, animals were assayed for chemotaxis to BZ. E and F, WT C. elegans, rgef-1(−/−) animals and animals expressing RGEF-1b-GFP under regulation of odr-1, odr-3 or gpa-3 promoters (rgef-1(−/−) background) were assayed for chemotaxis. Assays D–F were performed in triplicate; error bars are SEM. **P< 0.0001 compared with rgef-1 null animals, Bonferroni’s t test. Similar results were obtained in 3 experiments.

Figure 4. Mutation of Arg²⁹⁰ to Ala suppresses RGEF-1b activity and odorant-induced chemotaxis

A,cells were transfected with transgenes encoding FLAG-LET-60 and either RGEF-1b (lanes 1, 2) or RGEF-1b^R290A (lanes 3, 4). Cells were incubated with vehicle or PMA and assayed for LET-60-GTP, LET-60 and RGEF-1b content by immunoblotting. B, cells were co-transfected with plasmids encoding bombesin receptor (0.1 μg DNA), WT RGEF-1b (0.2 μg DNA), FLAG-LET-60 (1 μg DNA) and RGEF-1b^R290A (DNA varied). Cells incubated with 200 nM bombesin (10 min) and untreated cells were assayed for LET-60-GTP content. C and D, indicated C. elegans strains were assayed for chemotaxis to IAA. Error bars are SEM. **P< 0.0001 compared with rgef-1(−/−) animals, Bonferroni’s t test; *P< 0.0001 compared with WT, Dunnett’s t test. Similar results were obtained in 3 experiments.

Figure 5. RGEF-1b couples odorants to chemotaxis by activating of LET-60 and MEK-2

A, N terminal amino acid sequences of LET-60 and RAP-1. Mutation of Gly¹² to Val and Ser¹⁷ to Asn create constitutively active and dominant negative variants, respectively. B, cells were co-transfected with plasmids encoding RGEF-1b and either FLAG-LET-60^G12V or FLAG-RAP-1^G12V. Cells were incubated with 50 nM PMA as indicated and assayed for LET-60-GTP or RAP-1-GTP content. C, D, E, F and G, chemotaxis assays were performed on the indicated strains. In E and G, animals were incubated at 25ºC for 24 h prior to assays to incapacitate temperature sensitive variants of SOS-1 and AGE-1, respectively. Error bars are SEM. *P< 0.001 compared to WT C. elegans, Dunnett’s t test; **P< 0.001 compared to rgef-1(−/−) animals, Bonferroni’s t test; # P<0.001 compared to WT animals, Bonferroni’s t test. Similar results were obtained in 3 experiments.

Figure 6. RGEF-1b mediates activation of MPK-1 by odorant, EGL-30, EGL-8 and DAG

Specified animals were incubated with buffer (control) or buffer containing BZ (1:10,000 dilution), PMA (300 nM) or BZ plus PMA for 3 min at 21°C, prior to fixation and permeabilization. Endogenous phospho-MPK-1 in AWC neurons was visualized by immunostaining (Supplemental Experimental Procedures). Fluorescence signals proportional to the abundance of di-phosphorylated (activated) MPK-1 in AWC neurons were recorded from 15 animals for each experimental condition and quantified. Mean values are presented; error bars are SEM. Similar results were obtained in 3 experiments.

Figure 7. A C1 domain mediates RGEF-1b translocation and activation; loss of C1 function disrupts chemotaxis

A, cells were transfected with RGEF-1b-GFP, RGEF-1b^P503G-GFP and bombesin receptor transgenes as indicated. After incubation with vehicle, 50 nM PMA (10 min) or 200 nM bombesin (5 min), cells were fixed and GFP fluorescence was recorded via microscopy. B, amino acid sequences of C1 domains from RGEF-1b and RasGRPs 1 and 3 are aligned. Amino acids that generate A and B loops of the binding pocket are indicated by red and blue bars, respectively. β strands are marked by green arrows. A Pro residue predicted to mediate high affinity binding of PMA and DAG is enclosed in a red rectangle. Positions of conserved Cys (C) and His (H) residues are shown below the sequences. C, cells were co-transfected with transgenes encoding FLAG-LET-60 and either RGEF-1b or RGEF-1b^P503G. Some cells also expressed bombesin receptor (lanes 5–8). Cells were incubated with 50 nM PMA or 200 nM bombesin and assayed for LET-60-GTP, LET-60 and RGEF-1b content by immunoblotting. D, cells were co-transfected with transgenes encoding FLAG-LET-60 and either RGEF-1b^P503G or WT RGEF-1b. Cells were incubated with indicated amounts of PMA (10 min) and assayed for LET-60-GTP content by immunoblotting. E, indicated animals were assayed for chemotaxis using odorants detected by AWC (IAA) and AWA (diacetyl) neurons. Error bars are SEM. **P< 0.001 compared to rgef-1(−/−) animals, Bonferroni’s t test. F, cells were co-transfected with transgenes encoding FLAG-LET-60 and either RGEF-1b-Tb5 or RGEF-1b^P503G-Tb5. “Tb5” is a C-terminal, ER targeting domain that was appended to WT and mutant GEFs. Cells were incubated with PMA and assayed for LET-60-GTP content as described above.

Figure 8. Ca²⁺ binding by EF hands and a PKC phosphorylation site are dispensable for RGEF-1b mediated chemotaxis

A, specified C. elegans strains were tested for chemotaxis to odorants detected by AWC (IAA) and AWA (diacetyl) neurons. B, HEK293 cells were transfected with empty vector or an HA-RGEF-1b or HA-RGEF-1b^S135A transgene. Cells were incubated with 50 nM PMA (15 min) as indicated, prior to lysis. Anti-HA IgGs (1.5 μg) were added to aliquots of detergent-soluble proteins (300 μg). Antigen-antibody complexes were analyzed by Western immunoblot assays. Blots were probed with IgGs directed against P-Ser¹³⁵-RGEF-1b (upper panel) or a non-phosphorylated RGEF-1b epitope (lower panel). C, the indicated C. elegans strains were assayed for chemotaxis. Error bars are SEM. **P< 0.001 compared to rgef-1(−/−) animals, Bonferroni’s t test. Similar results were obtained in 3 experiments.