Elimination of catalytic activity or a moderate reduction in DAG binding affinity of the C1 domain disrupted RGEF-1b function in vivo. Chemotaxis was unaffected by mutations that inactivated Ca2+-binding EF hands or a conserved PKC phosphorylation site. Thus, RGEF-1b links external stimuli (odorants) and internal DAG to the control of behavior (chemotaxis) by differentially activating the LET-60-MPK-1 cascade in AWC neurons. A single gene, named rgef-1 (Ras GTP/GDP exchange factor-1), was identified by searching C. elegans genome, EST, and protein databases for RasGRP homologs. Cosmid F25B3 (GenBank)

contains the rgef-1 gene (4893 bp) and flanking DNA. RGEF-1 cDNA and protein Sirolimus price were not Imatinib supplier previously characterized. Thus, RGEF-1 cDNAs were amplified by RT-PCR and cloned into a mammalian expression vector pCDNA3.1 (see Supplemental Experimental Procedures available online). Sequencing revealed that alternative splicing generated two cDNAs as diagrammed in Figure S1A (available online). RGEF-1a and RGEF-1b ( Figure S1E) cDNAs encode proteins composed of 654 and 620 amino acids, respectively ( Figure S1B).The isoforms are 98% identical and diverge only in a segment of unknown function that links a C1 domain to

the C-terminal region. Quantitative real time PCR (qR-PCR) analysis disclosed that RGEF-1b mRNA accounts for >95% of rgef-1 gene transcripts ( Figure S1C). Thus, studies were focused on RGEF-1b. The predicted RGEF-1b protein (Mr ∼70,000) contains structural (REM),

catalytic (GEF), and regulatory (two EF hands and C1) domains that share substantial amino acid sequence identity and similarity with corresponding domains in human RasGRPs (Figure S1D). By analogy, the RGEF-1b GEF domain will promote opening of the GTP/GDP binding site in small G-proteins (Bos et al., 2007). Guanine nucleotides will equilibrate between G protein and cytoplasm. Since the GTP:GDP ratio is ∼10, the net result is exchange of GTP for GDP. EF-hands, which contain five Asp or Glu residues, often regulate enzymatic activity by binding Ca2+ (Gifford et al., 2007). The C1 domain is predicted to mediate RGEF-1b translocation by binding membrane associated DAG (Hurley and Misra, 2000). whatever Functions of RasGRP REM domains are unknown. Locations of domains along the RGEF-1b and RasGRP polypeptides are also preserved (Figure S1D). RGEF-1b translocates to membranes and catalyzes loading of GTP onto LET-60 in PMA-treated cells (see below). Together, these properties show that RGEF-1b is a new, but prototypical RasGRP. In C. elegans, unique genes encode a 21 kDa Ras homolog (LET-60) and a 21 kDa Rap1 polypeptide (RAP-1). LET-60 and RAP-1 cDNAs were inserted into a modified pCDNA3.1 plasmid that appends an N-terminal Flag epitope tag to encoded proteins. If RGEF-1b is a RasGRP, it will translocate to membranes and mediate loading of GTP onto colocalized LET-60 or RAP-1.