InteractiveFly: GeneBrief

Dopamine/Ecdysteroid receptor: Biological Overview | References

Gene name - Dopamine/Ecdysteroid receptor

Synonyms - CG18314

Cytological map position - 64B2-64B3

Function - G-protein coupled receptor

Keywords - neuromodulation, mushroom body, courtship memory, cAMP signaling, appetite control of sugar sensing, nongenomic responses to ecdysteroids and catecholamines

Symbol - DopEcR

FlyBase ID: FBgn0035538

Genetic map position - chr3L:4367640-4380376

Classification - G protein-coupled chemokine receptor-like protein

Cellular location - surface transmembrane

NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Petruccelli, E., Li, Q., Rao, Y. and Kitamoto, T. (2016). The unique dopamine/ecdysteroid receptor modulates ethanol-induced sedation in Drosophila. J Neurosci 36: 4647-4657. PubMed ID: 27098705
Steroids profoundly influence behavioral responses to alcohol by activating canonical nuclear hormone receptors and exerting allosteric effects on ion channels. Accumulating evidence has demonstrated that steroids can also trigger biological effects by directly binding G-protein-coupled receptors (GPCRs), yet physiological roles of such unconventional steroid signaling in controlling alcohol-induced behaviors remain unclear. The dopamine/ecdysteroid receptor (DopEcR) is a GPCR that mediates nongenomic actions of ecdysteroids, the major steroid hormones in insects. This study reports that Drosophila DopEcR plays a critical role in ethanol-induced sedation. DopEcR mutants take longer than control flies to become sedated during exposure to ethanol, despite having normal ethanol absorption or metabolism. RNAi-mediated knockdown of DopEcR expression reveals that this receptor is necessary after eclosion, and is required in particular neuronal subsets, including cholinergic and peptidergic neurons, to mediate this behavior. Additionally, flies ubiquitously overexpressing DopEcR cDNA have a tendency to become sedated quickly upon ethanol exposure. These results indicate that neuronal subset-specific expression of DopEcR in adults is required for normal sedation upon exposure to ethanol. It was also found that DopEcR may promote ethanol sedation by suppressing epidermal growth factor receptor/extracellular signal-regulated kinase signaling. Last, genetic and pharmacological analyses suggest that in adult flies ecdysone may serve as an inverse agonist of DopEcR and suppress the sedation-promoting activity of DopEcR in the context of ethanol exposure. These findings provide the first evidence for the involvement of nongenomic G-protein-coupled steroid receptors in the response to alcohol, and shed new light on the potential roles of steroids in alcohol-use disorders.

Lark, A., Kitamoto, T. and Martin, J. R. (2017). Modulation of neuronal activity in the Drosophila mushroom body by DopEcR, a unique dual receptor for ecdysone and dopamine. Biochim Biophys Acta [Epub ahead of print]. PubMed ID: 28554773
G-protein-coupled receptors (GPCRs) for steroid hormones mediate unconventional steroid signaling. Drosophila DopEcR is a GPCR that responds to both ecdysone (the major steroid hormone in insects) and dopamine, regulating multiple second messenger systems. Recent studies have revealed that DopEcR is preferentially expressed in the nervous system and involved in behavioral regulation. This study utilized the bioluminescent Ca2+-indicator GFP-aequorin to monitor the nicotine-induced Ca2+-response within the mushroom bodies (MB), a higher-order brain center in flies, and examined how DopEcR modulates these Ca2+-dynamics. The results show that in DopEcR knockdown flies, the nicotine-induced Ca2+-response in the MB was significantly enhanced selectively in the medial lobes. Application of DopEcR's ligands, ecdysone and dopamine, had different effects on nicotine-induced Ca2+-responses in the MB: ecdysone enhanced activity in the calyx and cell body region in a DopEcR-dependent manner, whereas dopamine reduced activity in the medial lobes independently of DopEcR. Finally, flies with reduced DopEcR function in the MB were shown to display decreased locomotor activity. This behavioral phenotype of DopEcR-deficient flies may be partly due to their enhanced MB activity, since the MB have been implicated in the suppression of locomotor activity. Overall, these data suggest that DopEcR is involved in region-specific modulation of Ca2+ dynamics within the MB, which may play a role in behavioral modulation.


