Multiple Subthreshold GPCR Signals Combined by the G-Proteins Gαq and Gαs Activate the Caenorhabditis elegans Egg-Laying Muscles.

Individual neurons or muscle cells express many G-protein-coupled receptors (GPCRs) for neurotransmitters and neuropeptides, yet it remains unclear how cells integrate multiple GPCR signals that all must activate the same few G-proteins. We analyzed this issue in the Caenorhabditis elegans egg-laying system, where multiple GPCRs on muscle cells promote contraction and egg laying. We genetically manipulated individual GPCRs and G-proteins specifically in these muscle cells within intact animals and then measured egg laying and muscle calcium activity. Two serotonin GPCRs on the muscle cells, Gαq-coupled SER-1 and Gαs-coupled SER-7, together promote egg laying in response to serotonin. We found that signals produced by either SER-1/Gαq or SER-7/Gαs alone have little effect, but these two subthreshold signals combine to activate egg laying. We then transgenically expressed natural or designer GPCRs in the muscle cells and found that their subthreshold signals can also combine to induce muscle activity. However, artificially inducing strong signaling through just one of these GPCRs can be sufficient to induce egg laying. Knocking down Gαq and Gαs in the egg-laying muscle cells induced egg-laying defects that were stronger than those of a SER-1/SER-7 double knockout, indicating that additional endogenous GPCRs also activate the muscle cells. These results show that in the egg-laying muscles multiple GPCRs for serotonin and other signals each produce weak effects that individually do not result in strong behavioral outcomes. However, they combine to produce sufficient levels of Gαq and Gαs signaling to promote muscle activity and egg laying.SIGNIFICANCE STATEMENT How can neurons and other cells gather multiple independent pieces of information from the soup of chemical signals in their environment and compute an appropriate response? Most cells express >20 GPCRs that each receive one signal and transmit that information through three main types of G-proteins. We analyzed how this machinery generates responses by studying the egg-laying system of C. elegans, where serotonin and multiple other signals act through GPCRs on the egg-laying muscles to promote muscle activity and egg laying. We found that individual GPCRs within an intact animal each generate effects too weak to activate egg laying. However, combined signaling from multiple GPCR types reaches a threshold capable of activating the muscle cells.

Serotonin and neuropeptides are both released by the HSN command neuron to initiate Caenorhabditis elegans egg laying.

Neurons typically release both a small-molecule neurotransmitter and one or more neuropeptides, but how these two types of signal from the same neuron might act together remains largely obscure. For example, serotonergic neurons in mammalian brain express the neuropeptide Substance P, but it is unclear how this co-released neuropeptide might modulate serotonin signaling. We studied this issue in C. elegans, in which all serotonergic neurons express the neuropeptide NLP-3. The serotonergic Hermaphrodite Specific Neurons (HSNs) are command motor neurons within the egg-laying circuit which have been shown to release serotonin to initiate egg-laying behavior. We found that egg-laying defects in animals lacking serotonin were far milder than in animals lacking HSNs, suggesting that HSNs must release other signal(s) in addition to serotonin to stimulate egg laying. While null mutants for nlp-3 had only mild egg-laying defects, animals lacking both serotonin and NLP-3 had severe defects, similar to those of animals lacking HSNs. Optogenetic activation of HSNs induced egg laying in wild-type animals, and in mutant animals lacking either serotonin or NLP-3, but failed to induce egg laying in animals lacking both. We recorded calcium activity in the egg-laying muscles of animals lacking either serotonin, NLP-3, or both. The single mutants, and to a greater extent the double mutant, showed muscle activity that was uncoordinated and unable to expel eggs. Specifically, the vm2 muscles cells, which are direct postsynaptic targets of the HSN, failed to contract simultaneously with other egg-laying muscle cells. Our results show that the HSN neurons use serotonin and the neuropeptide NLP-3 as partially redundant co-transmitters that together stimulate and coordinate activity of the target cells onto which they are released.

