Michael R. Koelle

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 Gαo and Gαq signaling pathways, including Gαo itself and two subunits of the regulator of G protein signaling (RGS) complex that inhibits Gαq. Our results indicate that extrasynaptic dopamine regulates C. elegans locomotion through D1- and D2-like receptors that activate the antagonistic Gαq and Gαo signaling pathways, respectively.

RGS-7 Completes a Receptor-Independent Heterotrimeric G Protein Cycle to Asymmetrically Regulate Mitotic Spindle Positioning in C. elegans.

Heterotrimeric G proteins promote microtubule forces that position mitotic spindles during asymmetric cell division in C. elegans embryos. While all previously studied G protein functions require activation by seven-transmembrane receptors, this function appears to be receptor independent. We found that mutating a regulator of G protein signaling, RGS-7, resulted in hyperasymmetric spindle movements due to decreased force on one spindle pole. RGS-7 is localized at the cell cortex, and its effects require two redundant Galphao-related G proteins and their nonreceptor activators RIC-8 and GPR-1/2. Using recombinant proteins, we found that RIC-8 stimulates GTP binding by Galphao and that the RGS domain of RGS-7 stimulates GTP hydrolysis by Galphao, demonstrating that Galphao passes through the GTP bound state during its activity cycle. While GTPase activators typically inactivate G proteins, RGS-7 instead appears to promote G protein function asymmetrically in the cell, perhaps acting as a G protein effector.

Two RGS proteins that inhibit Gαo and Gαq signaling in C. elegans neurons require a Gβ5-like subunit for function

Abstract Background: Gβ proteins have traditionally been thought to complex with Gγ proteins to function as subunits of G protein heterotrimers. The divergent Gβ 5 protein, however, can bind either Gγ proteins or r egulator of G protein s ignaling (RGS) proteins that contain a G g amma– l ike (GGL) domain. RGS proteins inhibit G protein signaling by acting as Gα GTPase activators. While Gβ 5 appears to bind RGS proteins in vivo, its association with Gγ proteins in vivo has not been clearly demonstrated. It is unclear how Gβ 5 might influence RGS activity. In C. elegans there are exactly two GGL-containing RGS proteins, EGL-10 and EAT-16, and they inhibit Gα o and Gα q signaling, respectively. Results: We knocked out the gene encoding the C. elegans Gβ 5 ortholog, GPB-2, to determine its physiological roles in G protein signaling. The gpb-2 mutation reduces the functions of EGL-10 and EAT-16 to levels comparable to those found in egl-10 and eat-16 null mutants. gpb-2 knockout animals are viable, and exhibit no obvious defects beyond those that can be attributed to a reduction of EGL-10 or EAT-16 function. GPB-2 protein is nearly absent in eat-16; egl-10 double mutants, and EGL-10 protein is severely diminished in gpb-2 mutants. Conclusions: Gβ 5 functions in vivo complexed with GGL-containing RGS proteins. In the absence of Gβ 5 , these RGS proteins have little or no function. The formation of RGS–Gβ 5 complexes is required for the expression or stability of both the RGS and Gβ 5 proteins. Appropriate RGS–Gβ 5 complexes regulate both Gα o and Gα q proteins in vivo.

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.

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.

Activation of EGL-47, a Gαo-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 Gαo and serves as a model for analyzing Gαo signaling. Mutations that alter egg-laying frequency have identified genes encoding a number of signaling proteins that act with Gαo, but the receptors that activate Gαo remain mostly uncharacterized. To further analyze Gαo 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 Gαo specifically in the HSNs was sufficient to inhibit egg-laying behavior. Our results suggest that EGL-47 regulates egg laying by activating Gαo in the HSN motor neurons to inhibit their activity. Because several neurotransmitters act through Gαo to inhibit HSN function, it appears that loss of any one receptor, such as EGL-47, causes only mild defects. Gαo apparently integrates signaling from multiple receptors in the HSNs, including EGL-47, to set the frequency of egg-laying behavior.

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 Gαo, 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.

Regulation of Serotonin Biosynthesis by the G Proteins Gαo and Gαq 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 Gαo and activated by the G protein Gαq. We found that Gαo and Gαq 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 Gαo and Gαq 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) Gαo and Gαq act in the same neurons to have opposing effects on behavior, in part, by antagonistically regulating transcription of specific genes. Gαo and Gαq 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.

Heterotrimeric G protein signaling: Getting inside the cell.

Heterotrimeric G proteins have traditionally been thought to transduce signals at the plasma membrane. In this issue, Slessareva et al. (2006) now show that a G protein alpha subunit acts at the endosome to stimulate a phosphatidylinositol 3-kinase to help yeast respond to mating pheromones.

Receptors and Other Signaling Proteins Required for Serotonin Control of Locomotion in Caenorhabditis elegans

A better understanding of the molecular mechanisms of signaling by the neurotransmitter serotonin is required to assess the hypothesis that defects in serotonin signaling underlie depression in humans. Caenorhabditis elegans uses serotonin as a neurotransmitter to regulate locomotion, providing a genetic system to analyze serotonin signaling. From large-scale genetic screens we identified 36 mutants of C. elegans in which serotonin fails to have its normal effect of slowing locomotion, and we molecularly identified eight genes affected by 19 of the mutations. Two of the genes encode the serotonin-gated ion channel MOD-1 and the G-protein-coupled serotonin receptor SER-4. mod-1 is expressed in the neurons and muscles that directly control locomotion, while ser-4 is expressed in an almost entirely non-overlapping set of sensory and interneurons. The cells expressing the two receptors are largely not direct postsynaptic targets of serotonergic neurons. We analyzed animals lacking or overexpressing the receptors in various combinations using several assays for serotonin response. We found that the two receptors act in parallel to affect locomotion. Our results show that serotonin functions as an extrasynaptic signal that independently activates multiple receptors at a distance from its release sites and identify at least six additional proteins that appear to act with serotonin receptors to mediate serotonin response.