DopEcR, a G-protein coupled receptor for ecdysteroids, is involved in activity- and experience-dependent plasticity of the adult central nervous system. Remarkably, a courtship memory defect in rutabaga (Ca2+/calmodulin-responsive adenylate cyclase) mutants is rescued by DopEcR overexpression or acute 20E feeding, whereas a memory defect in dunce (cAMP-specific phosphodiestrase) mutants is counteracted when a loss-of-function DopEcR mutation is introduced. A memory defect caused by suppressing dopamine synthesis is also restored through enhanced DopEcR-mediated ecdysone signaling, and rescue and phenocopy experiments revealed that the mushroom body (MB) - a brain region central to learning and memory in Drosophila - is critical for the DopEcR-dependent processing of courtship memory. Consistent with this finding, acute 20E feeding induced a rapid, DopEcR-dependent increase in cAMP levels in the MB. The multidisciplinary approach demonstrates that DopEcR mediates the non-canonical actions of 20E and rapidly modulates adult conditioned behavior through cAMP signaling, which is universally important for neural plasticity. This study provides novel insights into non-genomic actions of steroids, and opens a new avenue for genetic investigation into an underappreciated mechanism critical to behavioral control by steroids (Ishimoto, 2013).

Steroid hormones are essential modulators of a broad range of biological processes in a diversity of organisms across phyla. In the adult nervous system, the functions of steroids such as estrogens and glucocorticoids are of particular interest because they have significant effects on the resilience and adaptability of the brain, playing essential roles in endocrine regulation of behavior. Reflecting their importance in neural functions, steroid hormones are implicated in the etiology and pathophysiology of various neurological and psychiatric disorders, and are thus often targeted in therapies. The biological actions of steroids are mediated mainly by nuclear hormone receptors - a unique class of transcription factors that activate or repress target genes in a steroid-dependent manner. Substantial evidence suggests, however, that steroid hormones can also exert biological effects quickly and independently of transcriptional regulation, by modulating intracellular signaling pathways. Such 'non-genomic' effects might be induced by direct allosteric regulation of ion channels, including receptors for GABA and NMDA. Alternatively, in certain contexts, non-genomic steroid signaling could be mediated by classical nuclear hormone receptors acting as effector molecules in the cytosol (Ishimoto, 2013).

G-protein coupled receptors (GPCRs) that directly interact with steroids have the potential to play an important role in non-genomic steroid signaling. So far, however, only few GPCRs have been identified as bona fide steroid receptors in vertebrates. The G-protein coupled estrogen receptor 1 (GPER, formally known as GPR30) is the best studied GPCR that is responsive to steroids. Pharmacological and gene knockout approaches suggest that this protein has widespread roles in the reproductive, nervous, endocrine, immune and cardiovascular systems (Prossnitz, 2011). Although other G-protein coupled receptors were predicted to be responsive to steroids (e.g., the Gq-coupled membrane estrogen receptor and estrogen receptor-X), their molecular identity is not known (Qiu, 2006; ToranAllerand, 2002). Overall, the physiological roles of the GPCR-mediated actions of steroids and the underlying molecular mechanisms remain poorly understood, and sometimes controversial, in spite of their importance. In particular, it is unknown how this non-canonical steroid mechanism influences neural functions and complex behaviors (Ishimoto, 2013).

Drosophila genetics has been extensively used to study the roles and mechanisms of action of steroid hormones in vivo. The major steroid hormone in Drosophila is the molting hormone 20-hydroxy-ecdysone (20E), which orchestrates a wide array of developmental events, including embryogenesis, larval molting and metamorphosis. Recent studies revealed that 20E also plays important roles in adult flies, regulating: the innate immune response, stress resistance, longevity, the formation of long-term courtship memory and the active/resting state. In general, the functions of 20E during development and adulthood are thought to be executed by ecdysone receptors (EcRs), members of the evolutionarily conserved nuclear hormone receptor family (Ishimoto, 2013).

In addition to canonical ecdysone signaling via EcRs, Srivastava (2005) identified a novel GPCR called DopEcR, and showed that it propagates non-genomic ecdysone signaling in vitro. DopEcR shares a high level of amino-acid sequence similarity with vertebrate β-adrenergic receptors. In situ hybridization and microarray data revealed that DopEcR transcripts are preferentially expressed in the nervous system. In heterologous cell culture systems, DopEcR is localized to the plasma membrane and responds to dopamine as well as ecdysteroids (ecdysone and 20E), modulating multiple, intracellular signaling cascades (Srivastava, 2005). Furthermore, Inagaki (2012) recently detected DopEcR expression in the sugar-sensing gustatory neurons of adult flies, and showed that DopEcR-mediated dopaminergic signaling enhances the proboscis extension reflex during starvation. Nonetheless, little is known about whether DopEcR functions as a steroid receptor in vivo, and about how it drives responses in the central nervous system (CNS) to modulate complex behaviors. This study reports that DopEcR mediates non-genomic ecdysone signaling in the adult brain, and that it is critical for memory processing. It was also shown that, during memory processing, DopEcR transmits information via novel steroid signals that interact with the cAMP pathway, a signaling cascade that is universally important for neuronal and behavioral plasticity. This genetic study thus uncovers underappreciated GPCR-mediated functions and mechanisms of action that employ non-canonical steroid signaling to regulate the adult nervous system and, thereby, behavior (Ishimoto, 2013).

