Post-genomic approaches to resolve neuropeptide ...

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Invertebrate Neuropeptides and Hormones: Basic Knowledge and Recent Advances, 2006: ISBN: 81-7895-224-6 Editor: Honoo Satake

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Post-genomic approaches to resolve neuropeptide signaling in Drosophila Erik C. Johnson 222 Winston Hall, Department of Biology, Wake Forest University Winston-Salem, NC 27109, USA

Abstract The completion of the genome sequencing of Drosophila melanogaster has facilitated the identification of the complement of G protein-coupled receptors for this organism. There are 44 receptors that are predicted to have various neuropeptides as ligands and notably, these receptor molecules share common ancestors with mammalian neuropeptide receptors. This is strong evidence that the organizing principles of neuropeptidergic signaling have been highly conserved. Recent efforts have concentrated on pairing these receptors with their ligand partners, and significant progress has been made in identifying Correspondence/Reprint request: Dr. Erik C. Johnson, 222 Winston Hall, Department of Biology, Wake Forest University, Winston-Salem, NC 27109, USA. E-mail: [email protected]

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these receptor molecules. Initial descriptions of neuropeptides and their receptors suggest further experiments delving into the nature of these signaling systems. The application of Drosophila molecular and genetic tools has begun to provide insight into the functional properties of these receptor molecules. I review the current status of “de-orphaned” and “orphan” receptors, and where specific information is known, discuss specific expression patterns of peptides and receptors and their known or potential biological functions. Implications of these investigations are discussed, both in regards to advances in our understanding of the neural control of behavior and physiology, and as an important tool in comparative studies.

Introduction A major challenge in the field of neuroscience is to resolve how signaling events operating within single neurons impact cellular properties and how such alterations in cellular physiology resonate through neuronal networks, thereby regulating behavioral and physiological processes. This goal of modern neuroscience requires comprehensive descriptions of the signals and the receptor molecules that underlie neuronal signaling. Once this information is ascertained, anatomical distributions of these transmitters and receptors can be mapped out, essentially identifying the cellular substrates underlying specific behaviors and physiologies. Consequent to the definition of such neuroendocrine circuits, the arduous task of manipulating these elements and their biochemical properties and quantifying the impact of these manipulations on behavior becomes feasible. This body of information will also allow for a structural-functional analysis of these hormones and targets, which will allow for determination of the contribution and relative importance of discrete aspects of peptidergic signaling in behavioral phenotypes. In general, arthropods represent excellent models for the investigation of neuropeptidergic regulation of behavior and various physiologies. This is in part due to a number of rich behavioral repertoires, despite the relative simplicity of the nervous system. Here, I review recent advances, utilizing a variety of different experimental strategies, which have furthered our understanding of neuropeptide signaling in the genetically tractable model organism, Drosophila. The available genetic and molecular tools in this organism allows for targeted manipulations of specific neuronal populations, as well as generating mutations in genes of interest and the evaluation of behavioral phenotypes. Throughout the Metazoa, neuropeptides play important roles in many different behavioral, developmental, and physiological processes [1-3]. Indeed, this book is dedicated to review the roles of peptide hormones in the invertebrates. One important first task is to resolve the precise number of

Drosophila neuropeptides and receptors

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different neuropeptides present in Drosophila. Many peptide hormones have been identified utilizing traditional fractionation methods, MALDI-TOF spectroscopy, and bioinformatic analysis of the genome. Collectively, such efforts have identified a number of potential neuropeptide hormones in Drosophila [TABLE I]. Many of these peptides were isolated as active components in a bioassay, while recently others were isolated from the secreted peptide pool [4-5]. Still others were found through recent bioinformatic approaches [6]. These searches are based on conserved aspects of neuropeptide gene organization and subsequent precursor processing. Specifically, many neuropeptides exist in multiple copies and are proteolytically processed to give rise to mature neuropeptides, and many are biochemically modified [7]. Although a potential neurohormones can be identified based on these genomic and sequence predictions, their biological existence still requires validation as legitimate peptide hormones. Table I. Present or Predicted Peptide Hormones in Drosophila.

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Table I. Continued

How are these peptide transmitters operating? What are the specific behaviors and physiologies in which specific neuropeptides are operating? It has to be noted that many of these peptides were named based for activity in a single bioassay. For example, the diuretic hormones are able to increase secretion by the Malpighian tubule [8-9]; however, they have a larger set of functions than osmoregulation at the tubule [10]. Therefore, much work needs to be done to fully appreciate the scope and extent of peptidergic signaling. Following that argument, while many biological functions have been ascribed to the actions of specific neuropeptides, a complete understanding of their functional roles is contingent upon a comprehensive analysis of their dedicated receptor molecules. It is apparent that the signals elicited from a receptor can vary depending upon the cellular milieu, and such contextual differences can only be appreciated until receptor molecules are localized to distinct tissues. It is then possible to study the signaling from in vitro manipulated receptors and/or signaling components as well as that from loss of function alleles. Many, if not all, small peptide hormones bind to a well-defined group of receptor molecules with a signature structural motif: seven transmembrane domains, which are the hallmark of G protein-coupled receptors (GPCRs). GPCRs constitute one of the largest superfamilies of proteins, and are ubiquitous throughout the Metazoa [11-13]. This shared structural feature of these receptors has made it possible to identify the complete complement of receptor encoding genes once an annotated genome is in hand. Indeed,


