Egg laying is a simple behavior in C. elegans that extensive genetic studies have shown to be under the control of two heterotrimeric G proteins. The neurotransmitter receptors that act through these G proteins are not known.
egl-47 was identified by two dominant mutations that block egg laying, phenocopying ablation of the HSN neurons that stimulate egg laying (1). The HSN neurons are morphologically normal in
egl-47 mutants, suggesting the function, not the development of these neurons has been blocked. While the G proteins that control egg laying also affect many other behaviors,
egl-47 appears to only affect egg laying. We mapped
egl-47 to a 97 kb interval and found that a PCR product from within this interval generated from
egl-47 (gf) was able to block egg laying when transformed into wild-type worms. One gene in this PCR product contained mutations in the
egl-47(gf) mutants, as well as second-site mutations in two intragenic
egl-47 revertents, identifying the gene C50H2.2 as the
egl-47 gene. Analysis of
egl-47 mRNA revealed that
egl-47 uses two promoters and generates transcripts encoding proteins with different N termini and a common C terminus. The common C terminus appears to contain seven transmembrane spanning domains, suggesting
egl-47 encodes two G protein-coupled receptors with different extracellular N-terminal domains that may bind different ligands. Only a small percentage of G protein coupled receptors have known ligands, and EGL-47 is not similar enough to any of those to predict its ligand. The fact that EGL-47 appears to be a receptor that blocks egg laying suggests it may couple to GOA-1, the G protein that blocks egg laying.
goa-1(null);
egl-47(gf) double mutants show hyperactive egg laying, exactly like the
goa-1(null) single mutant, consistent with this model. Both
egl-47 (gf) mutants have an A to V change in a putative transmembrane domain of the EGL-47 protein. Mutations in analogous positions in several characterized receptors result in constitutive receptor activation. Deletion of the
egl-47gene causes no obvious phenotypic defects, suggesting that the normal function of EGL-47 may be redundant or subtle, although no close
egl-47 homologs are evident in the C. elegans genome sequence. The
egl-47 promoter was used to express full-length EGL-47::GFP fusion protein in C. elegans. Punctate fluorescence was seen in the HSNs, apparently at synapses these neurons make in the egg-laying system. A few other puncta were visible in the head. These observations support the model that EGL-47 couples to the G protein GOA-1 in the HSN to inhibit egg laying. They also suggest why EGL-47, being expressed in only a few cells, affects fewer behaviors than GOA-1, which is widely expressed.