This study used genetic, pharmacological, and behavioral approaches in Drosophila to demonstrate that the steroid hormone 20E rapidly regulates behavioral plasticity via a non-genomic mechanism that is mediated by the GPCR-family protein DopEcR. This non-canonical steroid signaling pathway was found to have strong functional interactions with the classical 'memory genes' rut and dnc, which encode the central components of the cAMP pathway. The identification of 20E as an important modulator of cAMP signaling in the adult Drosophila brain reveals an unprecedented opportunity - that of taking advantage of fly genetics to dissect the molecular and cellular mechanisms responsible for the non-genomic steroid signaling that underlies neuronal and behavioral plasticity (Ishimoto, 2013).

Electrophysiological analyses revealed that the adult giant-fiber (GF) pathway of DopEcR mutant flies is more resistant to habituation than that of control flies. Direct excitation of GF or its downstream elements would lead to a short-latency response of the dorsal longitudinal flight muscle (DLM), which could follow high-frequency stimuli up to several hundred Hz. In contrast, the afferent input to the GF leads to a long-latency response that is labile and fails to follow repetitive stimulation well below 100 Hz and displays habituation even at 2-5 Hz. Although there is the possibility that DopEcR-positive thoracic neurons may modulate thoracic motor outputs and contribute to certain parameters of the habituation process not characterized in this study, the more effective modulation would occur in the more labile element afferent to the GF circuit rather than the robust GF-PSI-DLMn downstream pathway (PSI referring to peripherally synapsing interneuron), which is responsible for the reliability of the escape reflex. Thus, the mutant phenotype in habituation indicates that DopEcR positively controls activity-dependent suppression of neuronal circuits afferent to the GF neurons in the brain (Ishimoto, 2013).

Moreover, the finding that DopEcR and rut mutants have a similar GF habituation phenotype raises the possibility that DopEcR positively regulates cAMP levels in the relevant neurons following repetitive brain stimulation. Besides GF habituation, Drosophila displays olfactory habituation, which is mediated by the neural circuit in the antennal lobe. Interestingly, Das (2011) found that olfactory habituation is induced by enhancement of inhibitory GABAergic transmission, and that rut function is required for this neuronal modulation. Similar modulation of GABAergic transmission may also be responsible for habituation of the GF pathway. It will be interesting to examine whether and how DopEcR contributes to the regulation of rut and enhanced GABAergic transmission in GF habituation (Ishimoto, 2013).

Several studies already suggested that 20E has rapid, EcR-independent effects in Drosophila and other invertebrate species. For example, 20E was shown to reduce the amplitude of excitatory junction potentials at the dissected Drosophila larval neuromuscular junction (NMJ), and to do so within minutes of direct application (Ruffner, 1999). Whereas treatment with 20E did not change the size and shape of the synaptic currents generated by spontaneous release, it led to a reduction in the number of synaptic vesicles released by the motor nerve terminals following electrical stimulation. A similar effect of 20E was observed in crayfish, and it was suggested that the suppression of synaptic transmission by 20E may account for the quiescent behavior of molting insects and crustaceans. These observations suggested that 20E suppresses synaptic efficacy under certain conditions by modulating presynaptic physiology through a non-genomic mechanism. It is possible that such actions of 20E are mediated by DopEcR. To detail the mechanisms underlying DopEcR-dependent neural plasticity, it will be worthwhile to determine if and how DopEcR contributes to 20E-induced, rapid synaptic suppression at the physiologically accessible larval NMJ, and to determine the extent to which non-genomic mechanisms of steroid actions are shared between the larval NMJ and the adult brain (Ishimoto, 2013).

One surprising finding made in this study is that ecdysone signaling can modify the phenotypes associated with mutations in the classic 'memory genes', namely rut and dnc, through the actions of DopEcR. rut and dnc encode central components of the cAMP pathway, which is required for memory processing in vertebrates as well as invertebrates. The demonstration that genetically and/or pharmacologically enhancing DopEcR-mediated ecdysone signaling restores the courtship memory phenotype of loss-of-function rut mutants suggests that 20E-mediated DopEcR activation triggers an outcome similar to rut activation, i.e., increased cAMP levels. This assumption is supported by the finding that loss-of-function dnc mutants restore courtship memory when DopEcR activity is suppressed. A similar restoration of the dnc memory phenotype also occurs in a dnc and rut double mutant, again supporting the idea that DopEcR positively regulates cAMP production (Ishimoto, 2013).

