InteractiveFly: GeneBrief

Cholecystokinin-like receptor at 17D1: Biological Overview | References

Gene name - Cholecystokinin-like receptor at 17D1

Synonyms - CCK-like receptor at 17D1

Cytological map position - 17D1-17D3

Function - G-protein coupled receptor

Keywords - growth of neuromuscular junction, CNS

Symbol - CCKLR-17D1

FlyBase ID: FBgn0259231

Genetic map position - chrX:18601727-18620646

Classification - 7TM_GPCR_Srx: Serpentine type 7TM GPCR chemoreceptor Srx

Cellular location - surface transmembrane

NCBI links: | EntrezGene

CCKLR-17D1 orthologs: Biolitmine
Recent literature
Yanagawa, A., Huang, W., Yamamoto, A., Wada-Katsumata, A., Schal, C. and Mackay, T. F. C. (2020). Genetic Basis of Natural Variation in Spontaneous Grooming in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 32727922
Spontaneous grooming behavior is a component of insect fitness. We quantified spontaneous grooming behavior in 201 sequenced lines of the Drosophila melanogaster Genetic Reference Panel and observed significant genetic variation in spontaneous grooming, with broad-sense heritabilities of 0.25 and 0.24 in females and males, respectively. Although grooming behavior is highly correlated between males and females, significant sex by genotype interactions were observed, indicating that the genetic basis of spontaneous grooming is partially distinct in the two sexes. Genome-wide association analyses of grooming behavior was performed, and 107 molecular polymorphisms associated with spontaneous grooming behavior were mapped, of which 73 were in or near 70 genes and 34 were over 1 kilobase from the nearest gene. The candidate genes were associated with a wide variety of gene ontology terms, and several of the candidate genes were significantly enriched in a genetic interaction network. Functional assessments were performed of 29 candidate genes using RNA interference, and 11 were found to affecte spontaneous grooming behavior. The genes associated with natural variation in Drosophila grooming are involved with glutamate metabolism (Gdh) and transport (Eaat); interact genetically with (CCKLR-17D1) or are in the same gene family as (PGRP-LA) genes previously implicated in grooming behavior; are involved in the development of the nervous system and other tissues; or regulate the Notch and Epidermal growth factor receptor signaling pathways. Several DGRP lines exhibited extreme grooming behavior. Excessive grooming behavior can serve as a model for repetitive behaviors diagnostic of several human neuropsychiatric diseases.

Neuropeptide signaling is integral to many aspects of neural communication, particularly modulation of membrane excitability and synaptic transmission. However, neuropeptides have not been clearly implicated in synaptic growth and development. This study demonstrates that cholecystokinin-like receptor (CCKLR), and Drosulfakinin (DSK), its predicted ligand, are strong positive growth regulators of the Drosophila melanogaster larval neuromuscular junction (NMJ). Mutations of CCKLR (CCKLR-17D1 but not CCKLR-17D3) or dsk produce severe NMJ undergrowth, whereas overexpression of CCKLR causes overgrowth. Presynaptic expression of CCKLR is necessary and sufficient for regulating NMJ growth. CCKLR and dsk mutants also reduce synaptic function in parallel with decreased NMJ size. Analysis of double mutants revealed that DSK/CCKLR regulation of NMJ growth occurs through the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element binding protein (CREB) pathway. These results demonstrate a novel role for neuropeptide signaling in synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural synaptic plasticity in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms (Chen, 2012).

Proper synaptic growth is essential for normal development of the nervous system and its function in mediating complex behaviors such as learning and memory. The Drosophila larval neuromuscular junction (NMJ) has become a powerful system for studying the molecular mechanisms underlying synaptic development and plasticity, and many of the key synaptic proteins are evolutionarily conserved (Chen, 2012).

Genetic and molecular analysis in Drosophila has uncovered numerous molecules and pathways that regulate NMJ growth, including proteins required for cell adhesion, endocytosis, cytoskeletal organization, and signal transduction via TGF-β, Wingless, JNK, cAMP, and other signaling molecules. For example, previous studies revealed that increased cAMP levels led to a down-regulation of the cell adhesion protein FasII at synapses, and the activation of the cAMP response element binding protein (CREB) transcription factor to achieve long-lasting changes in synaptic structure and function. Despite the identification and characterization of these various positive and negative regulators, understanding of the networks that govern synaptic growth is still incomplete, with many of the key components and mechanisms yet to be uncovered and analyzed (Chen, 2012).