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searches through the Drosophila genome [14] has identified the cohort of genes encoding GPCRs [15], and a comprehensive phylogenetic analysis of these genes has indicated the presence of 44 GPCRs likely to have peptide transmitters as their ligands [16]. In Drosophila, these peptide receptors fall into two families of GPCR; rhodopsin-like (Family A) and the secretin class (Family B) [15-16]. It is notable that prior to this genome identification, only a few neuropeptide receptor genes were known in Drosophila, despite the wealth of genetic information known about this organism [17-19]. These peptide GPCRs are largely derived from common ancestors with the mammalian peptide GPCRs, suggesting that the mechanisms of peptidergic signaling have been highly conserved [16]. This conservation suggests that the merits of a comprehensive analysis of peptidergic signaling in any system include practical biomedical applications. The molecular mechanisms that lie downstream of GPCR signaling are diverse, presenting major challenges to investigators focused on describing the biochemical and biophysical properties of the pathways that transducer neuropeptide action. As their name implies, GPCRs interact with any number of different types of specific heterotrimeric G proteins, the specific nature of which directs the specific signaling pathway(s) to be activated. In Drosophila, there are multiple alpha subunits coupling a receptor to either calcium increases (Gq/o) [20-21], and either positively or negatively impacting cAMP levels (Gs and Gi respectively) [22-23]. There are also apparently multiple subtypes of the βγ subunits as well, further increasing the complexity of intracellular signaling cascades [24-25]. Another issue to be considered is how this suite of peptide receptors is regulated in vivo and to relate such regulation to their signaling and functional properties. It is clear that regulatory elements can significantly impact GPCR function; for example, a human pathology is associated with a heritable variant in the vasopressin receptor that renders the receptor constitutively desensitized, evidenced by a basal association with the arrestins [26]. Typically, the means by which any given GPCR desensitizes depend upon the action of a group of regulatory proteins. Once a ligand is bound to a receptor, there is a conformational shift that allows for receptor interactions with the specific heterotrimeric G protein. Simultaneously, this active conformation is recognized by a specific G protein-coupled receptor kinase (GRK) which phosphorylates the receptor along intracellular residues, which typically (but not exclusively) reside in the carboxyl tail of the receptor [27-28]. This phosphorylated receptor then recruits another molecule, arrestin, which translocates from the cytoplasm to the membrane and binds to the receptor thereby preventing further receptor-G protein interactions [29]. In many cases, the arrestin/GRK complex serves to recruit the endocytotic machinery

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including dynamin and clathrin which leads to the removal of the receptor from the membrane [30, 31]. Internalization of GPCRs from the plasma membrane after prolonged activation by specific ligands is a common phenomenon serving to terminate peptide activation of the receptor complex [32, 33]. Surprisingly, signaling can occur from within the endosome from the receptor-arrestin complex – specifically interactions through the MAP kinase signaling cascade [34, 35]. While these molecular events have been identified utilizing mammalian GPCRs, the mechanisms by which Drosophila and other Arthropod receptors desensitize are likely to be conserved. The genome of Drosophila contains one non-visual arrestin gene called kurtz [36] and two GRKs [37]. Many of these events accurately describe how receptor desensitize, however, it must be noted that there are many different receptors which display significant variations in their desensitization kinetics and properties. A major question to be addressed is: how does this variation in receptor desensitization impact receptor signaling in vivo? Furthermore, there are significant variations in the requirements of peptidergic GPCR signaling, meaning the presence of a ligand and a receptor may not be all that is required for signal transduction to occur. For example, in mammals, the receptor for calcitonin gene related peptide (CGRP) and adrenomedullin (AM) requires additional protein components for receptor activation. The receptor activity modifying proteins (RAMPs) dictate ligand specificity of the CGRP receptor [38-39]. Another subunit, called the receptor component protein (RCP) is also necessary for high amplitude signaling from the receptor-RAMP complexes [40]. Another demonstration of the complexity of neuropeptide GPCR signaling is that high amplitude cAMP signaling from the mammalian PAC-1 receptor requires the activity of the Neurofimbrotosis (NF1) gene (41-42), which is apparently paralleled by the Drosophila PDF receptor [43]. Additionally, the many demonstrations of GPCR dimerization in mammals, consisting of both heterodimeric and homodimeric complexes [44], represent potentially unexplored regulatory controls of invertebrate neuropeptide receptor signaling.