The results of rescue and phenocopy experiments indicate that the MB is critical for the DopEcR-dependent processing of courtship memory. Although the endogenous expression pattern of DopEcR is not known, DopEcR is thus likely to modulate cAMP levels in the MB in response to 20E during courtship conditioning. A new Gal4 line has been generated in which a portion of the first coding exon of DopEcR is replaced with a DNA element that contains the Gal4 cDNA whose translation initiation codon is positioned exactly at the DopEcR translation start site. When this line was used to drive UAS-GFP, the reporter gene was widely expressed in the adult brain with prominent signals in the MB. This preliminary result strongly indicates the endogenous expression of DopEcR in the MB. It has also been directly shown that cAMP levels in the MB increase rapidly in flies fed 20E, and that this increase does not occur when DopEcR expression is down-regulated specifically in the MB. Taken together, these findings suggest that DopEcR expressed in the MB responds to 20E and acts upstream of cAMP signaling in a cell-autonomous manner (Ishimoto, 2013).

Surprisingly, enhancement of DopEcR-mediated ecdysone signaling restored courtship memory in flies harboring a strong hypomorphic allele of rut (rut1084). A similar result was obtained even in mutants harboring a presumptive rut null allele rut1. These results suggest that, upon stimulation by 20E, DopEcR may be able to signal via another adenylyl cyclase that can compensate for the lack of Rut. This interesting possibility requires further investigation (Ishimoto, 2013).

This study has focused on the roles and mechanisms of action of DopEcR-mediated, non-genomic ecdysone signaling. Since it has been found that 20E levels rise in the head during courtship conditioning (Ishimoto, 2009), the data presented in this study suggest that DopEcR is activated by 20E during conditioning, triggers a rise in cAMP levels and induces physiological changes that subsequently suppress courtship behavior. This interpretation assumes that 20E directly activates DopEcR to increase cAMP levels. Previous cell-culture studies suggested that DopEcR also responds to dopamine to modulate intracellular signaling (Srivastava, 2005). Furthermore, Inagaki (2012) has demonstrated that flies respond to starvation by sensitizing gustatory receptor neurons to sugar via dopamine/DopEcR signaling. It is therefore necessary to consider whether dopamine is directly involved in the processing of courtship memory through DopEcR. There is a possibility that 20E initially stimulates the production and/or release of dopamine, and that it in turn activates DopEcR and elevates cAMP levels to induce courtship memory. This possibility is thought unlikely because even when courtship memory is disrupted by pharmacological suppression of dopamine synthesis, 20E feeding can compensate for decreased dopamine and allow restoration of memory. Although dopamine plays a significant role in courtship memory, the results suggest that DopEcR does not act as the major dopamine receptor in this particular learning paradigm. The possibility is thus favored that dopamine contributes to courtship memory in parallel with, or upstream of, DopEcR-mediated ecdysone signaling. Consistent with this view, Keleman (2012) reported that the formation of courtship memory depends on the MB γ neurons, which express DopR1 dopamine receptors, receiving dopaminergic inputs. Notably, the current results indicate that the processing of courtship memory requires DopEcR expression in the αβ, but not γ, neurons of the MB, which makes it unlikely that DopEcR is directly influenced by the dopaminergic neurons innervating γ neurons (Ishimoto, 2013).

Ecdysone signaling through nuclear EcRs is necessary for forming long-term courtship memory that lasts at least 5 days, but appears not to have a significant effect on short-term courtship memory (Ishimoto, 2009). In contrast, we found that DopEcR-mediated ecdysone signaling is critical for habituation and 30-minute courtship memory. These findings suggest that DopEcR and EcRs control distinct physiological responses to courtship conditioning, and that the former regulates short-term memory, while the latter regulates long-term memory. Although non-genomic actions of steroid hormones have been implicated in vertebrate learning and memory, such actions have been attributed mainly to the classical nuclear hormone receptors that function outside of the nucleus and exert roles distinct from those of steroid-activated transcription factors. Although recent evidence has shown that membrane-bound receptors independent of the classical estrogen receptors are involved in estradiol-induced consolidation of hippocampal memory, the molecular identities of these proteins have not been established. The current findings in this study provide a novel framework for dissecting GPCR-mediated steroid signaling at the molecular and cellular levels. Furthermore, future analysis of the functional interplay between genomic and non-genomic steroid signaling pathways is expected to reveal novel mechanisms through which steroid hormones regulate plasticity of the nervous system and other biological phenomena (Ishimoto, 2013).

Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing

Behavior cannot be predicted from a 'connectome' because the brain contains a chemical 'map' of neuromodulation superimposed upon its synaptic connectivity map. Neuromodulation changes how neural circuits process information in different states, such as hunger or arousal. This study describes a genetically based method to map, in an unbiased and brain-wide manner, sites of neuromodulation under different conditions in the Drosophila brain. This method, and genetic perturbations, reveal that the well-known effect of hunger to enhance behavioral sensitivity to sugar is mediated, at least in part, by the release of dopamine onto primary gustatory sensory neurons, which enhances sugar-evoked calcium influx. These data reinforce the concept that sensory neurons constitute an important locus for state-dependent gain control of behavior and introduce a methodology that can be extended to other neuromodulators and model organisms (Inagaki, 2012).

The Tango system transforms a transient ligand/receptor interaction into a stable, anatomical readout of reporter gene expression. The reporter gene is activated by a 'private,' synthetic signal transduction pathway, using a bacterial transcription factor (lexA) that is covalently coupled (via a specific tobacco etch virus [TEV] protease-sensitive cleavage site) to the exogenous DA receptor expressed in the cells of interest. The transcription factor is cleaved from the DA receptor following ligand binding, by recruitment of an arrestin-TEV protease fusion protein, and translocates to the nucleus where it activates a lexAop-driven reporter. This system was originally developed to detect receptor activation in cultured mammalian cell lines, but it used to detect receptor activation in this system (Inagaki, 2012).

The data presented here provide proof-of-principle for the utility of a new method, called TANGO-map, to identify, in a brain-wide and relatively unbiased manner, circuit-level substrates of neuromodulation relevant to a particular state-dependent influence on behavior (Inagaki, 2012).

Sweet taste sensitivity in the labellum is enhanced with increasing duration of food deprivation in Drosophila. This observation confirms and extends previous reports in Drosophila and is consistent with observations in many other animal species. This phenomenon was used as a prototypic case of a state-dependent change in behavior to investigate the ability of TANGO-map to identify underlying neuromodulatory mechanisms (Inagaki, 2012).

The results indicate that starvation enhances endogenous DA release onto primary gustatory receptor neurons (GRNs) in the sub-esophageal ganglion, as detected by increased expression of the DopR-Tango reporter in vivo. In contrast, starvation did not increase the DopR-Tango reporter in the MB or AL, although L-dopa feeding did so. These data indicate that DopR-Tango is capable of revealing selective sites of endogenous DA release in a brain-wide manner, under specific behavioral conditions (Inagaki, 2012).

The results indicate that a mutation in the DA receptor DopEcR, as well as specific knockdown of this receptor in sugar-sensing GRNs, eliminates the effect of starvation to enhance the sucrose sensitivity of the proboscis extension reflex (PER). However, this phenotype was only observed at 6 hr of starvation; after 24 hr of food deprivation, these genetic manipulations no longer had an effect. This is not because these manipulations themselves became ineffective at later times, as the same manipulations did attenuate the increased PER sensitivity caused by L-dopa feeding for 24 hr. This suggests that at an early stage of starvation, DA is necessary to enhance the sugar sensitivity of the PER, whereas at later stages additional factors come into play. The slow kinetics of Tango reporter accumulation preclude the detection of statistically significant increases in signal as early as 6 hr following an experimental manipulation. However, the level of reporter expression detected in animals examined after 48 hr of treatment likely reflects the integration of increases in dopaminergic signaling occurring throughout the first 12-24 hr of the treatment period. Thus, although an increase was detected in DopR-Tango signal at a starvation time point when genetic reduction of DopEcR levels no longer impaired the behavioral effect of starvation and observed a behavioral phenotype at a time point too early to be evaluated directly by the TANGO-map method, this should not be taken to imply that no DA release occurred after 6 hr of starvation. Importantly, given the kinetics of the system, the DopR-Tango signals detected in vivo are likely to reflect primarily changes in tonic levels of DA signaling, rather than brief episodes of phasic DA release. Further improvements of the TANGO-map method are required to increase its temporal resolution. Nevertheless, the present methodology provides a powerful method to identify sites where dopaminergic modulation of a given behavior may occur, even if it cannot reveal precisely how quickly such regulation is exerted (Inagaki, 2012).

Several lines of evidence suggest that the dopaminergic modulation of sugar-sensing GRNs revealed in this study may involve an enhancement of Ca2+ influx at the nerve terminal. Both starvation and L-dopa feeding increased sucrose-evoked Ca2+ influx, without changing the frequency of action potentials measured extracellularly at GRN somata. Furthermore, it was found that direct exposure of the brain to DA increased Ca2+ influx at the presynaptic terminals of sugar-sensing GRNs in a DopEcR-dependent manner. A model consistent with these data is that starvation leads to increased DA release, which increases calcium influx into sugar-sensing GRNs via DopEcR, leading to increased neurotransmitter release. The fact that DopEcR signals via the cAMP/PKA pathway, and that this pathway has been reported to increase Ca2+ channel currents in Drosophila, is also consistent with this scenario. Nevertheless, the genetic data suggest that there are additional pathways through which starvation modulates feeding behavior in this system. The finding that DA modulates primary GRNs to control starvation-dependent changes in behavioral sensitivity to sugar echoes the observation of a similar influence of food deprivation on odorant sensitivity in Drosophila. Such neuromodulatory gain control at the level of primary sensory neurons has also been reported in a variety of other invertebrate as well as vertebrate species. Although the possibility that hunger also influences PER behavior at higher-order synapses in the circuit cannot be excluded, the data add to a growing body of information indicating that modulation of primary sensory neurons is a general mechanism for implementing state-dependent changes in behavioral responses to the stimuli detected by these neurons (Inagaki, 2012).

