Two chemoreceptors mediate developmental effects of dauer pheromone in C. elegans

Abstract

Intraspecific chemical communication is mediated by signals called pheromones. Caenorhabditis elegans secretes a mixture of small molecules (collectively termed dauer pheromone) that regulates entry into the alternate dauer larval stage and also modulates adult behavior via as yet unknown receptors. Here, we identify two heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) that mediate dauer formation in response to a subset of dauer pheromone components. The SRBC-64 and SRBC-66 GPCRs are members of the large Caenorhabditis-specific SRBC subfamily and are expressed in the ASK chemosensory neurons, which are required for pheromone-induced dauer formation. Expression of both, but not each receptor alone, confers pheromone-mediated effects on heterologous cells. Identification of dauer pheromone receptors will allow a better understanding of the signaling cascades that transduce the context-dependent effects of ecologically important chemical signals.

Fig. 1

srbc-64, srbc-66 and gpa-3 mutants exhibit defects in dauer formation in response to pheromone components. (A-D) Percent dauers formed by animals of the indicated genotypes when grown in the presence of different concentrations of ascarosides. Error bars are the s.e.m. * and ** indicate values that are different from the corresponding wild-type values at P<0.01 and 0.001, respectively. (E) Percent dauers formed in response to 600 nM C6. Ex refers to animals carrying the indicated expression constructs as extrachromosomal arrays. # indicates values that are different at P<0.01 between the values indicated by brackets. Shown are the averages from at least 4 independent experiments with 50-100 animals each. For each experiment, all strains were assayed in parallel.

Fig. 2

Mutations in srbc-64 and -66 decrease pheromone-mediated downregulation of gene expression. (A) Relative levels of str-3p::gfp expression in adult wild-type animals grown in the presence of the indicated concentrations of each ascaroside. str-3p::gfp expression levels in ethanol were assigned an arbitrary value (black line), and fluorescence levels in experimental conditions were quantified relative to this value. n > 30 for each; 2 independent experiments. (B) Expression of str-3p::gfp in wild-type and srbc-64 mutant adult animals grown in the presence of 6 μM C6. Lateral view; scale – 10 μm. Adult animals were examined using the same exposure time. (C) Relative levels of str-3p::gfp fluorescence in adult wild-type and srbc-64 mutant animals, grown in the presence of C6. n > 50 for each; 2 independent experiments. (D) Shown is the ratio of gfp fluorescence in animals of the indicated genotypes to gfp levels in wild-type animals grown on the same plates in the presence of ethanol, crude pheromone (1 μl/ml), or ascarosides (C3, C6, C9 - 6 μM; C7 - 60 μM). Adult animals were examined in all cases. n > 30 animals each; 2 independent experiments. Error bars are the s.e.m. * indicate values that are different from wild-type at P<0.05 in all panels (with the exception of (D), where values are compared to those of animals grown in ethanol).

Fig. 3

srbc-64 and srbc-66 are expressed in, and act via the GPA-3 Gα protein in the ASK chemosensory neurons. (A) Full-length GFP-tagged SRBC-64 and -66 proteins are localized to the cilia of the ASK neurons. Lateral view; scale - 10 μm. Images were acquired using confocal microscopy. (B-C) Percent dauers formed by animals of the indicated genotypes in response to C6 (C) and C3 (D). sra-9 regulatory sequences drive expression specifically in the ASK neurons (16). Shown are the averages from at least 4 independent experiments with 50-100 animals each. For each experiment, all strains were assayed in parallel. Error bars are the s.e.m. (D-E) Percent dauers formed when animals of the indicated genotypes were grown in the presence of C6. Shown are the averages from at least 4 independent experiments with 50-100 animals each. # and ## indicate values that are different at P<0.01 and <0.001 respectively, between the values indicated by brackets. * and ** indicate values that are different at P<0.01 and 0.001 from corresponding values of wild-type animals.

Fig. 4

Ascarosides inhibit forskolin-induced cAMP production in HEK293 cells co-expressing SRBC-64 and SRBC-66. (A-C) HEK293 cells were transiently transfected with the indicated expression constructs, and stimulated with 5 μM forskolin in the absence or presence of the indicated compounds. pME18S is a control vector. (C) Cells were co-transfected with SRBC-64 and SRBC-66. Representative data from one of four independent experiments are shown. Each data point is the average of three to eleven trials. † and †† indicate values that are different at P<0.05 and P<0.01, respectively, between the values indicated by brackets.