Determination of GPCR properties Many different experimental strategies have historically been employed to identify the potential ligand(s) of a given receptor. In the pre-genomic era, identification of a receptor stemmed from laborious biochemical purification techniques and such efforts were typically predicated upon the knowledge of a target tissue and being able to process large quantities of such receptorenriched tissues. These technical constraints were largely prohibitive in small organisms, such as Drosophila. One approach to identify DNA sequences encoding a receptor was based on low stringency hybridization techniques


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using probes from known receptors. This successfully identified many of the Drosophila homologs to amine receptors and identified two members of the Drosophila tachykinin receptors [17-18, 45-46]. An alternative approach was to expression clone the receptor of interest by isolating the specific message that encoded a receptor of interest [47-48]. However, these techniques are labor-intensive and require specific knowledge of either the receptor sequence or localization. Since the complete annotation of the Drosophila genome [14], many fields, including neuroendocrinology, significantly benefited from the wealth of this information. It became possible to adopt entirely new approaches to seek out receptor genes, specifically identifying candidate genes that possess the diagnostic character of G protein-coupled receptors (GPCRs): seven transmembrane domains. These GPCR genes can then be cloned and expressed in any number of several different expression systems and various parameters of potential receptor-ligand interactions can be measured. What specific feature or features need to be assessed to identify ligands for a candidate neuropeptide receptor? A common approach is to express the receptor in an expression system and quantify amounts of various second messengers as a function of neuropeptide presentation. A challenge to this experimental strategy is determining the nature of the second messenger to be monitored. A negative result may stem from measures of a second messenger or signaling pathway that is not activated by a specific receptor. Many studies have employed the use of the promiscuous G protein subunit, Gα16, which couples many disparate receptors to intracellular calcium release [49]. This reagent potentially circumvents the need to run multiple assays measuring different second messengers, although the use of this tool, while proven effective in being able to measure a receptor response [e.g., 50-52], obscures the nature of the signaling pathways elicited from activation of a receptor [53]. Additionally, efforts to identify the active domains that direct coupling to a second messenger system from a heterotrimeric G protein has lead to the generation of a number of G-protein chimeras [54]. Therefore, it is clear that manipulation of the signals elicited from peptide GPCRs is an important tool in the strategy to initially identify ligands at a given receptor. Intracellular calcium release has become the standard assay for assessing receptor activation, as it can readily be monitored through a number of different reagents. The aqueorin protein, isolated from a cnidarian, displays calcium-dependent bioluminescence [55] which can be detected by a luminometer. Additionally, a number of fluorescent dyes are available that can be used to monitor changes in intracellular calcium, e.g., FLUO3, or other dyes can be employed that determines absolute calcium levels, through ratiometric fluorescence [e.g., 56].

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The second messenger, cyclic AMP, is also an important intracellular signaling molecule that may be altered through receptor signaling. This may be reflected in coupling to adenylate cyclase, the enzyme that synthesizes this molecule, or through coupling to phosphodiesterases, which functions to degrade this molecule [57]. A number of different assays can be used to quantify cAMP levels either directly through immunoassays or indirectly through the use of downstream reporters. Multiple cAMP response element binding domains have been fused to the coding region of the luciferase gene; and such an element can readily be transfected with receptor cDNA and peptide-evoked luminescence can be measured [58]. Direct measures of peptide-receptor interactions can be assessed through binding assays. Employing an enzymatic, fluorescent, or radiolabeled ligand on either native tissues or on membranes derived from a cell line transfected with a receptor provides a gauge of the relative strength of peptide-receptor interactions [59]. These experiments can also offer insight into the relative binding affinities for multiple related ligands, which may or may not be derived from processing of the same peptide precursor. Also, determination of an IC50 or Kd for these interactions can be informative as these parameters are not subject to specific signaling contexts. However, ligand binding can be influenced by receptor interactions with the specific complement of heterotrimeric G proteins, as recent evidence suggests that there is precoupling of receptors to their respective G proteins [60]. Another method to examine specific receptor features is the use of a βarrestin-GFP chimera [61-63]. Simply, this construct is transfected along with the receptor of interest and cells expressing GFP are visualized under a microscope [61]. Peptides are added and a time-series of images are taken to witness the translocation of the arrestin-GFP from the cytoplasm to the membrane. This method can be used to identify ligands for an orphan receptor [63, 64], and it is also important as it enables evaluation of differences in the mechanisms of receptor desensitization. The major advantage of this assay is that GPCR function is observed in real time in living cells. Lastly, genetic approaches can be useful to implicate potential ligands for a receptor. The use of these technologies is also a critical tool to fully appreciate the functional significance of these peptidergic signaling systems. For example, the phenotypes of \ loss of function alleles of rickets, which encodes a Drosophila GPCR that is a member of the glycoprotein receptor subfamily, suggested that this gene may encode the receptor for the bursicon peptide [65-68]. Recent empirical observations have demonstrated that the bursicon peptide is indeed a ligand for the receptor encoded by rickets [69, 70]. Furthermore, the recent identification of the PDF receptor was largely facilitated by P-element insertions into the PDF-R gene [43, 71, 72]. These