TANGO-map affords a number of unique advantages to study neuronal modulation in the brain. First, and most importantly, it permits the detection of increases in endogenous neuromodulator release in vivo, in an organism in which the application of conventional methods is not feasible. Second, it provides an anatomical readout of neuromodulation at the neural circuit level. The use of a pan-neuronal GAL4 driver to express the sensor permits, in principle, an unbiased survey of potential sites of neuromodulatory activity throughout the brain. Third, the sensor has ligand specificity. The modular design of the Tango system affords the ability to develop in vivo Tango reporters for other biogenic amines and neuropeptides that work via G protein-coupled receptors (GPCRs). Importantly, because the method employs a synthetic, 'private' signal transduction pathway, the readout of the reporter should be relatively insensitive to interference from conventional signal transduction pathways activated by other endogenous receptors. Systematic and comprehensive application of this approach could, in principle, provide an overview of anatomic patterns of neuromodulation in the brain in a given behavioral setting. Finally, because the Tango system is transcriptionally based, in principle it permits the expression not only of neutral reporters but also of effectors such as RNAi’s or ion channels in the neurons receiving neuromodulatory input. Although the TANGO-map system can certainly benefit from improvements in its kinetics and SNR, it affords a means of identifying points-of-entry for studying circuit-level mechanisms of behaviorally relevant neuromodulation that are currently difficult to access in any otherway. The extension of thismethodology to other neuromodulators and model organisms should further understanding of state-dependent control of neural activity and behavior (Inagaki, 2012).

Rapid, nongenomic responses to ecdysteroids and catecholamines mediated by a novel Drosophila G-protein-coupled receptor

Nongenomic response pathways mediate many of the rapid actions of steroid hormones, but the mechanisms underlying such responses remain controversial. In some cases, cell-surface expression of classical nuclear steroid receptors has been suggested to mediate these effects, but, in a few instances, specific G-protein-coupled receptors (GPCRs) have been reported to be responsible. This study describes the activation of a novel, neuronally expressed Drosophila GPCR by the insect ecdysteroids ecdysone (E) and 20-hydroxyecdysone (20E). This is the first report of an identified insect GPCR interacting with steroids. The Drosophila melanogaster dopamine/ecdysteroid receptor (DmDopEcR) shows sequence homology with vertebrate beta-adrenergic receptors and is activated by dopamine (DA) to increase cAMP levels and to activate the phosphoinositide 3-kinase pathway. Conversely, E and 20E show high affinity for the receptor in binding studies and can inhibit the effects of DA, as well as coupling the receptor to a rapid activation of the mitogen-activated protein kinase pathway. The receptor may thus represent the Drosophila homolog of the vertebrate 'gamma-adrenergic receptors,' which are responsible for the modulation of various activities in brain, blood vessels, and pancreas. Thus, DmDopEcR can function as a cell-surface GPCR that may be responsible for some of the rapid, nongenomic actions of ecdysteroids, during both development and signaling in the mature adult nervous system (Srivastava, 2005).

It is unlikely that the effects described in this study are mediated by cell-surface expression of nuclear EcRs because, in both Sf9 and CHO cells, they only occur in transfected cells expressing DmDopEcR and not in nontransfected control cells. Although intact Sf9 cells contain cytoplasmic EcRs that can be detected by [3H]PoA binding, these receptors exhibit a different ecdysteroid-binding pharmacology. This contrasts with the current results. In addition, the bisacylhydrazine ecdysteroid agonists RH-5849 and RH-0345 (halofenozide), which bind effectively to EcRs from Lepidoptera and Drosophila, do not displace [3H]PoA binding to DmDopEcR at concentrations up to 1 μM. Thus, DmDopEcR shows a different pharmacology to insect nuclear EcRs. This conclusion is supported by the observation that [3H]PoA binds to plasma membranes from the anterior silk gland of the silkworm Bombyx mori (Srivastava, 2005).

