STR-33, a Novel G Protein-coupled Receptor That Regulates Locomotion and Egg Laying in Caenorhabditis elegans*

Despite their predicted functional importance, most G protein-coupled receptors (GPCRs) in Caenorhabditis elegans have remained largely uncharacterized. Here, we focused on one GPCR, STR-33, encoded by the str-33 gene, which was discovered through a ligand-based screening procedure. To characterize STR-33 function, we performed UV-trimethylpsolaren mutagenesis and isolated an str-33-null mutant. The resulting mutant showed hypersinusoidal movement and a hyperactive egg-laying phenotype. Two types of egg laying-related mutations have been characterized: egg laying-deficient (Egl-d) and hyperactive egg laying (Egl-c). The defect responsible for the egg laying-deficient Egl-d phenotype is related to Gαq signaling, whereas that responsible for the opposite, hyperactive egg-laying Egl-c phenotype is related to Gαo signaling. We found that the hyperactive egg-laying defect of the str-33(ykp001) mutant is dependent on the G protein GOA-1/Gαo. Endogenous acetylcholine suppressed egg laying in C. elegans via a Gαo-signaling pathway by inhibiting serotonin biosynthesis or release from the hermaphrodite-specific neuron. Consistent with this, in vivo expression of the serotonin biosynthetic enzyme, TPH-1, was up-regulated in the str-33(ykp001) mutant. Taken together, these results suggest that the GPCR, STR-33, may be one of the neurotransmitter receptors that regulates locomotion and egg laying in C. elegans.

Signal transduction through G protein-coupled receptors (GPCRs) 4 is conserved from yeast to mammals and mediates cellular processes as diverse as odorant detection, hormonal signaling, vision, and drug responses (1). GPCRs, which share a conserved seven-transmembrane domain structure, transduce environmental stimuli into intracellular signals by coupling to heterotrimeric G proteins. The subfamily of G proteins to which the receptor couples defines the specific linkage to intracellular effectors and determines the nature of the intracellular signal. In Caenorhabditis elegans, GPCRs are involved in a wide range of physiological functions, including perception of odorants and regulation of behaviors, such as locomotion (2), pharyngeal pumping (3), and male mating (4).
C. elegans locomotion involves sinusoidal movements that reflect neurotransmission at neuromuscular junctions regulated by dorsal and ventral motor neurons. The GPCR ligand acetylcholine (ACh) is a major neurotransmitter in C. elegans excitatory signaling pathways involved in regulating biological behaviors, including locomotion (5,6), whereas GABA (␥-aminobutyric acid) mediates inhibitory influences on locomotion and other behaviors (7). Appropriate movement behavior requires a balance between ACh and GABA (8). For example, mutation of the cholinergic biosynthetic enzyme CHA-1 produces a severe, uncoordinated locomotor phenotype characterized by deep body-bending movement (5), whereas mutation of the UNC-25 protein, which encodes a GABA biosynthetic enzyme, produces the opposite phenotype (7).
Egg-laying behavior is primarily regulated by two classes of motor neurons: hermaphrodite-specific neurons (HSNs) and ventral cord (VC) motor neurons. Of these, HSNs play the more critical role (9). HSNs make neuromuscular junctions with vulval muscles and VC motor neurons, which are cholinergic neurons that inhibit egg laying by inhibiting HSNs. ACh signaling through a putative GPCR coupled to the G protein GOA-1/G␣ o in HSNs suppresses the egg-laying behavior of C. elegans by inhibiting biosynthesis or release of newly synthesized 5-hydroxytryptamine (5-HT) (6,10). Expression of the tph-1 gene, which encodes tryptophan hydroxylase-1, is strongly suppressed by GOA-1/G␣ o ; conversely, tph-1 expression is activated by EGL-30/G␣ q . Thus, signaling through G o suppresses egg laying by inhibiting 5-HT biosynthesis in HSNs, whereas G q -mediated signaling stimulates 5-HT biosynthesis and egg laying (9,10).