Cellular Expression and Functional Roles of All 26 Neurotransmitter GPCRs in the C. elegans Egg-Laying Circuit.

Maps of the synapses made and neurotransmitters released by all neurons in model systems, such as Caenorhabditis elegans have left still unresolved how neural circuits integrate and respond to neurotransmitter signals. Using the egg-laying circuit of C. elegans as a model, we mapped which cells express each of the 26 neurotransmitter GPCRs of this organism and also genetically analyzed the functions of all 26 GPCRs. We found that individual neurons express many distinct receptors, epithelial cells often express neurotransmitter receptors, and receptors are often positioned to receive extrasynaptic signals. Receptor knockouts reveal few egg-laying defects under standard laboratory conditions, suggesting that the receptors function redundantly or regulate egg-laying only in specific conditions; however, increasing receptor signaling through overexpression more efficiently reveals receptor functions. This map of neurotransmitter GPCR expression and function in the egg-laying circuit provides a model for understanding GPCR signaling in other neural circuits.SIGNIFICANCE STATEMENT Neurotransmitters signal through GPCRs to modulate activity of neurons, and changes in such signaling can underlie conditions such as depression and Parkinson's disease. To determine how neurotransmitter GPCRs together help regulate function of a neural circuit, we analyzed the simple egg-laying circuit in the model organism C. elegans We identified all the cells that express every neurotransmitter GPCR and genetically analyzed how each GPCR affects the behavior the circuit produces. We found that many neurotransmitter GPCRs are expressed in each neuron, that neurons also appear to use these receptors to communicate with other cell types, and that GPCRs appear to often act redundantly or only under specific conditions to regulate circuit function.

RNA ligation in neurons by RtcB inhibits axon regeneration.

Activity of the RNA ligase RtcB has only two known functions: tRNA ligation after intron removal and XBP1 mRNA ligation during activation of the unfolded protein response. Here, we show that RtcB acts in neurons to inhibit axon regeneration after nerve injury. This function of RtcB is independent of its basal activities in tRNA ligation and the unfolded protein response. Furthermore, inhibition of axon regeneration is independent of the RtcB cofactor archease. Finally, RtcB is enriched at axon termini after nerve injury. Our data indicate that neurons have co-opted an ancient RNA modification mechanism to regulate specific and dynamic functions and identify neuronal RtcB activity as a critical regulator of neuronal growth potential.

Evolutionary Conservation of a GPCR-Independent Mechanism of Trimeric G Protein Activation.

Trimeric G protein signaling is a fundamental mechanism of cellular communication in eukaryotes. The core of this mechanism consists of activation of G proteins by the guanine-nucleotide exchange factor (GEF) activity of G protein coupled receptors. However, the duration and amplitude of G protein-mediated signaling are controlled by a complex network of accessory proteins that appeared and diversified during evolution. Among them, nonreceptor proteins with GEF activity are the least characterized. We recently found that proteins of the ccdc88 family possess a Gα-binding and activating (GBA) motif that confers GEF activity and regulates mammalian cell behavior. A sequence similarity-based search revealed that ccdc88 genes are highly conserved across metazoa but the GBA motif is absent in most invertebrates. This prompted us to investigate whether the GBA motif is present in other nonreceptor proteins in invertebrates. An unbiased bioinformatics search in Caenorhabditis elegans identified GBAS-1 (GBA and SPK domain containing-1) as a GBA motif-containing protein with homologs only in closely related worm species. We demonstrate that GBAS-1 has GEF activity for the nematode G protein GOA-1 and that the two proteins are coexpressed in many cells of living worms. Furthermore, we show that GBAS-1 can activate mammalian Gα-subunits and provide structural insights into the evolutionarily conserved determinants of the GBA-G protein interface. These results demonstrate that the GBA motif is a functional GEF module conserved among highly divergent proteins across evolution, indicating that the GBA-Gα binding mode is strongly constrained under selective pressure to mediate receptor-independent G protein activation in metazoans.