Expression studies suggest that DmDopEcR can be activated by both E and 20E. In most cases, the physiological actions of insect ecdysteroids are thought to involve 20E rather than E. The latter is usually considered to be a metabolic precursor of 20E. It is synthesized and released by the Drosophila ring glands, along with 20-deoxymakisterone A, and these steroids are metabolized in peripheral tissues to 20E and MaA, respectively . However, there are few known physiological actions of E and MaA. An exception is the stimulatory role of E in cell proliferation in the optic lobe of Manduca sexta. In addition, many arthropod rapid gustatory sensilla responses to ecdysteroids show selectivity in their responses to E and 20E (Srivastava, 2005).

An unusual property of the DmDopEcR receptor is its ability to respond to both catecholamines and to ecdysteroids. These ligands could bind competitively to overlapping topographical sites on the outer surface of the receptor or noncompetitively to two distinct nonoverlapping sites that could interact allosterically. It is extremely difficult to distinguish between these two possible forms of ligand interaction experimentally for GPCRs. However, it would appear in the present case that the interactions described in this study are not likely to be the result of a classical allosteric action of the ecdysteroids on the dopamine-binding site, because the ecdysteroids themselves are capable of activating the MAPK pathway through the receptor. The phenomenon of allosteric agonism appears to be very rare for GPCRs, and most allosteric ligands are either antagonists or modulators. However, a recent exception has been described for the chemokine receptor CXCR4. Additionally, allosteric modulation has been defined recently as requiring a reciprocal interaction between the sites, but this study could detect no effect of DA on PoA binding up to a concentration of 100 μM. Although it seems likely that the ecdysteroids and dopamine bind to overlapping sites, a definitive description of the extent of the overlap of these binding sites will require a combination of extensive in vitro mutagenesis studies, together with x-ray diffraction studies on purified receptor protein, which are beyond the scope of the present study (Srivastava, 2005).

The DmDopEcR receptor appears to demonstrate agonist-specific coupling. DA couples the receptor to the stimulation of adenylyl cyclase activity and activates PI3K, as assessed via Akt phosphorylation, whereas the ecdysteroids couple the receptor to the activation of the MAPK pathway, as assessed by ERK2 phosphorylation, presumably by each agonist inducing a different conformation of the receptor. Both the PI3K pathway and catecholamines are known to have important roles in the control of Drosophila development, and, in addition, the activation of the MAPK pathway is an important regulator of cellular differentiation in Drosophila. A wide range of other Drosophila GPCRs, including the OA/TYR receptor, the DopR99B D1-like DA receptor , and the short neuropeptide F receptor, have also been shown to exhibit various forms of agonist-specific coupling, as have several vertebrate adrenergic and neuropeptide GPCRs. It remains to be determined whether both DA and the ecdysteroids can signal or modulate each other's actions through DmDopEcR in vivo. Nevertheless, it is interesting to speculate that the many surges in ecdysteroid levels observed during insect development, which can reach concentrations as high as 1.35 μM, could serve to rapidly turn off DA-mediated signaling through this receptor. Such a suggestion would be consistent with the rapid ecdysteroid-mediated inhibition of nitric oxide signaling in Manduca optic lobe neurogenesis. It would allow the neural precursor cells to proceed into mitosis, because ecdysteroids induce a conformation of DmDopEcR that does not couple to the PI3K pathway activating nitric oxide synthesis. Equally, it is interesting to speculate that the expression of DmDopEcR in the developing eye imaginal disk may relate to the progression of the morphogenetic furrow, because this requires E, but not EcR, to function (Srivastava, 2005).

The in situ hybridization and the PCR expression studies on DmDopEcR suggest that the expression of the receptor is tightly controlled during development and is likely to have a number of well defined, stage-specific, functional roles. Thus, it is likely to be involved with the control of cell proliferation and differentiation in the salivary ducts and the midgut, although its specific role in these regions remains to be elucidated. The strong expression of DmDopEcR during the early development of the nervous system, when neuroblasts are dividing, suggests that the receptor could be involved with the control of cell proliferation. In addition, the strong expression of the receptor in adult heads suggests that the receptor may also be involved in the modulation of neuronal signaling because ecdysteroids are known to have rapid effects on neural activity in insect brains, paralleling the actions of vertebrate steroids on the brain (Srivastava, 2005).