To search for new regulators of synaptic growth, a forward genetic screen was conducted for mutants exhibiting altered NMJ morphology. In this screen, a new mutation was discovered that exhibits strikingly undergrown NMJs, which indicates disruption of a positive regulator of NMJ growth. The affected protein was identified as cholecystokinin (CCK)-like receptor (CCKLR), a putative neuropeptide receptor that belongs to the family of G-protein coupled receptors (GPCRs) sharing a uniform topology with seven transmembrane domains. When activated by their ligands, neuropeptide GPCRs affect levels of second messengers such as cAMP, diacylglycerol, inositol trisphosphate, and intracellular calcium. Through activation of their cognate receptors, secreted neuropeptides mediate communication among various sets of neurons as well as other cell types to regulate several physiological activities, including feeding and growth, molting, cuticle tanning, circadian rhythm, sleep, and learning and memory. In general, neuropeptides act by modulating neuronal activity through both short-term and long-term effects. Short-term effects include modifications of ion channel activity and alterations in release of or response to neurotransmitters. Long-term effects include changes in gene expression through activation of transcription factors and protein synthesis. In contrast with the well-known effects of neuropeptide signaling on neuronal activity and the strength of synaptic transmission, regulation of synaptic growth and development by neuropeptides has not previously been clearly established (Chen, 2012).

This study demonstrates that CCKLR is required presynaptically to promote NMJ growth. Moreover, mutations of drosulfakinin (dsk), which encode the predicted ligand of CCKLR, cause similar NMJ undergrowth and interact genetically with CCKLR mutations, indicating that DSK and CCKLR are components of a common signaling mechanism that regulates NMJ growth. In addition to the morphological phenotypes caused by mutations of CCKLR and dsk, mutant larvae also exhibit deficits in synaptic function. Through phenotypic analysis of double mutant combinations, it was shown that DSK/CCKLR signals through the cAMP-PKA-CREB pathway to regulate NMJ growth. The results suggest a novel role for neuropeptide signaling in regulation of synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural plasticity of synapses in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms (Chen, 2012).

Neuropeptides, whose effects have been extensively studied at NMJs, are usually described as neuromodulators because they modify the strength of synaptic transmission. For example, proctolin can potentiate the action of glutamate at certain NMJs in insects. However, involvement of neuropeptides in regulating neural development has not been well characterized. Recently, a C-type natriuretic peptide acting through a cGMP signaling cascade was found to be required for sensory axon bifurcation in mice (Schmidt, 2009), which suggests that neuropeptides may have a broader role in development than previously appreciated. The current studies demonstrate that DSK and its receptor, CCKLR, are strong positive regulators of NMJ growth in Drosophila (Chen, 2012).

DSK belongs to the family of FMRFamide-related peptides (FaRPs), which is very broadly distributed across invertebrate and vertebrate phyla. Originally identified in clams, FaRPs affect heart rate, blood pressure, gut motility, feeding behavior, and reproduction in invertebrates. They have been shown to enhance synaptic efficacy at NMJs in locust and to modulate presynaptic Ca2+ channel activity in crustaceans. In Drosophila, various neuropeptides derived from the FMRFa gene can modulate the strength of muscle contraction when perfused onto standard larval nerve-muscle preparations. To these previously described functions of FaRPs, this study adds a new role as a positive regulator of NMJ development (Chen, 2012).

