InteractiveFly: GeneBrief

Leucokinin receptor: Biological Overview | References


Gene name - Leucokinin receptor

Synonyms -

Cytological map position - 64D2-64D2

Function - G-protein coupled receptor

Keywords - behavior, brain, regulation of food intake, regulation of fluid production by Malpighian tubule, innervation of the gut

Symbol - Lkr

FlyBase ID: FBgn0035610

Genetic map position - chr3L:5505517-5522441

Classification - 7 transmembrane receptor (rhodopsin family)

Cellular location - surface transmembrane



NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Cavey, M., Collins, B., Bertet, C. and Blau, J. (2016). Circadian rhythms in neuronal activity propagate through output circuits. Nat Neurosci [Epub ahead of print]. PubMed ID: 26928065
Summary:
Twenty-four hour rhythms in behavior are organized by a network of circadian pacemaker neurons. Rhythmic activity in this network is generated by intrinsic rhythms in clock neuron physiology and communication between clock neurons. However, it is poorly understood how the activity of a small number of pacemaker neurons is translated into rhythmic behavior of the whole animal. To understand this, a screen was carried out for signals that could identify circadian output circuits in Drosophila melanogaster. Leucokinin neuropeptide (LK) and its receptor (LK-R) were found to be required for normal behavioral rhythms. This LK/LK-R circuit connects pacemaker neurons to brain areas that regulate locomotor activity and sleep. These experiments revealed that pacemaker neurons impose rhythmic activity and excitability on LK- and LK-R-expressing neurons. Pacemaker neuron-dependent activity rhythms were also found in a second circadian output pathway controlled by DH44 neuropeptide-expressing neurons. It is concluded that rhythmic clock neuron activity propagates to multiple downstream circuits to orchestrate behavioral rhythms.
Ohashi, H. and Sakai, T. (2018). Leucokinin signaling regulates hunger-driven reduction of behavioral responses to noxious heat in Drosophila. Biochem Biophys Res Commun 499(2): 221-226. PubMed ID: 29559237
Summary:
In the fruitfly Drosophila melanogaster, hunger has a significant impact on its sensory systems and brain functions, and consequently modifies related behaviors. However, it remains unclarified whether hunger affects nociceptive behavioral responses to heat stimuli. This study shows that food deprivation reduces responses to noxious heat in wild-type flies. The neuropeptide Leucokinin (Lk) and its receptor (Lkr) are essential for the reduction of responses to noxious heat. Temporal silencing of Lk-expressing neurons and a knockout mutation of Lkr generated using the CRISPR/Cas9 system inhibited the reduction of responses to noxious heat. Thus, these results reveal that hunger induces reduction of responses to noxious heat through the Lk/Lkr signaling pathway in Drosophila.
Rizzo, M. J., Evans, J. P., Burt, M., Saunders, C. J. and Johnson, E. C. (2018). Unexpected role of a conserved domain in the first extracellular loop in G protein-coupled receptor trafficking. Biochem Biophys Res Commun 503(3): 1919-1926. PubMed ID: 30064912
Summary:
G protein-coupled receptors are the largest superfamily of cell surface receptors in the Metazoa and play critical roles in transducing extracellular signals into intracellular responses. This action is mediated through conformational changes in the receptor following ligand binding. A number of conserved motifs have critical roles in GPCR function, and this study focused on a highly conserved motif (WxFG) in extracellular loop one (EL1). A phylogenetic analysis documents the presence of the WxFG motif in approximately 90% of Class A GPCRs and the motif is represented in 17 of the 19 Class A GPCR subfamilies. Using site-directed mutagenesis, the conserved tryptophan residue was mutagenized in eight receptors which are members of disparate class A GPCR subfamilies from different taxa. The modification of the Drosophila leucokinin receptor shows that substitution of any non-aromatic amino acid for the tryptophan leads to a loss of receptor function. Additionally, leucine substitutions at this position caused similar signaling defects in the follicle-stimulating hormone receptor (FSHR), Galanin receptor (GALR1), AKH receptor (AKHR), corazonin receptor (CRZR), and muscarinic acetylcholine receptor (mACHR1). Visualization of modified receptors through the incorporation of a fluorescent tag revealed a severe reduction in plasma membrane expression, indicating aberrant trafficking of these modified receptors. Taken together, these results suggest a novel role for the WxFG motif in GPCR trafficking and receptor function.