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genetic variants of the PDF receptor phenocopy PDF peptide mutations and PDF neuronal ablations [73]. Clearly, genetic analysis will be instrumental in resolving the functional roles of these peptide-receptor signals and how such signals control development, behavior, and physiology. These sets of experiments will also be critical to evaluate potential differences in cellular phenotypes and how they relate to receptor function. In sum, the numbers of different available assays that are able to assess specific ligand induced changes in receptor function offer the investigator a multiplicity of tools to identify ligands for orphan receptors. Perhaps even more substantial than the process of “deorphaning” receptors, is that these tools can provide a wealth of information for any peptide-receptor pair and suggest future experiments. For example, what are the signals that are liberated from a receptor when its specific peptide ligand binds? Additionally, what potential impact does the desensitization machinery have on receptor signaling? Are there multiple ligands for a given receptor, and if so, what are the binding differences between these peptides? What impact does the cell phenotype have upon receptor signaling? This information is required for each of these peptide receptors, as it will suggest how receptor signaling is essential for eliciting functional changes in target cell physiology.

Specific peptides and their receptors I review the current body of knowledge for each peptide-receptor pair that has been identified in Drosophila. I highlight the signaling pathways elicited by receptor activation, anatomical expression patterns, and potential functional roles of these peptidergic signaling systems [TABLE II]. This is not meant to be an extensive review of the physiological and behavioral phenomena that many of these receptors are involved in, but rather an introduction to the disparate and distinct roles of neuropeptides and their receptors.

Allatostatin A (AstA) Allatostatins are a group of peptides that affect the corpus allatum to inhibit the production and/or secretion of the ecdysteroid, Juvenile Hormone (JH). Given the importance and influence that JH exerts on many different biological processes, insight into the mechanisms of many developmental processes may be afforded with the identification of elements which specifically inhibit or activate the release of JH [74]. There are at least three different groups of peptides which recognize disparate receptors that have been termed allatostatic, although this function is dependent on the specific organism considered. The first group of allatostatic peptides, Allatostatin A (AstA), was initially isolated from the cockroach Diploptera punctata [75]. The diagnostic character of the AstA peptides is the YXFGL amidated sequence

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Table II. Neuropeptide GPCRs in Drosophila.


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Table II. Continued

at the C-termini and in Drosophila, the Allatostatin A precursor is cleaved to produce four N-terminal modified peptides [76]. This sequence motif shows some similarity to vertebrate bombesins, although the receptors for this peptide belong to the Galanin family of receptors, rather than to the Bombesin family (discussed below). This peptide is widely distributed through the central nervous system of Drosophila, specifically in four pairs of neurons in the larval brain and eight distinct pairs in the ventral ganglion [77]. There are also AstA immunolabeled cells in neuroendocrine cells of the midgut of larvae and adults [77]. Functional expression and reverse pharmacological approaches identified two related genes in Drosophila that encode the receptors for the Allatostatin A peptide. These genes, CG2872 and CG10001, are members of the Galanin receptor subfamily [16]. Through reverse pharmacological approaches, this peptide was purified on the basis of its activation of the CG2872 receptor when functionally expressed in Xenopus oocytes [78]. This receptor cDNA, when coexpressed with GIRK cDNA (a potassium channel), confers changes in the electrochemical gradient due to GIRK activation vis a vis activation of the allatostatin A receptor [78]. Consequent to this initial study, several other groups furthered the descriptions of these allatostatin A receptors and demonstrated that the receptor paralog, encoded by CG10001 also encoded an Allatostatin A receptor, as shown by expression in CHO cells with either a promiscuous G protein subunit [79] or through direct measures of altered Ca 2+ and a GTPγS assay [80]. These receptors show differences in their ligand sensitivity and G protein coupling, suggesting that they fulfill different functional aspects of AstA signaling. Specifically, the CG2872 (DAR1) differs in a number of receptor