Recent studies suggest that some mechanosensory neurons also regulate egg laying by inhibiting HSNs as well as VC motor neurons (11), although the specific signaling pathways and proteins involved have not yet been identified. Although diverse biological processes in C. elegans are mediated by GPCRs, many of these GPCRs are orphan receptors, and the specific function of only a few has been clarified. In this study, we sought to identify the function of nematode-specific GPCRs for which reverse genetics has failed to identify orthologs in other species. Here, we show that the newly isolated str-33, a member of the str family and one of the uncharacterized nematode-specific GPCRs, mediates locomotion and egg-laying behavior of C. elegans in mechanosensory neurons.
Isolation of str-33 Deletion Mutants and Phenotypic Analyses-The mutant str-33 gene was isolated by reverse genetics screening using UV-trimethylpsolaren (TMP) mutagenesis. Briefly, mutations in C. elegans were induced by exposure to UV irradiation in the presence of TMP (Sigma-Aldrich). Mutants were screened using a nested PCR-based method and sib selection. The following primers were designed based on the predicted N-terminal sequences of str-33: 5Ј-GCA TTG TGA ATG CAG TGA TCA G-3Ј (outer sense) and 5Ј-GTG CAT TGA TGC ACC ACA AAA CAG CA-3Ј (outer antisense); 5Ј-GAC CAG ATC TAT AGT TCC GGT CCT GT-3Ј (inner sense) and 5Ј-GCA GGT ATG AAG CAG GCG TAA GGT TA-3Ј (inner antisense). A homozygous line containing a 917-bp deletion was isolated and out-crossed with the wild type for six generations to eliminate other mutations that might have been formed by UV-TMP random mutagenesis.
For life span assays, synchronized stage L4 animals were transferred to a fresh plate and scored every 2 days. Animals were scored as dead when they no longer responded to gentle prodding with a platinum wire. Life span is defined as the time (t) from the L2 larval stage (t ϭ 0) until the day worms were scored as dead. Death by internal hatching of progeny, desiccation on the wall of the plate, extruded vulva, or loss during handling was not scored in life span assays. The brood sizes of N2 and str-33(ykp001) hermaphrodites were determined by placing late L4 stage individual worms on OP50-seeded plates and allowing them to self-fertilize at 20°C. The P o parental animal was then transferred to a fresh plate every 24 h for the following 5 days. The total number of F1 progeny on the plates was counted.
Behavioral Assays-Body bending rate assays were performed as described previously (15). Briefly, stage-synchro-nized young adult hermaphrodites were transferred from OP50-NGM agar plates onto a fresh, foodless NGM plate. Observation began 1 min after transfer. The number of body bends in 20-s intervals was recorded separately for each of the five animals on the assay plates. Forward run duration was counted as the time (s) from the initiation of forward movement to the initiation of backward movement. Backward run duration was counted using the same method. Spontaneous coiling was counted as the time (s) from initiation of coiling to the resumption of movement. Movement pattern was analyzed by calculating the ratio of each movement for 7 min, as described previously (16). The movement ratio for each movement was measured by transferring synchronized young adult hermaphrodites to a fresh plate and recording the time for every movement over a 1-min interval and calculating the ratio (%) 5 min after loading.