BIOLOGICAL OVERVIEW

Total food intake is a function of meal size and meal frequency, and adjustments to these parameters allow animals to maintain a stable energy balance in changing environmental conditions. The physiological mechanisms that regulate meal size have been studied in blowflies but have not been previously examined in Drosophila. This study shows that mutations in the leucokinin neuropeptide (leuc) and leucokinin receptor (lkr) genes cause phenotypes in which Drosophila adults have an increase in meal size and a compensatory reduction in meal frequency. Since mutant flies take larger but fewer meals, their caloric intake is the same as that of wild-type flies. The expression patterns of the leuc and lkr genes identify small groups of brain neurons that regulate this behavior. Leuc-containing presynaptic terminals are found close to Lkr neurons in the brain and ventral ganglia, suggesting that they deliver Leuc peptide to these neurons. Lkr neurons innervate the foregut. Flies in which Leuc or Lkr neurons are ablated have defects identical to those of leucokinin pathway mutants. These data suggest that the increase in meal size in leuc and lkr mutants is due to a meal termination defect, perhaps arising from impaired communication of gut distension signals to the brain. Leucokinin and the leucokinin receptor are homologous to vertebrate tachykinin and its receptor, and injection of tachykinins reduces food consumption. These results suggest that the roles of the tachykinin system in regulating food intake might be evolutionarily conserved between insects and vertebrates (Al-Anzi, 2011).

In mammals, nutrient intake is regulated to keep body weight constant over long periods of time. Most animals consume food in discrete bouts called meals, and total food intake is a function of both meal size and meal frequency. Identification of the pathways that regulate these meal related parameters is essential for the understanding of the relationships between body weight regulation and caloric intake (Al-Anzi, 2011).

Signals that control meal size and frequency fall into three categories: those that initiate a meal, those that maintain feeding once a meal has begun, and those that terminate a meal. In hungry mammals, the smell and taste of food initiate feeding. As feeding continues, the level of gastric distension is conveyed to the brain via stomach wall stretch receptors. When the extent of stomach distension passes a threshold, the meal is likely to terminate. Also, during the course of a meal, some nutrients are absorbed in the small intestine, allowing a post-gastric evaluation of the caloric content of ingested food that can also contribute to meal termination (Al-Anzi, 2011).

The steps involved in physiological regulation of feeding behavior in flies have been elucidated primarily through studies on the blowfly Phormia regina. As the hungry fly walks, taste hairs on its legs sample the surface. When a food source is detected, the fly extends its proboscis and begins to feed. During ingestion, liquid food passes through the foregut into a collapsible food-storage sac called the crop. Eventually, the fly becomes satiated and stops feeding. A number of factors contribute to termination of a feeding bout, and thus determine meal size. First, stretch receptors monitoring gut distension provide a negative feedback signal to the brain. Second, neurons in the taste hairs habituate and become less responsive to food (Al-Anzi, 2011).

Leucokinin (Leuc) is a myotropic neuropeptide found in most invertebrate species (Nässel, 2002). It was initially identified as a neurohormone that increases malpighian tubule fluid secretion and hindgut motility in some insect species. The biological activity of leucokinin requires an amidated C-terminal pentapeptide motif called FXXWG-amide, a feature that it shares with the related vertebrate tachykinin neuropeptides. The tachykinin family includes substance P, substance K/neurokinin A, and neuropeptide K /neurokinin B. Although the Drosophila genome encodes another peptide whose sequence is somewhat closer to vertebrate tachykinins than is leucokinin, the observation that the Drosophila leucokinin receptor, Lkr, is homologous to vertebrate tachykinin receptors confirms the homology between the leucokinin and tachykinin pathways (Radford, 2002; Al-Anzi, 2011).