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properties compared to that of the CG10001 (DAR2) receptor. First, the ligand sensitivity of the four different allatostatin A peptides appears to be different as evaluated from signaling and GTPγS assays [79-80]. Larsen et al., 2001 utilized treatments of Pertussis toxin, which can be used to discriminate between G protein subtypes, in conjunction with signaling and GTPγS assays. Without treatment of PTX, the CG10001 and CG2872 appear to be more selective for the AstA-3 peptide and less sensitive to the AstA-4 peptides [80]. However, inclusion of PTX abolished all signaling from CG10001 and reduced the amplitude of AstA signaling from CG2872. Additionally, this toxin altered the pharmacological profile of peptide sensitivity; specifically increasing sensitivity of the AstA-4 peptide compared to the responses in the absence of toxin [80]. Additionally, Larsen et al., 2001 proposes that the CG10001 receptor displays constitutive activity, and this receptor property may indeed have functional significance in vivo. While Lenz et al. 2001 agree that there are fundamental differences in peptide selectivity at the CG2872 gene; they disagree with the specific findings of Larsen et al. 2001. In both studies, receptor cDNA was introduced into Chinese Hamster Ovary (CHO cells), but one study utilized a promiscuous G protein subunit [79]. Given the suggestion that this receptor couples to disparate G proteins [80], such experimental discrepancies are likely to stem from differential G protein coupling issues. Indeed, authoritative experiments such as binding will be needed to fully appreciate which allatostatin A species is most effective at each of these receptors. The expression pattern of one of these receptor paralogs has been mapped in the larval brain. Using antisera directed against a portion of the N terminus of the CG10001 receptor, Bowser and Tobe (2004) observed labeling along the ventral ganglion corresponding to the distribution of the Ast-A peptide. Additionally, within the central brain, this antisera labels structures consistent with glial septa along with cell bodies in the ring gland [81]. The distribution of the other Ast-A receptor and any developmental changes of expression in CG10001 remain unknown, but such efforts will likely facilitate analysis of the functional roles of AstA signaling.

Allatostatin B (AstB) The type B allatostatins (AstB) were first identified in crickets and are characterized by the C-terminal motif W(X)6W-NH2 [82]. The allatostatins are generally referred to as brain–gut peptides due to the dual actions of central inhibition of JH production and peripheral inhibition of muscle contraction in the gut [83]. In Drosophila, the AstB precursor is cleaved to yield five different peptides and the gene encoding these peptides has been cloned [84]. Expression of this peptide, evaluated by in situ hybridization, is localized to


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twelve pairs of neurons within the brain and along the ventral ganglion, as well as endocrine cells within the gut [84]. The functions associated with this peptide in Drosophila are unknown, but it is presumed that there will be peripheral myoinhibitory effects as such functions have been associated with this peptide in Manduca and Locusta [85-86]. AstB also may be involved in the ecdysial program as it has been suggested for Manduca and Bombyx [87]. A candidate AstB receptor has been identified in Drosophila and is encoded by the CG14484 gene [63]. This receptor is one of two members of the Drosophila bombesin receptor subfamily [16]. Expression of the corresponding cDNA in HEK-293 cells with a βarrestin-GFP reporter leads to translocation of the arrestin to the membrane in specific response to application of the AstB peptide [63]. It is currently unknown which second messenger(s) are downstream of this receptor and the distribution profile of this receptor. Additionally, this assay does not readily allow for evaluation of ligand sensitivity, so differential sensitivities to the different AstB peptides at this receptor are currently unknown. This information is critical for the delineation of functional roles of AstB signaling in Drosophila.

Allatostatin C (AstC) The group of allatostatic peptides originally isolated from Manduca sexta, called Allatostatin C (AstC) is present in many different insect orders including other Diptera [88]. In Drosophila, the peptide has also been referred to as drostatin C and flatline, based on strong cardioinhibitory effects [89]. In addition, this peptide inhibits spontaneous contractions of the crop in Drosophila and is expressed in many different larval and adult neurons [8990]. Analysis of mRNA expression by in situ hybridization revealed expression in hundreds of cells throughout the gut, as well as six large clusters of neurons in the larval brain and several pairs of neurons in the ventral ganglion [89-90]. In the adult, AstC mRNA is located in many different clusters throughout the brain [89-90]. These cells appear to be the same as those labeled with a heterologous antibody against Manduca AstC [91]. Two receptors belonging to the somatostatin receptor family [16] are specifically activated by the Allatostatin C neuropeptide. Specifically, expression of CG7285 and CG13702 in Xenopus oocytes with a GIRK, leads to changes in transmembrane potentials when challenged with the AstC peptide in a dose-dependent fashion [92]. Both of these receptor paralogs have equal affinity for the AstC peptide, and estimates of the EC50 for this peptide at both receptors are in the nanomolar range [92]. The signaling from these Drosophila AstC receptors is similar to signals emanating from mammalian somatostatin receptors, furthering the notion that AstC and mammalian somatostatin are evolutionarily related [93].