Transgenic rescue experiments, RNAi expression, and overexpression of WT CCKLR demonstrate that CCKLR functions presynaptically in motor neurons to promote NMJ growth. Downstream components of this pathway were identified on the basis of known biochemistry of GPCRs and phenotypic interactions in double mutant combinations. GPCRs typically function by activating second messenger pathways via G proteins. Because loss-of-function mutations in dgs (which encodes the Gsα subunit in Drosophila) cause NMJ undergrowth, it is hypothesized that CCKLR signals through Gsα. Consistent with this idea, it was found that presynaptic constitutively active dgs overexpression rescues the NMJ undergrowth phenotype of CCKLR mutants. Conversely, dominant dose-dependent interactions were observed between CCKLR-null mutations and mutations of rut, which encodes an AC; or PKA-C1, which encodes a cAMP-dependent protein kinase, resulting in significant reductions in NMJ growth. These data place CCKLR together with the other genes in a common cAMP-dependent signaling pathway that regulates NMJ growth (Chen, 2012).

It is known that the AC encoded by rut is activated by Gsα, and on the basis of the results, it is proposed that Gsα is downstream of CCKLR signaling. However, the NMJ undergrowth in rut1, which is a presumptive null mutant, is not as severe as that of a CCKLR-null mutant. This is likely due to the fact that the Drosophila genome contains up to seven different AC-encoding genes, all of which are stimulated by Gsα. Presumably, one or more additional AC-encoding genes share some functional overlap with rut in regulation of NMJ growth. This idea is in good agreement with the results of Wolfgang (2004), who found that the NMJ undergrowth phenotype of rut1 is weaker than that of dgs mutants, which they also interpreted as an indication that multiple ACs are activated by the Gsα encoded by dgs (Chen, 2012).

The primary effector of this pathway is CREB2, a transcriptional regulatory protein that is activated upon phosphorylation by PKA. Consistent with the idea that CCKLR ultimately acts via activation of CREB, loss-of-function mutations of dCreb2 or neuronal overexpression of a dominant-negative dCreb2 transgene cause NMJ undergrowth similar to that of CCKLR-null mutants. Additionally, loss of one copy of dCreb2 in a CCKLR heterozygous background also causes NMJ undergrowth, and overexpression of WT dCreb2 fully rescues the NMJ undergrowth phenotype of CCKLR null, even leading to NMJ overgrowth. Thus, regulation of NMJ growth through the CCKLR signaling pathway is clearly mediated by dCreb2, whose activity is itself necessary and sufficient for regulating NMJ growth. This conclusion differs from another study that suggested that dCreb2 is required for NMJ function but not NMJ growth. One possible explanation for this discrepancy is that a weaker, inducible heat shock-driven transgene was used to express dCreb2 in the earlier work, whereas strong constitutive neuronal drivers were used this study. In any case, the current results demonstrate that in addition to its known role in NMJ function, CREB2 is also a strong positive regulator of NMJ growth and is likely to play a greater role in structural plasticity of synapses in learning and memory in Drosophila than previously suggested. This conclusion is consistent with a recent study (Koon, 2011) indicating that sprouting of type II larval NMJs in response to starvation is stimulated by a cAMP/CREB-dependent pathway via activation of an octopamine GPCR (Chen, 2012).

In addition to being undergrown, NMJs in CCKLR mutant larvae also exhibit a functional deficit. This is perhaps less straightforward than it might seem. Previous analyses of mutations affecting growth of the larval NMJ in Drosophila have shown that there is no simple correlation between the size and complexity of the NMJ and the amplitude of EJPs or amount of neurotransmitter release. These discrepancies arise because of various homeostatic compensatory mechanisms and because some of the affected signaling pathways alter synaptic growth and synaptic function in different ways via distinct downstream targets. For example, a mutation in highwire, which has the most extreme NMJ overgrowth phenotype described, is associated with a decrease in synaptic transmission. Wallenda mutations have been shown to fully suppress the overgrowth phenotype, but have no effect on the deficit in synaptic transmission (Chen, 2012).

In the case of CCKLR mutants, however, there appears to be a very good correspondence between the morphological phenotype and the electrophysiological phenotype: the reduction in the total number of active zones in CCKLR larvae as measured morphologically correlates very well with the reduction in quantal content that was observe. In addition, no difference in CCKLR mutants was detected in calcium sensitivity of transmitter release or in the size or frequency of spontaneous release events. Thus, the synaptic growth phenotype of CCKLR mutant NMJs is sufficient to account for the functional phenotype. However, the possibility cannot be ruled out that DSK/CCKLR signaling also exerts some modulatory effect on NMJ function that is distinct from its effect on NMJ development (Chen, 2012).