This paper reports that the leucokinin pathway is involved in meal size regulation in Drosophila. Flies with reduced leucokinin pathway signaling due to mutations in the genes encoding either the leucokinin ligand (leuc) or the leucokinin receptor (lkr) have an abnormal increase in meal size. This increase is associated with a reduction in meal frequency that causes mutant flies to consume the same total amount of food as wild-type flies. The functions of the leucokinin pathway in regulation of meal size are executed in neurons, since pan-neuronal expression of leuc or lkr rescues the phenotypes. leuc and lkr are expressed in distinct patterns of neurons, and ablation of these neurons phenocopies the effects of the leuc and lkr mutations (Al-Anzi, 2011).

To obtain insights into the molecular mechanisms involved in control of meal size, a screen was performed for mutations that cause adults of Drosophila to consume abnormally large amounts of food. A number of different assays have been used to monitor food consumption in Drosophila. For the screen used in this study, a two-dye feeding assay was developed, in which five-day old male flies in groups of twenty were starved for one day on 1% agarose, then transferred into a vial containing 1% sucrose in 1% agarose with acid red food dye. After 20 minutes, the flies were tapped into a new vial containing the same food but with acid blue dye instead of red dye, and left for 15 more minutes. Wild-type starved flies became satiated during their exposure to red food, and had an exclusively red abdomen, since they did not consume any of the blue food. Flies with a defect in meal size regulation either ate excessive amounts of red food, making them visibly bloated, and/or continued feeding during exposure to the blue food, which caused them to have a purple (red+blue) abdomen (Al-Anzi, 2011).

Since the primary interest of this study was in the neural control of feeding behavior, a set was screened of about 150 transposable element insertion mutations in genes encoding proteins involved in neuronal function, including neuropeptides and their receptors. Two PiggyBac elements were identified that caused strong meal termination defects when homozygous. One of these is leucc275, an insertion 929 base pairs (bp) 5' to the transcription start site of the leucokinin gene, which encodes the neuropeptide leucokinin. The other is lkrc003, an insertion in the third intron of the lkr gene encoding the leucokinin receptor. Both mutations produced abdominal bloating, usually associated with a red abdomen, when tested in the two-dye feeding assay, and dissection of the digestive tracts of bloated flies revealed overfilled crops (Al-Anzi, 2011).

Mutants with reduced expression of the leucokinin neuropeptide or its receptor both consume excess food immediately after starvation, but do not eat more than normal flies when continuously supplied with food. This finding is explained by the fact that leuc and lkr mutants consume abnormally large meals, but at a reduced frequenc (Al-Anzi, 2011).

Leucokinin is known to function as a hormone to regulate diuresis and hindgut motility, and lkr is expressed in the Malpighian tubules, the fly excretory organ (Terhzaz, 1999). However, the effects of leucokinin on meal size regulation are likely to be due to its action as a peptide neurotransmitter rather than to humoral effects on malpighian tubule Lkr, because the leuc and lkr meal size phenotypes are fully rescued by pan-neuronal expression of these genes. This shows that control of meal size by lkr is due to reception of a leucokinin signal by neurons and does not involve Lkr signaling in malpighian tubules (Al-Anzi, 2011).

The expression patterns of leuc and lkr was examined by antibody staining and by constructing promoter-Gal4 fusions. Both genes are expressed in small subsets of neurons in the brain and ventral ganglia, and Lkr is also expressed in the foregut, which is known to be involved in meal termination. Ablation of leuc neurons using cell death genes produces the same meal size phenotype as loss of leucokinin, indicating that this neuronal circuit is essential for control of food intake (Al-Anzi, 2011).

What are the mechanisms by which leucokinin and Lkr regulate meal size? Since ablation of Lkr neurons causes the same phenotype as a reduction in Lkr expression, the data suggest that the activities of Lkr neurons are reduced in leuc and lkr mutants. Also, since reductions in either leucokinin or Lkr cause the same phenotype, it is likely that the Lkr neurons that are relevant to the phenotype include the brain and/or ventral ganglion neurons that are near leucokinin-positive synaptic boutons. Direct or indirect input of Lkr neurons to the foregut could modulate the signals emanating from gut stretch receptors, so that when Lkr neurons are absent or fire less frequently the fly's brain becomes less sensitive to gut stretch signals that indicate satiety (Al-Anzi, 2011).