Construction of GFP-Reporter Plasmids-A polymerase chain reaction (PCR)-amplified DNA fragment encompassing the full-length str-33 gene was ligated into the GFP cassette pPD114.108, kindly provided by A. Fire (Department of Pathology and Genetics, Stanford University School of Medicine). The Pstr-33::GFP construct was prepared by amplifying C. elegans genomic DNA by PCR to obtain a 5,064-bp promoter sequence of str-33 and ligating the resulting DNA fragment into BamHI/ SmaI restriction sites in the multicloning site of the pPD114.108 GFP vector. The primers used were 5Ј-GGA TCC CGG GCC ATA ATA ATT TCG GG-3Ј (sense) and 5Ј-CCC GGG GCG AAG GTT CAC CGT CAT-3Ј (antisense). The construct (100 ng/l) was co-injected with the rol-6(su1006) plasmid pRF4 (50 ng/l) into wild-type C. elegans, and transgenic worms were observed using an AXIO fluorescence microscope (Zeiss). The Ptph-1::GFP construct was prepared by PCR amplification of a 2,514-bp promoter sequence upstream of the open reading frame for tph-1 using C. elegans genomic DNA as a template. The resulting DNA fragment was ligated into HindIII/BamHI restriction sites in the multicloning site of the pPD95.79 GFP vector. The Ptph-1::GFP construct (100 ng/l) was injected into wild-type C. elegans and transferred to str-33(ykp001) mutants by mating to generate str-33(ykp001);Ex[Ptph-1::GFP] transgenic animals. Synchronized L4 stage animals were transferred to fresh NGM plates and incubated at 20°C. After 36 h, tph-1 gene expression was compared in animals with wild-type and str-33(ykp001) mutant backgrounds. Quantitative fluorescence microscopy was performed on a Zeiss AXIO fluorescent microscope, and image analysis was carried out using AxioVision software. Pmec-4::DsRED was created to confirm the expression loci of STR-33 as described previously (17). Briefly, a touch receptor neuron-specific construct was prepared by PCR, yielding a 1,140-bp promoter sequence of mec-4. The resulting DNA fragment was ligated into restriction sites within the multicloning site of the pPD95.79 DsRED vector, which had been produced by substitution of a GFP-encoding region into DsRED. This final construct was co-injected into animals with the Pstr-33::GFP reporter construct.
Egg-laying Assay-The average number of unlaid eggs and the percentage of early stage eggs laid were quantified as described previously (6,19). Staged adults were obtained by collecting late L4 animals and culturing at 20°C for 36 h. In the unlaid egg assay, 30 staged adults were individually dissolved in 10% sodium hypochlorite, and their eggs were counted (eggs survive this procedure because of their shells). Five staged adults were placed on an NGM agar plate and allowed to lay eggs for 1 h, after which adults were killed. Each egg was observed under an Olympus SZ-40 dissecting microscope and categorized as having eight or fewer cells. Eggs with eight or fewer cells were classified as "early stage." Egg-laying Stimulation Assay-The effects of drugs on egglaying behavior were determined by measuring the egg-laying rate on plates containing different concentrations of levamisole. Egg-laying rates were determined by placing five adult (L4) animals on each plate and, 36 h later, counting the number of eggs laid in 1 h. For egg-laying assays performed in liquid, stage-synchronized adults were placed into 20 l of M9 solution (20 mM KH 2 PO 4 , 40 mM Na 2 HPO 4 , 88 mM NaCl, 1 mM MgSO 4 ) without or with 5-HT creatinine sulfate (Sigma-Aldrich). The number of eggs laid in each well was counted after 90 min.
Paralysis Assay-The aldicarb treatment assay was done as previously described (20). Briefly, L4 stage larvae were transferred to plates seeded with E. coli OP50 and incubated at 20°C for 12 h. In all experiments, the time course of paralysis was measured by scoring the percentage of paralyzed animals on the plates every 20 min over a 3-h period. An animal was counted as paralyzed if no movement was observed after gentle prodding with a platinum wire. The inhibition of aldicarb effects by exogenous 5-HT was assayed by first incubating young adults for 2 h on plates containing 12.5 mM 5-HT creatinine sulfate (Sigma-Aldrich) and then transferring them to plates containing both 1 mM aldicarb and 12.5 mM 5-HT creatinine sulfate for assay of the time course of paralysis. To avoid bias, behavioral assays, including egg-laying, egg stimulation, and paralysis assays, were carried out by investigators blinded to genotype.