DSK is identified as the Drosophila orthologue of CCK, the ligand of CCKLR in vertebrates, on the basis of sequence analysis. The genetic analysis strongly supports the conclusion that DSK is the ligand of CCKLR at the larval NMJ. First, mutations of dsk and expression of dsk RNAi result in NMJ undergrowth phenotypes similar to that of CCKLR mutants. Second, loss of one copy of both dsk and CCKLR in double heterozygotes results in NMJ undergrowth. Third, heterozygosity for dsk does not further enhance the phenotype of a CCKLR-null mutant as expected if DSK regulates NMJ growth through its action on CCKLR. Fourth, overexpression of UAS-dsk does not rescue the undergrowth phenotype of a CCKLR-null mutant, but CCKLR overexpression can rescue NMJ undergrowth of a dsk hypomorphic mutant (Chen, 2012).

The discovery of an entirely novel role for neuropeptide signaling in NMJ growth raises several questions about how this signaling is regulated and the biological significance of this mechanism. Although answers to these questions will require much additional work, an immediate question is whether a paracrine or autocrine mechanism is involved. In the case of octopamine-mediated synaptic sprouting in response to starvation, both autocrine and paracrine signaling are involved in the sprouting of type II and type I NMJs, respectively (Koon, 2011). In an early immunohistochemical investigation, it was reported that DSK was detected in medial neurosecretory cells in the larval CNS that extended projections anteriorly into the brain and posteriorly to the ventral ganglion (Nichols, 1992). As it was not possible to obtain the original DSK antiserum and raising a new antiserum was not successful, the previous report has not been extended or confirmed. Instead, tissue-specific RNAi experiments were performed to examine the spatial requirement for DSK. Pan-neuronal dsk RNAi expression indicates that DSK expression in neurons is required to promote NMJ growth. In addition, C739-Gal4-driven dsk RNAi also causes NMJ undergrowth, whereas OK-Gal4-driven dsk RNAi in motor neurons does not. The expression pattern of C739-Gal4 overlaps with the DSK-positive cells previously identified by immunohistochemistry, which suggests that DSK produced by those neurosecretory cells is required for normal NMJ growth. Thus, from available data, it seems most likely that DSK is acting in paracrine fashion to regulate NMJ growth. However, further investigation will be necessary to determine the exact source of the DSK that promotes NMJ growth to fully understand how this neuropeptide regulates NMJ development (Chen, 2012).

Studies have demonstrated a role for CREB in long-term synaptic plasticity-structural changes in synaptic morphology that underlie the formation of long-term memories (Benito, 2010). This study shows that in addition to CREB's role in structural modification of synapses in response to experience after development is complete, it is also a key regulator of growth and morphology during development of the larval NMJ. Moreover, although CREB is the transcriptional effector for many GPCRs, the fact that NMJs in CCKLR mutants are as undergrown as those of CREB mutants suggests that DSK/CCKLR signaling is a major input to CREB during NMJ growth. Many of the genes encoding intermediate components of the pathway such as dnc, rut, and PKA also have effects on NMJ growth and development as well as on synaptic plasticity and learning and memory, further emphasizing an overlap between the mechanisms that regulate synaptic growth during development and those that regulate postdevelopmental structural synaptic plasticity. These results raise the possibility that DSK/CCKLR signaling also plays a role in long-term synaptic plasticity and learning as well as in synaptic development (Chen, 2012).

Drosulfakinin signaling in fruitless circuitry antagonizes P neurons to regulate sexual arousal in Drosophila

Animals perform or terminate particular behaviors by integrating external cues and internal states through neural circuits. Identifying neural substrates and their molecular modulators promoting or inhibiting animal behaviors are key steps to understand how neural circuits control behaviors. This study identified the Cholecystokinin-like peptide Drosulfakinin (DSK) that functions at single-neuron resolution to suppress male sexual behavior in Drosophila. Dsk neurons physiologically interact with male-specific P neurons, part of a command center for male sexual behaviors, and function oppositely to regulate multiple arousal-related behaviors including sex, sleep and spontaneous walking. This study further found that the DSK- peptide functions through its receptor CCKLR-17D to suppress sexual behaviors in flies. Such a neuropeptide circuit largely overlaps with the fruitless-expressing neural circuit that governs most aspects of male sexual behaviors. Thus DSK/CCKLR signaling in the sex circuitry functions antagonistically with P neurons to balance arousal levels and modulate sexual behaviors (Wu, 2019).