Other neuropeptides and neuronal circuits have been demonstrated to affect feeding in Drosophila. However, the current analysis suggests that their functions are distinct from those of the leucokinin pathway. In adult flies, inhibiting hugin expressing neurons causes rapid meal initiation and crop bloating, and ablating NPF neurons affects larval feeding. This study examined meal size in adults with ablated hugin or NPF neurons, but found no changes from wild-type. Two distinct neuronal populations, defined by the expression patterns of the Fru-GAL4 and c673a-GAL4 drivers, control long-term energy homeostasis. Flies in which these neurons are silenced store excess fat, while those in which they are hyperactivated lose fat. c673a-GAL4 silenced flies also consume more food than controls. Sulfakinins and allatostatins inhibit contraction of insect visceral muscles, and these peptides can inhibit feeding when injected int (Al-Anzi, 2011 and references therein).

A variety of mammalian peptides have been implicated in food intake regulation. Some, like leptin, measure the status of the body's energy stores and are believed to influence long term food intake. Other neuronal and gastrointestinal tract peptides regulate meal related parameters such as initiation, size, and frequency. Neuronally produced neuropeptide Y, endocannabinoid, and orexin, along with gastrically secreted ghrelin, are thought to be involved in meal initiation, while gastrointestinal tract peptides such as cholecystokinin (CCK), pancreatic peptide Y (3-36), and glucagon-like peptide 1 are believed to regulate meal size and frequency (Murphy, 2006; Woods, 2008; Al-Anzi, 2011 and references therein).

In mice, a reduction in CCK pathway signaling causes feeding defects (meal size increases associated with compensatory reductions in meal frequency) that are similar to those seen in leuc and lkr mutants. This probably does not represent a conserved pathway, since leucokinin and its receptor have little sequence homology with mammalian CCK pathway components. However, Drosophila CCK-related peptides called sulfakinins do inhibit feeding when injected into flies (Al-Anzi, 2011).

Leucokinin and its receptor are homologous to vertebrate tachykinin and tachykinin receptor, and tachykinins cause reductions in food intake when injected into vertebrates (Achapu, 1992; Dib, 1999; Kalra, 1991; Sahu, 1988, Volkoff, 2004). Tachykinins and their receptors are expressed within or near brain centers that regulate body weight and food intake, such as the arcuate nucleus (Cvetkovic, 2003). The findings in Drosophila suggest that the roles of tachykinins in regulating food intake might be evolutionarily conserved between insects and vertebrates (Al-Anzi, 2011).

Enteric neurons and systemic signals couple nutritional and reproductive status with intestinal homeostasis

The gastrointestinal tract is emerging as a key regulator of appetite and metabolism, but daunting neuroanatomical complexity has hampered identification of the relevant signals. Invertebrate models could provide a simple and genetically amenable alternative, but their autonomic nervous system and its visceral functions remain largely unexplored. This study developed a quantitative method based on defecation behavior to uncover a central role for the Drosophila intestine in the regulation of nutrient intake, fluid, and ion balance. A key homeostatic role has been identified for autonomic neurons and hormones, including a brain-gut circuit of insulin-producing neurons modulating appetite, a vasopressin-like system essential for fluid homeostasis, and enteric neurons mediating sex peptide-induced changes in intestinal physiology. These conserved mechanisms of visceral control, analogous to those found in the enteric nervous system and hypothalamic/pituitary axis, enable the study of autonomic control in a model organism that has proved instrumental in understanding sensory and motor systems (Cognigni, 2011).

These experiments provide evidence for significant regulation of nutrient utilization independent of intake. For example, intestinal transit was found to be differentially modulated by a low-calorie diet and reproductive state: two conditions known to increase food intake. In contrast to the faster emptying rate associated with a low-calorie diet, the action of a reproductive hormone (the sex peptide) leads to concentration of intestinal contents and slower intestinal transit in mated females. This effect is strikingly similar to that of progesterone, oxytocin, and estrogen on intestinal passage, secretion, and water absorption, which cause bloating and constipation during pregnancy. These reproductive gastrointestinal changes may be associated with enhanced nutrient absorption: a possible competitive advantage at a time of high nutritional demands (Cognigni, 2011).