Two types of chloride transporters are required for GABA(A) receptor-mediated inhibition in C. elegans.

Chloride influx through GABA-gated Cl(-) channels, the principal mechanism for inhibiting neural activity in the brain, requires a Cl(-) gradient established in part by K(+)-Cl(-) cotransporters (KCCs). We screened for Caenorhabditis elegans mutants defective for inhibitory neurotransmission and identified mutations in ABTS-1, a Na(+)-driven Cl(-)-HCO(3)(-) exchanger that extrudes chloride from cells, like KCC-2, but also alkalinizes them. While animals lacking ABTS-1 or the K(+)-Cl(-) cotransporter KCC-2 display only mild behavioural defects, animals lacking both Cl(-) extruders are paralyzed. This is apparently due to severe disruption of the cellular Cl(-) gradient such that Cl(-) flow through GABA-gated channels is reversed and excites rather than inhibits cells. Neuronal expression of both transporters is upregulated during synapse development, and ABTS-1 expression further increases in KCC-2 mutants, suggesting regulation of these transporters is coordinated to control the cellular Cl(-) gradient. Our results show that Na(+)-driven Cl(-)-HCO(3)(-) exchangers function with KCCs in generating the cellular chloride gradient and suggest a mechanism for the close tie between pH and excitability in the brain.

Postsynaptic ERG potassium channels limit muscle excitability to allow distinct egg-laying behavior states in Caenorhabditis elegans.

Caenorhabditis elegans regulates egg laying by alternating between an inactive phase and a serotonin-triggered active phase. We found that the conserved ERG [ether-a-go-go (EAG) related gene] potassium channel UNC-103 enables this two-state behavior by limiting excitability of the egg-laying muscles. Using both high-speed video recording and calcium imaging of egg-laying muscles in behaving animals, we found that the muscles appear to be excited at a particular phase of each locomotor body bend. During the inactive phase, this rhythmic excitation infrequently evokes calcium transients or contraction of the egg-laying muscles. During the serotonin-triggered active phase, however, these muscles are more excitable and each body bend is accompanied by a calcium transient that drives twitching or full contraction of the egg-laying muscles. We found that ERG-null mutants lay eggs too frequently, and that ERG function is necessary and sufficient in the egg-laying muscles to limit egg laying. ERG K(+) channels localize to postsynaptic sites in the egg-laying muscle, and mutants lacking ERG have more frequent calcium transients and contractions of the egg-laying muscles even during the inactive phase. Thus ERG channels set postsynaptic excitability at a threshold so that further adjustments of excitability by serotonin generate two distinct behavioral states.

AGS-3 alters Caenorhabditis elegans behavior after food deprivation via RIC-8 activation of the neural G protein G αo.

Proteins containing the G protein regulator (GPR) domain bind the major neural G protein Gα(o) in vitro. However, the biological functions of GPR proteins in neurons remain undefined, and based on the in vitro activities of GPR proteins it is unclear whether these proteins activate or inhibit G protein signaling in vivo. We found that the conserved GPR domain protein AGS-3 activates Gα(o) signaling in vivo to allow Caenorhabditis elegans to alter several behaviors after food deprivation, apparently so that the animals can more effectively seek food. AGS-3 undergoes a progressive change in its biochemical fractionation upon food deprivation, suggesting that effects of food deprivation are mediated by modifying this protein. We analyzed one C. elegans food-regulated behavior in depth; AGS-3 activates Gα(o) in the ASH chemosensory neurons to allow food-deprived animals to delay response to the aversive stimulus octanol. Genetic epistasis experiments show the following: (1) AGS-3 and the guanine nucleotide exchange factor RIC-8 act in ASH in a mutually dependent fashion to activate Gα(o); (2) this activation requires interaction of the GPR domains of AGS-3 with Gα(o); and (3) Gα(o)-GTP is ultimately the signaling molecule that acts in ASH to delay octanol response. These results identify a biological role for AGS-3 in response to food deprivation and indicate the mechanism for its activation of Gα(o) signaling in vivo.