Male courtship in Drosophila melanogaster is one of the best-understood innate behaviors, and largely controlled by the fruitless (fru) gene and doublesex (dsx) gene, which encode sex-specific transcription factors (FRUM and DSXM in males and DSXF in females). FRUM is responsible for most aspects of male courtship, and DSXM is important for the experience-dependent acquisition of courtship in the absence of FRUM, and courtship intensity and sine song production in the presence of FRUM. FRUM is expressed in a dispersed subset of ca. 2000 neurons including sensory neurons, interneurons, and motor neurons that are potentially interconnected to form a sex circuitry controlling sexual behaviors. In contrast, DSXM is expressed in ca. 700 neurons in males, the majority of which also express FRUM, and are crucial for male courtship. Recently, substantial progress has been made into how external sensory cues are perceived and integrated by fruM and/or dsxM neurons to initiate male courtship, in particular, how a subset of male-specific fruM- and dsxM-expressing P1 neurons integrate olfactory and gustatory cues from female or male targets to initiate or terminate courtship. Such a neuronal pathway is also conserved in other Drosophila species (Wu, 2019).

Behavioral decisions depend on both excitatory and inhibitory modulations. P1 neurons represent an excitatory center that integrates multiple (both excitatory and inhibitory) sensory cues and initiates courtship. However, whether there is an inhibitory counterpart that operates against P1 neurons to balance sexual activity is still unknown. Indeed, males do not absolutely court virgin females even if these females may provide the same visual, olfactory, and gustatory cues, depending on the male's internal states and past experiences. It has been previously shown that neuropeptide SIFamide acts on fruM-positive neurons and inhibits male-male but not male-female courtship, and SIFamide neurons also integrate multiple peptidergic neurons to orchestrate feeding behaviors, but whether SIFamide inhibits internal arousal states for sexual behaviors is not clear. Recently, it was found that sleep and sex circuitries interact mutually and demonstrate how DN1 neurons in the sleep circuitry and P1 neurons in the courtship circuitry function together to coordinate behavioral choices between sleep and sex. However, very little is known about the inhibitory pathway(s) that may represent internal arousal states and inhibit courtship toward females (Wu, 2019).

This study sets out to identify courtship inhibitory neurons that express neuropeptides in Drosophila, as neuropeptides play key roles in adjusting animal behaviors based on environmental cues and internal needs. This study identifies that the neuropeptide Drosulfakinin (DSK), the fly ortholog of Cholecystokinin (CCK) in mammals, functions through its receptor CCKLR-17D3 in the fruM-expressing sex circuitry to inhibit male courtship toward females. It is further demonstrate dthat Dsk neurons and P1 neurons interact and oppositely regulate male sexual behaviors (Wu, 2019).

The results identify, at single-neuron resolution, four pairs of fruM-expressing Dsk neurons (MP1 and MP3) that suppress male and female sexual behaviors. The suppression of male and female sexual behaviors depends on the secretion of the neuropeptide DSK-2, which then acts on one of its receptors CCKLR-17D3 that is expressed in many fruM neurons including P1 neurons and the mushroom bodies. Dsk neurons function antagonistically with courtship promoting P1 neurons to co-regulate male courtship, as well as sleep and spontaneous walking (Wu, 2019).

Cholecystokinin (CCK) signaling appears well conserved over evolution and modulates multiple behaviors. In Drosophila, the CCK-like sulfakinin (DSK) is multifunctional and has been reported to be involved in regulating aspects of food ingestion and satiety, aggression, as well as escape-related locomotion and synaptic plasticity during neuromuscular junction development. In mammals, CCK generated from the intestine acts on its receptors in the nucleus of the solitary tract of the brain to transmit satiety signaling and thus inhibit feeding. Furthermore, CCK signaling in the nucleus accumbens modulates dopaminergic influences on male sexual behaviors in rats. CCK is also involved in nociception, learning and memory, aggression and depressive-like behaviors (Wu, 2019).