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Interestingly, expression of AstC receptor cDNAs in mammalian cells does not lead to a similar signaling profile. Expression of these receptors leads to a constitutive trafficking, which can be stopped with the addition of endocytotic inhibitors [63]. Upon the redistribution of the pool of AstC receptors, responses to the AstC peptides could be measured in HEK cells [63]. This curious behavior of these receptors is most certainly not indicative of the situation in native AstC receptor expressing neurons in Drosophila. Johnson et al., 2003b, concluded that this feature likely mapped to the large stretches of continuous serine and threonine residues, which are putative recognition sequences for receptor kinases [e.g., 94]. While the constitutive association of the AstC receptors with βarrestin in HEK cells is not applicable in a natural setting, it does speak to the possibility of receptor regulation through cellular context. AstC receptor signaling may be tightly controlled through the amounts and activation of the desensitization machinery. This notion is supported by the demonstration of differential affinities of mammalian somatostatin receptor subtypes for the arrestin molecule [95] and underscores the importance of the cell phenotype in receptor function.

Adipokinetic hormone (AKH) Adipokinetic hormone (AKH) is an endocrine regulator of mobilization of energy stores. This peptide is an octomer and possesses a pyroglutamine at its N terminus. Within the arthropods there is incredible diversity of the number and sequence of AKH members for a given insect species [96]. Specifically, there are at least three different AKH hormones with variations in their primary sequences in the locust, Locusta migratoria, [97] and in Drosophila it appears there is only a single member of the AKH peptide (4,5). The functions of AKH include cardioactivity in Drosophila [98], and the mobilization of energy reserves from the fat body [99]. It has been suggested, based on these roles of AKH, that this peptide is the arthropod equivalent of the glucagon peptide, however the identification of the AKH receptor suggests that this peptide is evolutionarily related to the Gonadotropin releasing hormones (GnRH) of mammals [16, 52, 100]. AKH distribution in the central nervous system in Drosophila is limited to a small group of cells that comprise the corpus cardiacum and ablation of these cells leads to altered behaviors under starvation protocols and concomitant changes in sugar metabolism [101-102]. Specifically, the loss of this neuronal population leads to increased survival when these flies are challenged with starvation conditions [101-102]. Additionally, manipulation of these neurons leads to a decrease in trehalose levels, and these neurons have been implicated in the process of glucose sensing [103].


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A specific receptor for the Adipokinetic Hormone has been identified and is encoded by the CG11325 gene in Drosophila [52, 104]. This receptor is one of the two members of the Drosophila Gonadotropin Releasing Hormone (GnRH) receptor family [18, 100]. Expression of this cDNA in mammalian CHO cells lead to AKH evoked increases in free calcium [52], although the promiscuous G protein subunit, Gα16, was also employed in this study, thereby limiting interpretations of coupling partners for the receptor. Initially, AKH was purified as the bioactive agent acting upon these receptor transfected cell lines [52] and these results were mimicked with application of synthetic AKH on CG11325 expressing cells. Expression of this cDNA in Xenopus oocytes leads to the activation of the calcium-activated chloride conductance, suggesting that the signaling from AKH through CG11325 is to increase cytosolic calcium concentrations [104]. Collectively, these two studies propose that CG11325 is an endogenous target for the AKH neuropeptide, and the estimations of sensitivity from these two expression systems are in agreement. Specifically, both studies estimate an EC50 in the subnanomolar range, thus this receptor is highly specific for the AKH peptide [52, 104]. The anatomical distribution of the AKH-R is unknown and mapping of these targets will be informative to appreciate the full complement of AKH signaling in Drosophila. Given that this hormone acts to temper the effects of starvation and is a major regulatory element in sugar homeostasis, such efforts to define AKH receptor properties will advance our understanding of such critical physiologies.

Corazonin Corazonin was originally isolated as a cardioactive factor in the cockroach, Periplaneta americana [105]. Subsequently, it has been implicated as a participant in the neuroendocrinology underlying the ecdysis (shedding of the exoskeleton) behavioral program in Manduca sexta [106], and is also expressed in presumptive circadian pacemaker neurons in this same insect [107]. The level of this peptide correlates with the gregarious pigmentation patterns in both Locusta and Shistocerca [108]. In Drosophila, corazonin is expressed in approximately twenty four larval neurons mapping to two different anatomical loci – a cluster of eight pairs along the ventral nerve cord and a set of four cells within each hemisphere. Curiously, the larval corazonin neurons along the ventral nerve cord are absent in the adult brain, suggesting that these neurons may undergo programmed cell death during metamorphosis [109]. This observation of peptidergic cell death is consistent with the described functional roles of corazonin as an ecdysial factor as adult flies obviously do not undergo molts. Peptidergic cell death has been established for the CCAP neurons in Drosophila and Manduca [110,111] and CCAP has been