Using NeuroPAL Multicolor Fluorescence Labeling to Identify Neurons in C. elegans.

NeuroPAL (Neuronal Polychromatic Atlas of Landmarks) is a recently developed transgene that labels each of the 118 classes of neurons in C. elegans with various combinations of four fluorescent proteins. This neuron-type-specific labeling helps identify neurons that could otherwise be confused with neighboring neurons. Neuron identification enables researchers to combine new data that they generate on a C. elegans neuron with existing datasets on that same neuron, such as its synaptic connections, neurotransmitters, and transcriptome. An impediment to using NeuroPAL, however, is overcoming the steep learning curve for interpreting three-dimensional (3D) fluorescence images of crowded neural ganglia within which different neurons may be similarly colored, some neurons are only very faintly labeled, and the positions of some neurons are variable. Here, we provide protocols that allow researchers to learn to accurately identify neurons within 3D images of NeuroPAL-labeled animals. We provide 3D reference images that illustrate NeuroPAL labeling of each body region, and additional 3D images as training exercises to learn to accurately carry out C. elegans neuron identifications. We also provide tools to annotate images in 3D, and suggest that such 3D annotated images should be the standard for documenting C. elegans neuron identifications for publication. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Using Imaris software to view and annotate images of NeuroPAL-labeled animals in 3D Alternate Protocol: Using FIJI/ImageJ software to view and annotate images of NeuroPAL-labeled animals in 3D Basic Protocol 2: Identifying tail neurons-an introduction to identifying neurons Basic Protocol 3: Identifying midbody neurons Basic Protocol 4: Identifying anterior head neurons Basic Protocol 5: Identifying posterior head neurons Basic Protocol 6: Identifying ventral head and retrovesicular ganglion neurons.

C. elegans G protein regulator RGS-3 controls sensitivity to sensory stimuli.

Signal transduction through heterotrimeric G proteins is critical for sensory response across species. Regulator of G protein signaling (RGS) proteins are negative regulators of signal transduction. Herein we describe a role for C. elegans RGS-3 in the regulation of sensory behaviors. rgs-3 mutant animals fail to respond to intense sensory stimuli but respond normally to low concentrations of specific odorants. We find that loss of RGS-3 leads to aberrantly increased G protein-coupled calcium signaling but decreased synaptic output, ultimately leading to behavioral defects. Thus, rgs-3 responses are restored by decreasing G protein-coupled signal transduction, either genetically or by exogenous dopamine, by expressing a calcium-binding protein to buffer calcium levels in sensory neurons or by enhancing glutamatergic synaptic transmission from sensory neurons. Therefore, while RGS proteins generally act to downregulate signaling, loss of a specific RGS protein in sensory neurons can lead to defective responses to external stimuli.

A conserved protein interaction interface on the type 5 G protein beta subunit controls proteolytic stability and activity of R7 family regulator of G protein signaling proteins.

Regulators of G protein signaling (RGS) proteins of the R7 subfamily limit signaling by neurotransmitters in the brain and by light in the retina. They form obligate complexes with the Gß5 protein that are subject to proteolysis to control their abundance and alter signaling. The mechanisms that regulate this proteolysis, however, remain unclear. We used genetic screens to find mutations in Gß5 that selectively destabilize one of the R7 RGS proteins in Caenorhabditis elegans. These mutations cluster at the binding interface between Gß5 and the N terminus of R7 RGS proteins. Equivalent mutations within mammalian Gß5 allowed the interface to still bind the N-terminal DEP domain of R7 RGS proteins, and mutant Gß5-R7 RGS complexes initially formed in cells but were then rapidly degraded by proteolysis. Molecular dynamics simulations suggest the mutations weaken the Gß5-DEP interface, thus promoting dynamic opening of the complex to expose determinants of proteolysis known to exist on the DEP domain. We propose that conformational rearrangements at the Gß5-DEP interface are key to controlling the stability of R7 RGS protein complexes.