Isolation and Basic Characterization of the str-33
Single Deletion Mutant-During the course of studying daumone signaling-associated proteins, we discovered STR-33, a novel GPCRlike protein that lacks homology to known mammalian proteins 5 . To characterize this protein, we constructed a specific str-33 single mutant strain, termed str-33(ykp001), using UV-TMP random mutagenesis. DNA sequencing revealed that the str-33 mutant gene contained a 917-bp deletion in a region spanning from the upstream promoter (Ϫ57) to the second intronic region (ϩ860). We anticipated that this mutation would be functionally null because it lacked a start codon ( Fig. 1A and supplemental Fig. S1). We next confirmed the mutation in the str-33 gene locus using external and internal primer sets (Fig.  1B). Basic physiological features, including adult life span, total brood size, pharyngeal pumping rates, and defecation rhythm, were unchanged in str-33(ykp001) mutants (data not shown).
str-33(ykp001) Mutants Show an Abnormal Locomotor Phenotype-Unlike wild-type animals, which move forward through regular sinusoidal movements, str-33(ykp001) mutants displayed locomotor defects, exhibiting exaggerated bodybending locomotion that resulted in a steeper slope of sinusoidal movement (Fig. 1C). The bending rate of str-33(ykp001) mutant (13.9 Ϯ 1.59, n ϭ 15) was lower than that of wild-type animals (21.8 Ϯ 1.62, n ϭ 15; Fig. 1D). Moreover, the frequency of forward movement was lower, whereas that of backward movement was higher in str-33(ykp001) mutants. Adoption of a spontaneous coiling state, which was not observed in wild-type animals, was also detected frequently ( Fig. 1, D-F). The net result of these properties was that str-33(ykp001) mutants showed deep body-bending movement but slower forward progress.
str-33(ykp001) Mutants Exhibit a Hyperactive Egg-laying Phenotype-str-33(ykp001) mutants showed a hyperactive egglaying phenotype in which worms are unable to properly inhibit egg laying. Whereas wild-type animals retained an average of 12.8 Ϯ 1.8 eggs within their uterus and laid eggs that had reached the gastrulation stage, str-33(ykp001) mutants laid early stage eggs, resulting in the retention of fewer eggs (4.6 Ϯ 1.6; n ϭ 30; Fig. 2, A and B), regardless of adult reproductive stage (supplemental Fig. S2).
STR-33 Is Involved in Cholinergic Signaling in Mechanosensory Neurons-To examine STR-33 expression patterns, we injected a Pstr-33::GFP reporter construct into wild-type animals. Unexpectedly, str-33 was not expressed in HSNs or VC neurons, which are directly involved in egg-laying behavior (6, 10). However, it was detected in ALM and PLM mechanosensory neurons and head neurons (Fig. 3A). To confirm that FIGURE 1. Molecular characterization and abnormal locomotor phenotype of the str-33(ykp001) mutant. A, the str-33 gene is composed of four exons (boxes) and three introns. A 917-bp region from before the start codon to the second intron was deleted by UV-TMP mutagenesis. a and b represent the primer set used to confirm deletion of the str-33 gene. B, PCR bands obtained from single worm PCR analyses of wild-type and str-33(ykp001) mutants using external (a) and internal (b) primers. C, wild-type animals showed normal sinusoidal locomotion (a). str-33(ykp001) mutants displayed hypersinusoidal locomotion (b). The locomotory defect of str-33(ykp001) was rescued by introducing a str-33 genetic fragment with its own promoter (c) or a mechanosensory neuron-specific promoter (d). A stage-synchronized adult animal of each genotype was placed on a plate for 5 min, and its tracks were photographed. D, the bending rate of str-33(ykp001) mutants compared with wild-type animals was measured as described under "Experimental Procedures." Bending rates of the indicated genotypes are shown as the mean Ϯ S.E. (error bars). *, p Ͻ 0.001 versus wild-type animals; n ϭ 15 for each genotype