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implicated as a component of the ecdysis behavioral program. These observation lend support for the hypothesis that corazonin may be likewise participating in Drosophila ecdysis. The receptor for corazonin has been identified and is encoded by the CG10698 gene [63, 104,112, 113]. This receptor is ancestrally related to mammalian gonadotropin releasing hormone (GnRH) receptors [16]. Functional expression of this receptor cDNA in Xenopus oocytes confers specific activity, as measured by the activation of the endogenous calciumactivated chloride conductance in response to application of corazonin [104]. Additionally, expression in CHO cells with the promiscuous G protein subunit (Gα16) leads to specific increases in calcium as measured by aquoerin luminescence [112]. Expression in HEK-293 cells of CG10698 leads to increases in intracellular calcium as directly measured by the fluorescent dye (FLUO-3) [113]. Thus, these experiments suggest that the endogenous signaling pathway elicited by receptor activation is likely to be the phospholipase C pathway, which is a signaling pathway commonly used by mammalian GnRH receptors [114]. Expression of this receptor in HEK-293 cells with a βarrestin-GFP reporter confirms that CG10698 is a corazonin receptor [63]. This independent measure also reveals the desensitization properties of the corazonin receptor. Specifically, recruitment of the βarrestin-GFP chimeric protein to the membrane was rapid and specific to the corazonin neuropeptide. Following recruitment, the receptor appears to rapidly internalize with the βarrestin-GFP, which is also a feature of mammalian GnRH receptors and is further evidence of the similarities between the corazonin and GnRH signaling pathways [27, 115]. This pattern of association of the receptor and βarrestin is thought to be indicative of activation of the MAP-kinase signaling cascade [116]. Thus, this potential activation of both this signaling pathway and the PLC pathway from the corazonin receptor will have to be addressed in vivo. What are the functional roles of corazonin in Drosophila? While there are many different actions ascribed to this peptide in various insects, in Drosophila, no reports of its activity have been published to date. However, the entire cohort of the corazonin neurons in Drosophila expresses receptors for two different diuretic hormones [10]. It may be that these neurons are integrating osmotic state of the animal into unrelated behaviors or physiologies that are downstream of corazonin signaling. Alternatively or additionally, corazonin may play a direct role in control of fluid secretion. Localization of the corazonin receptor to central and peripheral cellular populations may suggest the functional roles of this widespread neuropeptide. Northern blots indicate that this transcript is widespread throughout developmental stages and in different tissues [112]. Identification of the cellular populations that express


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the corazonin receptor will undoubtedly provide clues to the functional significance of corazonin signaling in Drosophila.

Crustacean cardioactive peptide (CCAP) Subsequent to the initial discovery of CCAP in the pericardial organs of the crab, Cancer [117], this peptide has been isolated in a number of insect species, including Locusta migrotoria [118], Manduca sexta [119], and Tenebrio molitor [120]. Remarkably, the primary sequence (PVCFNAFTCamide) of this neuropeptide is invariant over large phylogenetic distances and genes encoding this peptide have been identified in Drosophila, Anopheles, and Manduca [16,121,122]. CCAP is a multifunctional transmitter and has different physiological roles; it has been implicated in the ecdysis behavioral program [118, 123-125], the release of Adipokinetic hormone [126,127], in addition to its established role as a myotropic and cardioactive agent [e.g., 128, 129]. CCAP is found throughout the central nervous system, specifically in neuroendocrine cells along the ventral nerve cord and within the corpora cardiaca, suggesting dual roles as both a synaptic and hormonal modulator. In Drosophila, a number of peripheral neurons express this peptide, specifically cardiac innervating neurons within the adult and CCAP is cardioactive at larval, pupal, and adult stages [130, 131]. The observation that this neuropeptide is cardioactive in Drosophila at developmental stages that lack cardiac innervation suggests that the action of CCAP is direct on the pacemaker and/or myocardium itself. CCAP is expressed in a well-defined subset of peptidergic neurons, specifically, approximately twenty cells along the ventral nerve cord and several within the brain proper. Many of these neurons undergo programmed cell death during metamorphosis [110]. Ablation of these cells leads to deficits in the circadian control of ecdysis [125] and uninflated wings, although some of these phenotypes may be attributable to the CCAP co-transmitter, bursicon [132]. Specifically, many of the stereotyped behaviors involved in the ecdysis program are lengthened [125] and such data are consistent with models placing CCAP as an initiator of ecdysis behavior. Two different research groups have identified the sole Drosophila member of the oxytocin/vasopressin receptor family as the CCAP receptor. Specifically, expression of the open reading frame of the CG6111 cDNA in Xenopus oocytes leads to activation of the calcium-activated chloride conductance in specific response to application of CCAP [104]. Furthermore, expression of this receptor in CHO cells with the promiscuous G protein subunit leads to CCAP dependent increases in cytoplasmic calcium [133]. In the Xenopus oocyte expression system, this receptor was found to be