The neural G protein Gαo tagged with GFP at an internal loop is functional in Caenorhabditis elegans.

Gαo is the alpha subunit of the major heterotrimeric G protein in neurons and mediates signaling by every known neurotransmitter, yet the signaling mechanisms activated by Gαo remain to be fully elucidated. Genetic analysis in Caenorhabditis elegans has shown that Gαo signaling inhibits neuronal activity and neurotransmitter release, but studies of the molecular mechanisms underlying these effects have been limited by lack of tools to complement genetic studies with other experimental approaches. Here, we demonstrate that inserting the green fluorescent protein (GFP) into an internal loop of the Gαo protein results in a tagged protein that is functional in vivo and that facilitates cell biological and biochemical studies of Gαo. Transgenic expression of Gαo-GFP rescues the defects caused by loss of endogenous Gαo in assays of egg laying and locomotion behaviors. Defects in body morphology caused by loss of Gαo are also rescued by Gαo-GFP. The Gαo-GFP protein is localized to the plasma membrane of neurons, mimicking localization of endogenous Gαo. Using GFP as an epitope tag, Gαo-GFP can be immunoprecipitated from C. elegans lysates to purify Gαo protein complexes. The Gαo-GFP transgene reported in this study enables studies involving in vivo localization and biochemical purification of Gαo to compliment the already well-developed genetic analysis of Gαo signaling.

The potassium chloride cotransporter KCC-2 coordinates development of inhibitory neurotransmission and synapse structure in Caenorhabditis elegans.

Chloride influx through GABA-gated chloride channels, the primary mechanism by which neural activity is inhibited in the adult mammalian brain, depends on chloride gradients established by the potassium chloride cotransporter KCC2. We used a genetic screen to identify genes important for inhibition of the hermaphrodite-specific motor neurons (HSNs) that stimulate Caenorhabditis elegans egg-laying behavior and discovered mutations in a potassium chloride cotransporter, kcc-2. Functional analysis indicates that, like mammalian KCCs, C. elegans KCC-2 transports chloride, is activated by hypotonic conditions, and is inhibited by the loop diuretic furosemide. KCC-2 appears to establish chloride gradients required for the inhibitory effects of GABA-gated and serotonin-gated chloride channels on C. elegans behavior. In the absence of KCC-2, chloride gradients appear to be altered in neurons and muscles such that normally inhibitory signals become excitatory. kcc-2 is transcriptionally upregulated in the HSN neurons during synapse development. Loss of KCC-2 produces a decrease in the synaptic vesicle population within mature HSN synapses, which apparently compensates for a lack of HSN inhibition, resulting in normal egg-laying behavior. Thus, KCC-2 coordinates the development of inhibitory neurotransmission with synapse maturation to produce mature neural circuits with appropriate activity levels.

Regulation of serotonin biosynthesis by the G proteins Galphao and Galphaq controls serotonin signaling in Caenorhabditis elegans.

To analyze mechanisms that modulate serotonin signaling, we investigated how Caenorhabditis elegans regulates the function of serotonergic motor neurons that stimulate egg-laying behavior. Egg laying is inhibited by the G protein Galphao and activated by the G protein Galphaq. We found that Galphao and Galphaq act directly in the serotonergic HSN motor neurons to control egg laying. There, the G proteins had opposing effects on transcription of the tryptophan hydroxylase gene tph-1, which encodes the rate-limiting enzyme for serotonin biosynthesis. Antiserotonin staining confirmed that Galphao and Galphaq antagonistically affect serotonin levels. Altering tph-1 gene dosage showed that small changes in tph-1 expression were sufficient to affect egg-laying behavior. Epistasis experiments showed that signaling through the G proteins has additional tph-1-independent effects. Our results indicate that (1) serotonin signaling is regulated by modulating serotonin biosynthesis and (2) Galphao and Galphaq act in the same neurons to have opposing effects on behavior, in part, by antagonistically regulating transcription of specific genes. Galphao and Galphaq have opposing effects on many behaviors in addition to egg laying and may generally act, as they do in the egg-laying system, to integrate multiple signals and consequently set levels of transcription of genes that affect neurotransmitter release.