and three independent experiments; Student's t test. E, movement-ratio analysis of each genotype. Error bars, S.E. F, movement-pattern analysis of each genotype. Error bars, S.E. *, p Ͻ 0.001 versus wild-type animals; n ϭ 15 for each genotype. N.D., not detected. expression of str-33 was located in PLM neurons, we co-injected a str-33 reporter construct with the mechanosensory neuron-specific marker, mec-4::Ds-RED, which revealed expression in ALM and PLM neurons (Fig. 3B). The primary function of ALM (L/R) and PLM (L/R) neurons is to respond to anterior and posterior touch, respectively, via interneurons (21). However, this unexpected result suggested that some mechanosensory neurons play a role in regulating egg laying, as has been noted previously (11), and implicated STR-33 in this function. To demonstrate that str-33 acts in these mechanosensory neurons, we performed a rescue experiment using a Pmec-4::str-33 fusion construct. As shown in Fig. 3C, microinjecting Pmec-4::str-33 into str-33(ykp001) mutants rescued the hyperactive egg-laying behavior of these mutants, increasing the number of unlaid eggs in uterus from 4.6 Ϯ 1.6 to 11.7 Ϯ 2.1 (n ϭ 15). This transgene was expressed in several mechanosensory neurons, including PLMs, which regulate egg laying by transiently inhibiting HSN activity (11), suggesting that STR-33 could function to regulate egg laying in PLM neurons.
Because ACh signaling is known to mediate egg-laying behavior, we tested whether the cholinergic agonist levamisole stimulated egg laying in str-33(ykp001) mutants. Levamisole stimulated egg laying at a low concentration but exerted no concentration-dependent effect and failed to restore egg laying to wild-type levels (Fig. 3E). These results suggest that STR-33 may be one of the GPCRs involved in cholinergic signaling that regulate egg-laying and locomotion in C. elegans. Given the fact that cholinergic neurotransmission is known to affect dauer larva formation (25), and several cholinergic mutants exhibit a dauer-  NOVEMBER 18, 2011 • VOLUME 286 • NUMBER 46 defective phenotype under certain assay conditions 6 , we anticipated that str-33(ykp001) mutants might show defects in dauer formation. However, str-33(ykp001) mutants entered the dauer state very well in standard dauer formation assays (26,27) and showed normal dauer recovery (data not shown), suggesting that STR-33 may not be directly involved in the dauer signaling pathway, although it could be related to cholinergic signaling.

STR-33 Suppresses 5-HT Biosynthesis in HSNs through
Inhibition of tph-1 Transcription-GOA-1 signaling is known to regulate egg laying by reducing the expression of the tph-1 gene in HSNs. Consistent with this, we found that tph-1 expression was increased in goa-1(n1134) mutants (data not shown), as previously reported (10). As a first step toward confirming whether the hyperactive egg-laying behavior of str-33(ykp001) mutants was due to changed tph-1 expression in HSNs, we transfected these animals with a tph-1::gfp construct. Similar to the results obtained in goa-1(n1134) mutants, tph-1 expression in HSNs was increased ϳ2-fold in str-33(ykp001) mutants, indicating that loss of STR-33 function up-regulates 5-HT biosynthesis in HSNs (Fig. 4). The endogenous serotonin-deficient mutant tph-1(mg280) exhibited an Egl-d phenotype in which more eggs were observed in the uterus compared to wild type, indicating that 5-HT is a critical regulator of egg laying in C. elegans (28). We constructed tph-1(mg280);str-33(ykp001) double mutants and confirmed the Egl-d phenotype of tph-1(mg280) (supplemental Fig. S5), suggesting that the egg-laying defect phenotype of str-33(ykp001) was dependent on serotonin signaling. Taken together, our results imply that STR-33 signaling strongly suppresses HSN 5-HT levels, which are dependent on GOA-1/G␣ o and are important for regulating egg laying in C. elegans.