The differential enteric physiology of mated females and diet-restricted flies also points to a link between internal diuresis and life span, whereby longevity would be associated with faster intestinal transit and/or diets with a higher water/calorie ratio. This would explain the recently reported deleterious effects of water-poor dietary regimes , and why the impact on life span of dietary restriction (positive) and mating (deleterious in females) is not entirely attributable to calorie intake or egg production. Consistent with this idea, it was also found that sterile ovoD1 mutant females, which do not increase their long-term food intake after mating but still experience mating survival costs, also concentrate excreta. It will be interesting to establish how intestinal physiology is affected by the amino acid imbalance recently found to account for the life-shortening effects of certain diets (Cognigni, 2011).

Finally, it will be instructive to investigate how intestinal flora affects or is affected by the reproductive changes in enteric physiology triggered by mating, diet, and internal metabolic state. Increasing evidence points to a differential role for specific phyla of gut bacteria in nutrient acquisition, energy regulation, and obesity. Given the relative simplicity of the intestinal microbial consortium of lab-reared Drosophila strains, the behavioral and physiological readouts could easily be exploited to investigate the interactions between this bacterial diversity and organismal homeostasis (Cognigni, 2011).

The organizational principles of mammalian enteric nervous systems are broadly conserved in Drosophila. In contrast to the gut of nematode worms (which is devoid of direct innervation), this study found the Drosophila intestine to be extensively innervated by sensory and efferent fibers confined to three discrete portions containing smooth muscle sphincters or valves: a neural architecture suggestive of 'checkpoints' where intestinal transit may be sensed and modulated. Interestingly, it was also observed that a subset of enteric neurons innervate the underlying intestinal epithelium, and it was found that at least one such lineage effects changes in water balance associated with reproduction. This kind of innervation has not been previously described in invertebrates, and is reminiscent of the fibers of the submucosal plexus, which regulate epithelial crypt cell secretion in mammals (Cognigni, 2011 and references therein).

The direct innervation of the adult intestine by insulinergic fibers suggests a novel mode of action for Ilps. Although Ilp2 appears to act systemically to regulate larval growth, endogenous Ilps have not so far been detected in the circulation. Hence, it is possible that Ilps modulate intestinal physiology locally to regulate food intake (and perhaps some of the previously reported mNSC functions in glucose homeostasis and energy storage). In humans and other mammals, neural and hormonal signals originating from the intestine can promote satiety and modulate pancreatic insulin secretion. Insulin could, in turn, convey information about nutritional state to the intestine, which would integrate additional signals (such as those found to emanate from reproductive tissues or diuretic centers) to regulate nutrient processing or the production of intestinal satiety signals. Such intestinal roles would be consistent with the finding of isolated insulin-producing secretory cells in the digestive tract of other invertebrates and protochordates. In this regard, the positioning of the ring gland (which is profusely innervated by median neurosecretory cell fibers) in close proximity to the esophagus and anterior midgut of adult flies is strikingly reminiscent of the islet organ of primitive vertebrates such as hagfish: a discrete aggregation of insulin-producing cells found in the same anatomical location. Hence, the acquisition of insulinergic fate by endocrine organs of different evolutionary origin may reflect a shared requirement for a local source of insulin release close to the intestine. In any event, it suggests that a differential developmental origin (brain and gut insulins) does not necessarily imply functional diversification (Cognigni, 2011).

This study has uncovered an essential role for a very restricted group of central neuroendocrine cells in the systemic regulation of diuresis: a role strikingly similar to the effect on the kidney of vasopressin, a hormone synthesized in the hypothalamus and released from the pituitary gland into the blood stream. Loss of Leucokinin signaling has acute effects: fluid retention is such that adult wet weight almost doubles within a few days, eventually leading to death. This effect contrasts with the relatively modest weight alterations resulting from interfering with adult feeding, energy storage, or developmental growth. The importance of fluid regulation, both during development and in adult homeostasis, may therefore have been underestimated. The connections between water consumption, energy intake, and body weight in humans are poorly understood. This work and a recent study (Al-Anzi, 2010) point to a model wherein one neurohormone (LK) acts on the same receptor centrally to regulate food intake and peripherally to maintain fluid balance (Cognigni, 2011).