Despite its significant and conserved roles in modulating multiple innate and learned behaviors, how CCK or DSK signaling responds to environment and/or internal changes and acts on specific neurons expressing its receptors to modulate multiple behaviors, is still rarely known. The finding that DSK/CCKLR signaling functions in the fruM- and/or dsx-expressing sex circuitry to inhibit male courtship is an effort to use Drosophila as a model to investigate how this conserved signaling modulates animal behaviors (Wu, 2019).

The results uncovered a functional circuitry from many dsx neurons (including courtship-promoting P1 neurons) to four pairs of Dsk MP neurons via direct synaptic transmission, and these MP neurons then modulate CCKLR-17D3 neurons including many fruM and/or dsx neurons via secretion of DSK peptides. It is of particular interest to reveal how the four pairs of MP neurons integrate sensory information (in any), physiological states and past experiences in the future to better understand how this neuropeptide signaling modulate arousal states. It is still not known if these MP neurons receive sensory inputs, but since they receive inputs from many dsx neurons, including P1 neurons that integrate multiple chemosensory information, these sensory inputs will at least relay to Dsk MP neurons via P1. Whether there are other pathways from sensory inputs to MP neurons awaits further study. It is also noted that multiple physiological changes including feeding states, aging and sleep deprivation, as well as past housing conditions affect expression of DSK/CCKLR-17D3, but how they affect DSK signaling and behaviors is still unclear. This study showed that male-male group-housing increases DSK expression and thereby reduces male courtship at least under a restricted condition, and previous findings also revealed opposite effects of group-housing on the excitability of P1 neurons in males that have fruM function or lack fruM function. Such male-male housing experience may mildly reduce sexual arousal in a persistent manner, perhaps by increasing DSK expression, but how such housing condition affects physiological roles of Dsk MP neurons and P1 neurons awaits further functional imaging studies on a potential P1-Dsk-P1 functional loop (and a much complex dsx-Dsk-17D3 pathway) with sensory stimulation under different physiological states. In terms of the time scale that DSK functions to inhibit male courtship, the results indicate an immediate behavioral effect upon Dsk neuronal activation. It is also noted that activation of Dsk neurons inhibits male courtship and lasts for minutes, and previous findings also showed that activation of P1 neurons promoted wing extension and aggression and lasts for minutes. These persistent behavioral effects may represent persistent arousal states regulated by Dsk and P1 neurons in this study; however, how this persistency is generated both in the circuit level and behavioral level still needs further investigation (Wu, 2019).

Dsk mutants do not have obvious courtship abnormality under the courtship assays. There are at least two possibilities: (1) DSK may function only in specific conditions (e.g., group-housing) that increase its expression to inhibit courtship; and (2) There are redundant inhibitory signals for courtship, such as another neuropeptide SIFamide that acts on fruM neurons, although they specifically inhibit male-male courtship. Further studies on how DSK/CCKLR signaling is activated under certain conditions, as well as how DSK, SIFamide and other inhibitory signals (if any) jointly modulate male courtship are needed to fully understand this. Nevertheless, that courtship inhibition by activation of Dsk neurons depends on DSK/CCKLR-17D3 signaling, and increasing such signaling through Cas9 activators in an otherwise wild-type male efficiently inhibited courtship, unambiguously reveal the role of DSK/CCKLR-17D3 signaling in suppressing sexual behaviors (Wu, 2019).

As DSK signaling modulates multiple behaviors, one may argue that its role in male courtship is not specific, e.g., activation of Dsk neurons may drive a competing behavior that phenotypically shunting male courtship. Although such possibility cannot be excluded, a number of evidences are listed to support DSK's role with specificity in courtship inhibition: (1) DSK functions in four pairs of fruM-expressing neurons to inhibit courtship; (2) males with activated Dsk neurons rarely court virgin females, while they follow rotating visual objects normally; (3) Dsk neurons receive synaptic transmission from courtship promoting P1 neurons (and many other dsx-expressing neurons) in an experience-dependent manner; (4) Dsk and P1 neurons antagonistically modulate sexual behaviors and wakefulness; and (5) DSK receptor CCKLR-17D3 inhibits male courtship and expresses in many fruM and/or dsx neurons including P1 neurons. It is noted that CCKLR-17D3 is expressed broadly in the CNS including not only P1 neurons, but also mushroom bodies that regulate a range of behaviors including learning, locomotion and sleep. That DSK signaling is multifunctional is possibly due to broad expression of its receptors, and further studies on dissection of CCKLR function in subsets of neurons will help to understand how DSK/CCKLR signaling modulates multiple behaviors (Wu, 2019).