STR-33 Mediates Egg-laying Behavior through Regulation of Endogenous 5-HT Levels-The observed increase in tph-1 expression in HSNs of str-33(ykp001) mutants suggests that these animals might release more 5-HT onto neuromuscular junctions, causing them to lay early stage eggs that would not normally be laid by wild-type animals. As the final neuromodulator in the egg-laying pathway, 5-HT, whether endogenously elevated or exogenously applied, would be expected to increase egg laying in C. elegans under conditions in which 5-HT levels are limiting. As expected, inclusion of 5-HT in hypertonic buffer, which normally suppresses egg laying (29), increased the number of eggs laid by wild-type C. elegans from fewer than 1 to 6.7 Ϯ 1.2 (n ϭ 50; Fig. 5, A and B). In contrast, str-33(ykp001) mutants laid more eggs in hypertonic buffer than wild-type animals and were resistant to exogenous 5-HT (Fig. 5, A and B). These results suggest that 5-HT levels in str-33(ykp001) mutants are already sufficiently elevated to maximally stimulate egg laying and suggest that wild-type STR-33 normally acts through inhibition of endogenous 5-HT biosynthesis to inhibit egg laying in C. elegans.
In wild-type animals, exogenous 5-HT treatment generally causes resistance to the paralytic effect of the acetylcholinesterase inhibitor, aldicarb, due to 5-HT-mediated inhibition of ACh release by motor neurons (20). To further characterize the role of STR-33 in the regulation of 5-HT synthesis in HSNs suggested by the above results, we assessed ACh release and accumulation in neuromuscular junctions by testing the aldicarb sensitivity of wild-type and str-33(ykp001) mutant worms pre-exposed to exogenous 5-HT. As expected, wild-type worms exhibited an aldicarb-resistant phenotype; however, 5-HT treatment did not reduce aldicarb sensitivity in str-33(ykp001) mutants (Fig. 5, C and  D). These results provide additional evidence that STR-33 is involved in regulating 5-HT synthesis, suggesting that the str-33(ykp001) mutation disrupts 5-HT-induced modulation of ACh neurotransmission at neuromuscular junctions as well as 5-HTmediated regulation of the egg-laying circuit.

DISCUSSION
Approximately 1,500 putative chemoreceptor genes or pseudogenes, or ϳ7% of all genes in C. elegans, are predicted to be members of the seven-transmembrane superfamily (30, 31). 6 Jeeyong Lee and Young-Ki Paik, unpublished data. The str family is one of the subfamilies of the Str superfamily, which is the largest superfamily, consisting of several subfamily genes, such as str, srh, srd, etc. Studies on the GPCR families revealed that the str family consists of about 200 genes and 75 pseudogenes (32). Of particular interest to us was STR-33, a previously uncharacterized nematode-specific GPCR identified in initial screening with ligand binding assays (data not shown). str-33(ykp001) mutants exhibited severely uncoordinated locomotion and a hyperactive egg-laying phenotype. Although other GPCRs are known to contribute to egg laying in C. elegans, ours is the first study to describe a GPCR involved in both locomotion and egg-laying behavior.