The recent discovery of axonal terminals emanating from water-sensing neurons in the subesophageal ganglion, where leucokinin-positive dendrites arborize (de Haro, 2010), suggests sensory input into this vasopressin-like system. Phenotypes like the one resulting from LK neuron silencing provide a behavioral readout with which to test this idea or investigate the nature of possible internal osmolality sensors (Cognigni, 2011).

In C. elegans, the genetic analysis of defecation rate has proved to be an excellent system with which to identify developmental or metabolic genes. The present assay allows quantification of additional aspects of diuresis, enteric function, and food intake. It is thus the first integrative behavioral readout for metabolism in an invertebrate, which can be used in high-throughput screens for genes or compounds regulating diuresis, gastrointestinal physiology, ion transport, and their neural, nutritional, and reproductive control. In particular, having uncovered at least two distinct mechanisms of nutritional modulation of acid-base homeostasis (only one of which is foxo dependent), it will now be of interest to use the pH of excreta as a readout for genetic screens aimed at identifying the metabolic pathways involved, the intestinal mechanisms of pH regulation, and the contribution and site of action of the large number of previously reported foxo targets. In parallel, the assay will also enable future studies aimed at establishing the contribution of specific neurons, peptides, and cell populations, such as gut stem cells and enteroendocrine cells, to gastrointestinal function and organismal homeostasis (Cognigni, 2011).

Detailed analysis of leucokinin-expressing neurons and their candidate functions in the Drosophila nervous system

The distribution of leucokinin (LK) neurons in the central nervous system (CNS) of Drosophila melanogaster was described by immunolabelling many years ago. However, no detailed underlying information of the input or output connections of their neurites was then available. This study provides a more accurate morphological description by employing a novel LK-specific GAL4 line that recapitulates LK expression. In order to analyse the possible afferent and efferent neural candidates of LK neurons, this lk-GAL4 line was used together with other CNS-Gal4 lines, combined with antisera against various neuropeptides or neurotransmitters. Four kinds of LK neurons in the brain were found. (1) The lateral horn neurons connect the antennal glomerula to the mushroom bodies. (2) The s neurons connect the gustatory receptors to the subesophageal ganglia and ventral nerve cord. (3) The anterior neurons innervate the corpus cardiacum of the ring gland but LK expression is surprisingly not detectable from the third instar onwards in these neurons. (4) A set of abdominal ganglion neurons connect to the dorsal median tract in larvae and send their axons to a segmental muscle 8. Thus, the methods employed in this study can be used to identify individual neuropeptidergic neurons and thereby characterize functional cues or developmental transformations in their differentiation (de Haro, 2010).

Peptidergic paracrine and endocrine cells in the midgut of the fruit fly maggot

Endocrine cells in the larval midgut of Drosophila melanogaster are recognized by antisera to seven regulatory peptides: the allatostatins A, B, and C, short neuropeptide F, neuropeptide F, diuretic hormone 31, and the tachykinins. These are the same peptides that are produced by the endocrine cells of the adult midgut, except for short neuropeptide F, which is absent in adult midgut endocrine cells. The anterior larval midgut contains two types of endocrine cells. The first produces short neuropeptide F, which is also recognized by an antiserum to the receptor for the diuretic hormone leucokinin. The second type in the anterior midgut is recognized by an antiserum to diuretic hormone 31. The latter cell type is also found in the junction between the anterior and middle midgut; an additional type of endocrine cell in this region produces allatostatin B, a peptide also known as myoinhibitory peptide. Both types of endocrine cells in the junction between the anterior and middle midgut can express the homeodomain transcription factor Labial. The copper cell region contains small cells that either produce allatostatin C or a combination of neuropeptide F, allatostatin B, and diuretic hormone 31. The latter cell type is also found in the region of the large flat cells. The posterior midgut possesses strongly immunoreactive allatostatin C endocrine cells immediately behind the iron cells. In the next part of the posterior midgut, two cell types have been found: one produces diuretic hormone 31, and a second is strongly immunoreactive to antiserum against the leucokinin receptor and weakly immunoreactive to antisera against allatostatins B and C and short neuropeptide F. The last part of the posterior midgut again has two types of endocrine cells: those that produce allatostatin A, and those that produce tachykinins. Many of the latter cells are also weakly immunoreactive to the antiserum against diuretic hormone 31. As in the adult, the insulin-like peptide 3 gene appears to be expressed by midgut muscles, but not by midgut endocrine cells (Veenstra, 2009).