The decision for male flies to court or not depends on not only environmental cues such as availability and suitability of potential mates (males, virgin females, or mated females), but also their internal states (e.g., thirsty or sleepy). It is proposed that there are at least four factors affecting such a decision: (1) external cues that inhibit courtship, referred to as Ex-In factor, such as the male-specific pheromone cVA4; (2) external cues that are excitatory for courtship, referred to as Ex-Ex factor, such as courtship song; (3) internal states that inhibit courtship, referred to as In-In factor; and (4) internal states that are excitatory for courtship, referred to as In-Ex factor. These factors dynamically change and jointly determine males' decision to court or not (Wu, 2019).

Substantial progress has been made on how Ex-In and Ex-Ex factors jointly modulate the activity of male-specific P1 neurons, which is crucial for courtship initiation. In contrast, much less is understood on In-In and In-Ex factors. Recently, Zhang (2016; 2018) found that dopaminergic modulation of P1 neurons drives male courtship not only by desensitizing P1 to inhibition, but also by promoting recurrent P1 stimulation, thus may act as an In-Ex factor for male courtship. Note that all the three factors mentioned above converge on P1 neurons, making them a decision-making center for male courtship. The DSK/CCKLR signaling identified in this study is of particular interest, as it is likely to act as an In-In factor for male courtship, and above all, it does not simply act on P1 neurons like three other factors, but instead forms a potential functional loop with P1 neurons and antagonizes P1 function in modulating male courtship and wakefulness. That Dsk neurons receive synaptic transmission from P1 neurons and other dsx-expressing neurons in an experience-dependent manner further highlights a central role that the DSK/CCKLR signaling plays. These factors, excitatory vs. inhibitory, external vs. internal, jointly control appropriate performance of sexual behaviors, and further studies will reveal how P1 and other dsx-expressing neurons physiologically interact with Dsk neurons to balance behavioral output (Wu, 2019).

A prominent feature of the neuronal control of male and female sexual behaviors in Drosophila is that, despite large similarity in sensory systems, central integrative neurons are sex-specific in the two sexes, with dsx-expressing pC1 neurons integrating olfactory and auditory cues and promoting receptivity to courting males, and fruM-expressing P1 neurons (largely overlapped with dsx-expressing pC1) integrating olfactory, gustatory, and auditory cues and promoting courtship to females. In contrast, the four pairs of Dsk-expressing MP neurons investigated in this study are sexually monomorphic and inhibit both male courtship and female receptivity. Thus DSK/CCKLR signaling may inhibit sexual behaviors in response to physiological changes that are common to both sexes, while sex-promoting central neurons integrating distinct sensory cues are sexually dimorphic. Interestingly, these Dsk neurons common to both sexes receive synaptic transmission from sexually dimorphic dsx neurons in both males and females, providing a simple solution to link sex-specific excitatory and sexually non-specific inhibitory control of sexual behaviors in males and females (Wu, 2019).

The 5-amino acid N-terminal extension of non-sulfated drosulfakinin II is a unique target to generate novel agonists

The ability to design agonists that target peptide signaling is a strategy to delineate underlying mechanisms and influence biology. A sequence that uniquely characterizes a peptide provides a distinct site to generate novel agonists. Drosophila melanogaster sulfakinin encodes non-sulfated drosulfakinin I (nsDSK I; FDDYGHMRF-NH2) and nsDSK II (GGDDQFDDYGHMRF-NH2). Drosulfakinin is typical of sulfakinin precursors, which are conserved throughout invertebrates. Non-sulfated DSK II is structurally related to DSK I, however, it contains a unique 5-residue N-terminal extension; drosulfakinins signal through G-protein coupled receptors, DSK-R1 (Cholecystokinin-like receptor at 17D3) and DSK-R2 (Cholecystokinin-like receptor at 17D1) (Leander, 2016).