The GPCR membrane receptor, DopEcR, mediates the actions of both dopamine and ecdysone to control sex pheromone perception in an insect

Olfactory information mediating sexual behavior is crucial for reproduction in many animals, including insects. In male moths, the macroglomerular complex (MGC) of the primary olfactory center, the antennal lobe (AL) is specialized in the treatment of information on the female-emitted sex pheromone. Evidence is accumulating that modulation of behavioral pheromone responses occurs through neuronal plasticity via the action of hormones and/or catecholamines. A G-protein-coupled receptor (GPCR), AipsDopEcR, with its homologue known in Drosophila for its double affinity to the main insect steroid hormone 20-hydroxyecdysone (20E), and dopamine (DA), has been shown to be present in the ALs, and is involved in the behavioral response to pheromone in the moth, Agrotis ipsilon. This study tested the role of AipsDopEcR as compared to nuclear 20E receptors in central pheromone processing, combining receptor inhibition with intracellular recordings of AL neurons. The sensitivity of AL neurons for the pheromone in males decreases strongly after AipsDopEcR-dsRNA injection but also after inhibition of nuclear 20E receptors. Moreover the involvement of 20E and DA was tested in the receptor-mediated behavioral modulation in wind tunnel experiments, using ligand applications and receptor inhibition treatments. Both ligands are necessary and act on AipsDopEcR-mediated behavior. Altogether these results indicate that the GPCR membrane receptor, AipsDopEcR, controls sex pheromone perception through the action of both 20E and DA in the central nervous system, probably in concert with 20E action through nuclear receptors (Abrieux, 2004).


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Abrieux, A., Duportets, L., Debernard, S., Gadenne, C. and Anton, S. (2014). The GPCR membrane receptor, DopEcR, mediates the actions of both dopamine and ecdysone to control sex pheromone perception in an insect. Front Behav Neurosci 8: 312. PubMed ID: 25309365

Das, S., Sadanandappa, M. K., Dervan, A., Larkin, A., Lee, J. A., Sudhakaran, I. P., Priya, R., Heidari, R., Holohan, E. E., Pimentel, A., Gandhi, A., Ito, K., Sanyal, S., Wang, J. W., Rodrigues, V. and Ramaswami, M. (2011). Plasticity of local GABAergic interneurons drives olfactory habituation. Proc Natl Acad Sci U S A 108: E646-654. PubMed ID: 21795607

Inagaki, H. K., Ben-Tabou de-Leon, S., Wong, A. M., Jagadish, S., Ishimoto, H., Barnea, G., Kitamoto, T., Axel, R. and Anderson, D. J. (2012). Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing. Cell 148: 583-595. PubMed ID: 22304923

Ishimoto, H., Sakai, T. and Kitamoto, T. (2009). Ecdysone signaling regulates the formation of long-term courtship memory in adult Drosophila melanogaster. Proc Natl Acad Sci U S A 106: 6381-6386. PubMed ID: 19342482

Ishimoto, H., Wang, Z., Rao, Y., Wu, C. F. and Kitamoto, T. (2013). Novel Role for Ecdysone in Drosophila Conditioned Behavior: Linking GPCR-Mediated Non-canonical Steroid Action to cAMP Signaling in the Adult Brain. PLoS Genet 9: e1003843. PubMed ID: 24130506

Keleman, K., Vrontou, E., Kruttner, S., Yu, J. Y., Kurtovic-Kozaric, A. and Dickson, B. J. (2012). Dopamine neurons modulate pheromone responses in Drosophila courtship learning. Nature 489: 145-149. PubMed ID: 22902500

Prossnitz, E. R. and Barton, M. (2011). The G-protein-coupled estrogen receptor GPER in health and disease. Nat Rev Endocrinol 7: 715-726. PubMed ID: 21844907

Qiu, J., Bosch, M. A., Tobias, S. C., Krust, A., Graham, S. M., Murphy, S. J., Korach, K. S., Chambon, P., Scanlan, T. S., Ronnekleiv, O. K. and Kelly, M. J. (2006). A G-protein-coupled estrogen receptor is involved in hypothalamic control of energy homeostasis. J Neurosci 26: 5649-5655. PubMed ID: 16723521

Ruffner, M. E., Cromarty, S. I. and Cooper, R. L. (1999). Depression of synaptic efficacy in high- and low-output Drosophila neuromuscular junctions by the molting hormone (20-HE). J Neurophysiol 81: 788-794. PubMed ID: 10036278

Toran-Allerand, C. D., Guan, X., MacLusky, N. J., Horvath, T. L., Diano, S., Singh, M., Connolly, E. S., Jr., Nethrapalli, I. S. and Tinnikov, A. A. (2002). ER-X: a novel, plasma membrane-associated, putative estrogen receptor that is regulated during development and after ischemic brain injury. J Neurosci 22: 8391-8401. PubMed ID: 12351713

Srivastava, D. P., Yu, E. J., Kennedy, K., Chatwin, H., Reale, V., Hamon, M., Smith, T. and Evans, P. D. (2005). Rapid, nongenomic responses to ecdysteroids and catecholamines mediated by a novel Drosophila G-protein-coupled receptor. J Neurosci 25: 6145-6155. PubMed ID: 15987944

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date revised: 30 November 2013

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