Systematic G-protein-coupled receptor analysis in Drosophila melanogaster identifies a leucokinin receptor with novel roles

Leucokinins are insect neuropeptides that stimulate hindgut motility and renal fluid secretion. Drosophila has a single leucokinin gene, (Leucokinin or Lk), encoding the longest known leucokinin, Drosokinin. To identify its receptor, a genome-wide scan for G-protein-coupled receptors was performed in silico and candidate receptors identified by similarity to known tachykinin receptors. The deduced peptides were expressed, with a transgene for the calcium reporter aequorin, in S2 cells and only one gene (CG10626) encoded a protein that responded to Drosokinin. The properties of the heterologously expressed receptor match closely those reported for the action of Drosokinin on Malpighian (renal) tubules. Antibodies raised against the receptor identified known sites of leucokinin action: stellate cells of the Malpighian tubule, two triplets of cells in the pars intercerebralis of the adult central nervous system, and additional cells in larval central nervous system. Western blots and RT-PCR confirmed these locations, but also identified expression in male and female gonads. These tissues also displayed elevated calcium in response to Drosokinin, demonstrating novel roles for leucokinin. A functional genomic approach has thus yielded the first complete characterization of a leucokinin receptor in an insect (Radford, 2002).

The leucokinin family of insect diuretic peptides has attracted great interest, both from a basic scientific viewpoint, and as possible lead compounds for insecticide development. However, although a plausible insect candidate had been identified, this was based on an asserted distant similarity of the snail neuropeptide (which did not perfectly match the canonical leucokinin SWGamide C-terminal), to the insect peptide family, and there was no supporting functional evidence for this assignment. This study has shown that of the GPCRs that were closely similar both to lymnokinin and other tachykinins, only a single gene within the whole Drosophila genome, CG10626, really is a leucokinin receptor. The functional properties of the receptor, when expressed heterologously in cell lines, are so similar to those that were previously inferred from physiological studies of leucokinin action on the Malpighian tubule that it can be confidently said that the cognate receptor has been obtained. This does not, of course, exclude the possibility that further leucokinin receptors are among the other 100 or so GPCRs in the Drosophila genome. However, multiple receptors for given ligands, even where they act through different second messengers, tend to co-segregate within the dendrogram (e.g., serotonin receptors), so it is thought that the possibility of there being other receptors in this species is remote (Radford, 2002).

Now that the functional similarity between leucokinin-like receptors in snail, tick, and insect has been established, it can also be inferred that leucokinin signaling is phylogenetically widely distributed among invertebrate phyla (Radford, 2002).

Leucokinins were originally characterized as affecting hindgut motility. Consistent with this, the receptor is expressed in the hindgut. Stimulation of circular muscles in the hindgut would lead to an increase in peristalsis, as originally reported in L. maderae hindgut. However this paper has identified two areas of gene expression, by RT-PCR or immunocytochemistry, that had not previously been expected. The first is an extensive staining of the central nervous system, particularly in areas associated with neuropeptide secretion, and the second is in both male and female genital tracts. Expression in both of these domains is functional, as it was possible to show significant stimulation of calcium upon leucokinin application. This is thus the first demonstration that leucokinins act on the insect genital tract and may be of significance in fertility or in the peristaltic transfer of sperm or eggs (Radford, 2002).

However, the tissue in which leucokinin signaling has been characterized in most detail is in the Malpighian tubule. In Drosophila, the EC50 for stimulation of fluid production has previously been shown to match that for elevation of intracellular calcium, and leucokinin was additionally shown not to raise tubule cAMP or cGMP, allowing calcium to be identified as the second messenger (Terhzaz, 1999). This study has shown that the EC50 for the heterologously expressed receptor is exactly concordant with these previously determined values. Thus the action of Drosokinin on Malpighian tubules can be explained by its interaction with the receptor characterized in this study (Radford, 2002).