Numerous GPCRs expressed in sensory neurons transmit signals in response to environmental stimuli, and a variety of olfactory receptors in these neurons are able to sense volatile or water-soluble chemicals and thereby control attraction and avoidance behavior of C. elegans. However, we found that chemotactic responses to chemicals recognized by AWA olfactory neurons (diacetyl and pyrazine) and AWC olfactory neurons (benzaldehyde, isoamyl alcohol, butanone, and 2,3-pentanedione) were unchanged in str-33(ykp001) mutants (supplemental Fig. S6). Because the expression of str-33 was observed in mechanosensory neurons, such as ALMs and PLMs, we performed standard gentle touch assays using an eyelash hair, as described previously (33). str-33(ykp001) mutants showed a weak defect for sensing gentle touch, but their phenotype was modest compared with that of other touch-insensitive mutants, such as mec-4(e1339), mec-4(d), and mec-10(e1515) (supplemental Fig. S4). Moreover, reversal to nose touch and avoidance of high osmolarity solution (osmosensation response) (34) characteristic of wild-type animals were fully retained in str-33(ykp001) mutants (data not shown). These observations suggest that STR-33 does not function as a sensory GPCR to transduce environmental stimuli. The absence of sensory defects in str-33(ykp001) mutants may reflect the fact that STR-33 is not expressed in chemosensory or osmosensory neurons (Fig. 3A). It is also likely that dysfunction of STR-33 alone did not produce an overt sensory phenotype in response to mechanosensation, suggesting that modulation of egg laying is enacted independently or possibly downstream of mechanosensation. This would help explain why str-33(ykp001) mutants exhibited normal responses to mechanical stimuli, despite evidence that STR-33 is normally expressed in mechanosensory neurons, such as ALM and PLM. The fact that str-33(ykp001) mutants do not respond to treatment with the cholinergic agonist levamisole (Fig. 3E) suggests that STR-33 may be involved in cholinergic signaling. Whether STR-33 actually binds ACh and mediates ACh signaling remains to be determined. It is not clear how a response to the nicotinic cholinergic receptor agonist, levamisole, is compatible with STR-33 as a GPCR. However, previous studies reported that levamisole-activated nicotinic cholinergic receptors required G protein function (4). We suggest that the GPCR STR-33 could transmit a signal through its specific G protein that is required for nicotinic cholinergic receptors.
Among other receptors known to regulate egg laying in C. elegans is the muscarinic ACh receptor GAR-2, which signals through the GOA-1/G␣ o pathway in HSNs (6). Another GPCR expressed in HSNs that regulates egg laying is EGL-47 (13). Phe-Met-Arg-Phe-NH 2 (FMRFamide) neuropeptides acting on EGL-6 in HSNs synergize with ACh to modulate egg laying in C. elegans; EGL-6, which is composed of two GPCR isoforms, also signals through GOA-1/G␣ o (35). However, which molecules in PLM mechanosensory neurons regulate the egg-laying circuitry of HSNs has remained unknown. On the basis of our results, we suggest that STR-33 is a novel GPCR that regulates the egg-laying behavior of C. elegans through a mechanism different from that of other GPCRs known to regulate egg laying. Because STR-33 lacks sequence homology to other well known neurotransmitter receptors (e.g. GAR-2, GAR-3, SER-1, and EGL-47) (4, 6, 13), it is not clear which molecules bind STR-33 to transmit downstream signals.
Locomotion and egg-laying behavior are not regulated by a single signaling pathway but are affected by multiple pathways involving a variety of neurotransmitters and neuropeptides, including ACh, 5-HT, octopamine, and some Arg-Phe-NH 2 (RFamides) (6,29,35,36). Prominent among these are 5-HT and ACh. 5-HT is a monoamine neurotransmitter that contributes to the regulation of locomotion, defecation, and pharyngeal pumping in C. elegans (14,15,29), but perhaps the best studied role of 5-HT signaling is in the control of egg laying. 5-HT released from HSNs stimulates egg laying of C. elegans via egg-laying muscle. In HSNs, ACh signaling regulates egg laying by inhibiting transcription of tph-1, which encodes the ratelimiting enzyme for 5-HT biosynthesis (6,20). The G protein GOA-1/G␣ o is known to transmit this inhibitory signal. Accordingly, we predicted that STR-33 could be one of the neurotransmitter receptors that transmit ACh signaling through GOA-1. Consistent with this, tph-1 expression was FIGURE 6. Proposed functional models of the putative GPCR, STR-33. A, STR-33 in PLM mechanosensory neurons mediates regulatory signaling that suppresses egg laying by inhibiting GOA-1/G␣ o signaling-dependent activity in HSNs. B, GOA-1/G␣ o signaling also might regulate egg-laying behavior in PLM mechanosensory neurons, which mediate signaling directly with STR-33.