Drosulfakinin II distinctly influences adult and larval gut motility and larval locomotion; yet, its structure-activity relationship was unreported. It was hypothesized that substitution of an N-terminal extension residue may alter nsDSK II activity. By targeting the extension analogs were identified that mimicking nsDSK II, yet, surprisingly, novel agonists with were also discovered that increased (super) and opposite (protean) effects. It was determined [A3] nsDSK II increased larval gut contractility rather than, like nsDSK II, decrease it. [N4] nsDSK II impacted larval locomotion, although nsDSK II was inactive. In adult gut, [A1] nsDSK II, [A2] nsDSKII, and [A3] nsDSK II mimicked nsDSK II, and [A4] nsDSK II and [A5] nsDSK II were more potent; [N3] nsDSK II and [N4] nsDSK II mimicked nsDSK II. This study reports nsDSK II signals through DSK-R2 to influence gut motility and locomotion, identifying a novel role for the N-terminal extension in sulfakinin biology and receptor activation; it also led to the discovery of nsDSK II structural analogs that act as super and protean agonists (Leander, 2016).


Search PubMed for articles about Drosophila CCKLR

Benito, E. and Barco, A. (2010). CREB's control of intrinsic and synaptic plasticity: implications for CREB-dependent memory models. Trends Neurosci 33: 230-240. PubMed ID: 20223527

Chen, X. and Ganetzky, B. (2012). A neuropeptide signaling pathway regulates synaptic growth in Drosophila. J Cell Biol 196: 529-543. PubMed ID: 22331845

Koon, A. C., Ashley, J., Barria, R., DasGupta, S., Brain, R., Waddell, S., Alkema, M. J. and Budnik, V. (2011). Autoregulatory and paracrine control of synaptic and behavioral plasticity by octopaminergic signaling. Nat Neurosci 14: 190-199. PubMed ID: 21186359

Leander, M., Heimonen, J., Brocke, T., Rasmussen, M., Bass, C., Palmer, G., Egle, J., Mispelon, M., Berry, K. and Nichols, R. (2016). The 5-amino acid N-terminal extension of non-sulfated drosulfakinin II is a unique target to generate novel agonists. Peptides 83: 49-56. PubMed ID: 27397853

Nichols, R. (1992). Isolation and expression of the Drosophila drosulfakinin neural peptide gene product, DSK-I. Mol Cell Neurosci 3: 342-347. PubMed ID: 19912877

Schmidt, H., Stonkute, A., Juttner, R., Koesling, D., Friebe, A. and Rathjen, F. G. (2009). C-type natriuretic peptide (CNP) is a bifurcation factor for sensory neurons. Proc Natl Acad Sci U S A 106: 16847-16852. PubMed ID: 19805384

Wolfgang, W. J., Clay, C., Parker, J., Delgado, R., Labarca, P., Kidokoro, Y. and Forte, M. (2004). Signaling through Gs alpha is required for the growth and function of neuromuscular synapses in Drosophila. Dev Biol 268: 295-311. PubMed ID: 15063169

Wu, S., Guo, C., Zhao, H., Sun, M., Chen, J., Han, C., Peng, Q., Qiao, H., Peng, P., Liu, Y., Luo, S. D. and Pan, Y. (2019). Drosulfakinin signaling in fruitless circuitry antagonizes P neurons to regulate sexual arousal in Drosophila. Nat Commun 10(1): 4770. PubMed ID: 31628317

Zhang, S. X., Rogulja, D. and Crickmore, M. A. (2016). Dopaminergic circuitry underlying mating drive. Neuron 91(1): 168-181. PubMed ID: 27292538

Zhang, S. X., Miner, L. E., Boutros, C. L., Rogulja, D. and Crickmore, M. A. (2018). Motivation, perception, and chance converge to make a binary decision. Neuron 99(2): 376-388 e376. PubMed ID: 29983326

Biological Overview

date revised: 15 December 2020

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