Leucokinins are known to stimulate fluid production by increasing the chloride shunt conductance through the epithelium. Other workers have ascribed this to paracellular pathways in other Diptera (39). However, this study has shown the presence of high conductance chloride channels in tubules and has demonstrated that chloride current hotspots invariably map to stellate cells. Additionally, tubules were used in which aequorin had been targeted specifically to either principal cells or stellate cells to show that only the latter responds to calcium by an increase in intracellular calcium. Consistent with this, the data show that the Drosokinin receptor is expressed only in stellate cells. It can now be said with confidence that, in Drosophila at least, leucokinin acts by raising calcium in only stellate cells and that this is sufficient to activate chloride shunt conductance. Now that a prototypic insect leucokinin receptor has been identified and characterized in detail, the scope of this model in other Diptera and other insect orders should be easy to establish (Radford, 2002).


REFERENCES

Search PubMed for articles about Drosophila Leucokinin receptor

Achapu, M., Pompei, P., Polidori, C., de Caro, G. and Massi, M. (1992). Central effects of neuropeptide K on water and food intake in the rat. Brain Res. Bull. 2: 299-303. PubMed ID: 1596748

Al-Anzi, B., Armand, E., Nagamei, P., Olszewski, M., Sapin, V., Waters, C., Zinn, K., Wyman, R. J. and Benzer, S. (2010). The leucokinin pathway and its neurons regulate meal size in Drosophila. Curr, Biol. 20(11): 969-78. PubMed ID: 20493701

Cognigni, P., Bailey, A.P. and Miguel-Aliaga, I. (2011). Enteric neurons and systemic signals couple nutritional and reproductive status with intestinal homeostasis. Cell Metab. 13(1): 92-1. PubMed ID: 21195352

Cvetkovic, V., et al. (2003). Diencephalic neurons producing melanin-concentrating hormone are influenced by local and multiple extra-hypothalamic tachykininergic projections through the neurokinin 3 receptor. Neuroscience 119(4): 1113-45. PubMed ID: 12831868

de Haro, M., Al-Ramahi, I., Benito-Sipos, J., López-Arias, B., Dorado, B., Veenstra, J. A., Herrero, P. (2010). Detailed analysis of leucokinin-expressing neurons and their candidate functions in the Drosophila nervous system. Cell Tissue Res. 339(2): 321-336. PubMed ID: 19941006

Dib, B. (1999). Food and water intake suppression by intracerebroventricular administration of substance P in food- and water-deprived rats. Brain Res. 830(1): 38-42. PubMed ID: 10350558

Kalra, S., Sahu, A., Dube, G. and Kalra, P. (1991). Effects of various tachykinins on pituitary LH secretion, feeding, and sexual behavior in the rat. Ann. N. Y. Acad. Sci. 632: 332-8. PubMed ID: 1719876

Murphy, K. and Bloom, S. (2006). Gut hormones and the regulation of energy homeostasis. Nature 444(7121): 854-9. PubMed ID: 17167473

Nässel, D. R. (2002). Neuropeptides in the nervous system of Drosophila and other insects: multiple roles as neuromodulators and neurohormones. Prog Neurobiol. 2002 Sep;68(1):1-84. PubMed ID: 12427481

Radford, J. C., Davies, S. A. and Dow, J. A. T. (2002), Systematic G-protein-coupled receptor analysis in Drosophila melanogaster identifies a leucokinin receptor with novel roles. J. Biol. Chem. 277(41): 38810-38817. PubMed ID: 12163486

Sahu A., et al. (1988), Neuropeptide K suppresses feeding in the rat. Regul. Pept. 23(2): 135-43. PubMed ID: 3231743

Terhzaz, S., et al. (1999). Isolation and characterization of a leucokinin-like peptide of Drosophila melanogaster. J. Exp. Biol. 202(24): 3667-3676. PubMed ID: 10574744

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Biological Overview

date revised: 3 September 2012

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