A specific subset of transient receptor potential vanilloid-type channel subunits in Caenorhabditis elegans endocrine cells function as mixed heteromers to promote neurotransmitter release.

Transient receptor potential (TRP) channel subunits form homotetramers that function in sensory transduction. Heteromeric channels also form, but their physiological subunit compositions and functions are largely unknown. We found a dominant-negative mutant of the C. elegans TRPV (vanilloid-type) subunit OCR-2 that apparently incorporates into and inactivates OCR-2 homomers as well as heteromers with the TRPV subunits OCR-1 and -4, resulting in a premature egg-laying defect. This defect is reproduced by knocking out all three OCR genes, but not by any single knockout. Thus a mixture of redundant heteromeric channels prevents premature egg laying. These channels, as well as the G-protein G alpha(o), function in neuroendocrine cells to promote release of neurotransmitters that block egg laying until eggs filling the uterus deform the neuroendocrine cells. The TRPV channel OSM-9, previously suggested to be an obligate heteromeric partner of OCR-2 in sensory neurons, is expressed in the neuroendocrine cells but has no detectable role in egg laying. Our results identify a specific set of heteromeric TRPV channels that redundantly regulate neuroendocrine function and show that a subunit combination that functions in sensory neurons is also present in neuroendocrine cells but has no detectable function in these cells.

Mechanism of extrasynaptic dopamine signaling in Caenorhabditis elegans.

D1-like and D2-like dopamine receptors have synergistic and antagonistic effects on behavior. To understand the mechanisms underlying these effects, we studied dopamine signaling genetically in Caenorhabditis elegans. Knocking out a D2-like receptor, DOP-3, caused locomotion defects similar to those observed in animals lacking dopamine. Knocking out a D1-like receptor, DOP-1, reversed the defects of the DOP-3 knockout. DOP-3 and DOP-1 have their antagonistic effects on locomotion by acting in the same motor neurons, which coexpress the receptors and which are not postsynaptic to dopaminergic neurons. In a screen for mutants unable to respond to dopamine, we identified four genes that encode components of the antagonistic Galpha(o) and Galpha(q) signaling pathways, including Galpha(o) itself and two subunits of the regulator of G protein signaling (RGS) complex that inhibits Galpha(q). Our results indicate that extrasynaptic dopamine regulates C. elegans locomotion through D1- and D2-like receptors that activate the antagonistic Galpha(q) and Galpha(o) signaling pathways, respectively.

Neurotransmitter signaling through heterotrimeric G proteins: insights from studies in C. elegans.

Neurotransmitters signal via G protein coupled receptors (GPCRs) to modulate activity of neurons and muscles. C. elegans has ∼150 G protein coupled neuropeptide receptor homologs and 28 additional GPCRs for small-molecule neurotransmitters. Genetic studies in C. elegans demonstrate that neurotransmitters diffuse far from their release sites to activate GPCRs on distant cells. Individual receptor types are expressed on limited numbers of cells and thus can provide very specific regulation of an individual neural circuit and behavior. G protein coupled neurotransmitter receptors signal principally via the three types of heterotrimeric G proteins defined by the G alpha subunits Gαo, Gαq, and Gαs. Each of these G alpha proteins is found in all neurons plus some muscles. Gαo and Gαq signaling inhibit and activate neurotransmitter release, respectively. Gαs signaling, like Gαq signaling, promotes neurotransmitter release. Many details of the signaling mechanisms downstream of Gαq and Gαs have been delineated and are consistent with those of their mammalian orthologs. The details of the signaling mechanism downstream of Gαo remain a mystery. Forward genetic screens in C. elegans have identified new molecular components of neural G protein signaling mechanisms, including Regulators of G protein Signaling (RGS proteins) that inhibit signaling, a new Gαq effector (the Trio RhoGEF domain), and the RIC-8 protein that is required for neuronal Gα signaling. A model is presented in which G proteins sum up the variety of neuromodulator signals that impinge on a neuron to calculate its appropriate output level.

