Cells were cotransfected with RGEF-1b and either FLAG-tagged LET-60 or RAP-1 transgenes. After incubation with 50 nM PMA or vehicle for 15 min, cells were lysed and amounts of LET-60-GTP or RAP-1-GTP were assayed by western immunoblot analysis. RGEF-1b promoted modest accumulation of LET-60-GTP in untreated cells (Figure 1A, lane 3).

In contrast, RGEF-1b activity increased ∼6-fold when cells were incubated with PMA (Figure 1A, lane 4). If RGEF-1b has a functional C1 domain, it will be regulated by endogenous DAG. Cells were transfected with bombesin receptor, RGEF-1b and FLAG-LET-60 transgenes. Bombesin receptor, which has seven transmembrane domains and couples with heterotrimeric Gq protein, promotes DAG production KU-57788 mouse (Feng et al., 2007). When bombesin peptide

binds, the receptor elicits PLCβ activation via Gαq-GTP. PLCβ generates DAG and IP3 by cleaving PI4,5P2 in membranes. Incubation of cells with bombesin increased RGEF-1b-mediated LET-60 activation ∼4-fold (Figure 1B, lanes 3 and 4). Stimulation by both bombesin and PMA (a DAG surrogate) suggests that DAG is a major regulator of RGEF-1b catalytic activity. Modest basal and PMA-stimulated accumulation of RAP-1-GTP was evident in HEK293 cells lacking Selleck Selisistat RGEF-1b because of endogenous GEFs (Figure 1C, lanes 1 and 2). Expression of RGEF-1b elicited increased accumulation of RAP-1-GTP in the absence of stimuli (Figure 1C, lane 3). Moreover, PMA further enhanced RGEF-1b catalyzed loading of GTP onto RAP-1 (Figure 1C, lane 4). Thus, LET-60 and RAP-1 are RGEF-1b substrates. A fragment of genomic DNA (2670 bp) that precedes exon 1 of the rgef-1 gene was amplified by PCR. This DNA, which contains promoter-enhancer elements, was inserted upstream from a green fluorescent protein (GFP) reporter gene in a C. elegans expression plasmid (pPD 95.77). Animals stably expressing the rgef-1::GFP transgene were generated by microinjection. Cells producing GFP were identified by fluorescence microscopy and reference to the WORMATLAS anatomy database. rgef-1 promoter activity was evident in a high proportion of neurons ( Figures 2A and 2C) in four independently Terminal deoxynucleotidyl transferase isolated

strains. GFP was not detected in nonneuronal cells. Terminal divisions and differentiation of neurons were completed before rgef-1 promoter activity was switched on during late embryonic development ( Figure 2E). Panneuronal GFP fluorescence was sustained from the end of embryogenesis (hatching) through adulthood. We characterized a gene deletion mutant (rgef-1(ok675)) acquired from the C. elegans Knockout Consortium. Gene fragments were amplified by PCR ( Figure S2). DNA sequencing revealed that nucleotides 1493–2594 were deleted from the rgef-1 gene. This eliminated exons 5–7 and part of exon 8 ( Figures S1A and S2), which encode the RGEF-1b catalytic domain. Splicing of exon 4 to exon 9 would yield a mutant protein lacking GTP exchange activity. Thus, the disrupted rgef-1 gene is a null mutant.