Neural Architecture of Hunger-Dependent Multisensory Decision Making in C. elegans.

Little is known about how animals integrate multiple sensory inputs in natural environments to balance avoidance of danger with approach to things of value. Furthermore, the mechanistic link between internal physiological state and threat-reward decision making remains poorly understood. Here we confronted C. elegans worms with the decision whether to cross a hyperosmotic barrier presenting the threat of desiccation to reach a source of food odor. We identified a specific interneuron that controls this decision via top-down extrasynaptic aminergic potentiation of the primary osmosensory neurons to increase their sensitivity to the barrier. We also establish that food deprivation increases the worm's willingness to cross the dangerous barrier by suppressing this pathway. These studies reveal a potentially general neural circuit architecture for internal state control of threat-reward decision making.

Activity of the C. elegans egg-laying behavior circuit is controlled by competing activation and feedback inhibition.

Like many behaviors, Caenorhabditis elegans egg laying alternates between inactive and active states. To understand how the underlying neural circuit turns the behavior on and off, we optically recorded circuit activity in behaving animals while manipulating circuit function using mutations, optogenetics, and drugs. In the active state, the circuit shows rhythmic activity phased with the body bends of locomotion. The serotonergic HSN command neurons initiate the active state, but accumulation of unlaid eggs also promotes the active state independent of the HSNs. The cholinergic VC motor neurons slow locomotion during egg-laying muscle contraction and egg release. The uv1 neuroendocrine cells mechanically sense passage of eggs through the vulva and release tyramine to inhibit egg laying, in part via the LGC-55 tyramine-gated Cl- channel on the HSNs. Our results identify discrete signals that entrain or detach the circuit from the locomotion central pattern generator to produce active and inactive states.

Activation of EGL-47, a Galpha(o)-coupled receptor, inhibits function of hermaphrodite-specific motor neurons to regulate Caenorhabditis elegans egg-laying behavior.

Caenorhabditis elegans egg-laying behavior is inhibited by neurotransmitter signaling through the neural G-protein Galpha(o) and serves as a model for analyzing Galpha(o) signaling. Mutations that alter egg-laying frequency have identified genes encoding a number of signaling proteins that act with Galpha(o), but the receptors that activate Galpha(o) remain mostly uncharacterized. To further analyze Galpha(o) signaling, we cloned the egl-47 gene, which was identified by two dominant mutations that severely inhibit egg laying. egl-47 encodes two orphan G-protein-coupled receptor isoforms, which share all seven transmembrane domains but have different extracellular N termini. Both dominant mutations change the same alanine to valine in the sixth transmembrane domain, resulting in constitutively activated receptors. Deletion of the egl-47 gene caused no detectable egg-laying defects, suggesting that EGL-47 functions redundantly, or it inhibits egg laying under specific circumstances as yet unidentified. Using promoter::green fluorescent protein transgenes, we found that EGL-47 is expressed in a number of neurons, including the hermaphrodite-specific neurons (HSNs) that innervate the egg-laying muscles to stimulate contraction. Transgenic expression of constitutively active EGL-47 or constitutively active Galpha(o) specifically in the HSNs was sufficient to inhibit egg-laying behavior. Our results suggest that EGL-47 regulates egg laying by activating Galpha(o) in the HSN motor neurons to inhibit their activity. Because several neurotransmitters act through Galpha(o) to inhibit HSN function, it appears that loss of any one receptor, such as EGL-47, causes only mild defects. Galpha(o) apparently integrates signaling from multiple receptors in the HSNs, including EGL-47, to set the frequency of egg-laying behavior.