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responsive to AKH, albeit with half the sensitivity of CCAP evoked responses (240 nM EC50 value for AKH as compared with 130 nM EC50 value for CCAP) [104]. In contrast, introduction of CG6111 cDNA in CHO cells leads to a different pharmacological profile both in terms of sensitivity and specificity. Specifically, Cazzamali et al., found that the receptor encoded by CG6111 is highly specific for the CCAP peptide as no other peptide (of twenty tested) produced a significant change in calcium levels. Likewise, the receptor in this cell line also appears to have greater sensitivity with an EC50 value in the subnanomolar range (~ 0.54 nM ) [133]. These differences may stem from uses of different expression systems, technical differences in the experimental setups, or alternatively, differences in the nucleotide sequences of the CG6111 cDNA as has been suggested by [133]. Further experiments will be informative to determine the sensitivity, specificity, and the signaling of this CCAP receptor. Evaluation of the localization of the CCAP-R transcripts indicates potential different developmental and tissue distributions. Northern blots suggest the receptor is highly expressed in embryonic tissues, wane in larval stages, and begin accumulating through the pupal to adult stages [133]. Additionally, the transcript is highly expressed in the adult head, whereas CCAP-R mRNA is either absent or expressed at low levels in thoracic and abdominal tissues [133]. The use of more sensitive methods, including immunocytochemistry and/or in situ hybridization is needed to identify specific cellular populations that are targets of CCAP modulation.

Drosulfakinin (Dsk) Sulfakinins are a group of peptides that share structural characters with mammalian gastrin/cholecystokinin peptides and possess the XXY*XXRF (where the * refers to a sulfated tyrosine) sequence at their C-terminus. In Drosophila, sulfakinin has been reported to act as a myotropic peptide. In other arthropods, this peptide has been implicated as an anti-feeding agent and such actions are consistent with descriptions of DSK activity in the Drosophila crop and gut [134]. Such functions and sequence similarities imply that the sulfakinins are evolutionarily related to mammalian CCK/gastrin signaling [134,135]. In Drosophila and other representative Diptera, this peptide is expressed throughout the central nervous system, with immunolabeling in the optic lobes and in several soma along the ventral nerve cord [136]. A receptor for the DSK peptide has been identified and is highly similar to the gastrin/cholecystokinin receptors of mammals [16]. The receptor encoded by the CG6857 gene specifically responds to DSK peptides as monitored through calcium increases in CHO, HEK-293, and Sh-EP cells stably expressing this receptor cDNA [137]. This activity was found to be insensitive


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to the G protein toxin, pertussis toxin, implicating Go signaling from the DSKR1 receptor but only in CHO and HEK cells. In contrast, introduction of the DSK-R1 in Sh-EP cells, DSK signaling was found to have both Pertussis sensitive and insensitive components, suggesting coupling to multiple G proteins in this cell line [137]. This result underscores the fact that receptor behavior can be modified due to cellular context and experiments aimed at determining in vivo DSK signaling will need to address potential differences in G protein coupling. Additionally, the receptor is highly selective to the sulfated tyrosine residues within the sulfakinin peptide, as analogues that lack this feature are crippled in their ability to elicit DSK signals [137]. The receptor expression has not been evaluated and such information is necessary to appreciate the functions associated with sulfakinin signaling. Additionally, sequence analysis suggests that the DSK receptor paralog encoded by CG6881 is an additional sulfakinin receptor, although this remains to be empirically determined.

Tachykinin (TK): Multifunctional hormone The insect tachykinins are multifunctional peptides that possess myostimulatory, diuretic, and neuromodulatory activities [138]. The gene Tk (CG14734) encodes a peptide precursor that is cleaved to gives rise to six different Drosophila tachykinin related peptides (see Satake and Kawada, Chapter 10 for peptide nomenclature), DTK-1 to 6 [139]. These peptides are expressed in interneurons within the central nervous system and within endocrine cells of the intestine [140]. The roles for these signaling molecules throughout the Arthropods are quite extensive: tachykinin related peptides have been implicated in olfactory behaviors in Drosophila [141], as cardiac regulators in various Coleopterans (beetles) [142], diuretic factors in Manduca [143], and as mediators of gut contractions in the cockroach [144]. How do these peptides affect these disparate physiologies? What are the signals that impact the release of these peptides? Insights into the answers for these important questions will stem from further genetic manipulations of tachykinin related peptide signaling. In Drosophila, two receptors encoded by CG6515 (NKD or Takr86C) and CG7887 (DTKR or Takr99D), were cloned prior to the completion of genome sequencing based on sequence similarities to mammalian tachykinin receptors [17-18]. The CG6515 receptor was demonstrated to be responsive to heterologous insect tachykinin related peptides from Locusta [18], whereas CG7887 was demonstrated to be sensitive to relatively high concentrations of mammalian substance P [17]. These receptors were recently re-evaluated with authentic Drosophila tachykinin related peptides, and multiple lines of evidence support the notion that these two receptor paralogs are bona fide tachykinin receptors.

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The CG7887 receptor displays activation by all tachykinin isoforms, as measured by elevations in both cAMP and Ca2+ levels in a heterologous expression system [145]. This observation suggests that the CG7887 receptor couples to multiple signaling pathways, which has been shown for a number of different peptide GPCRs including other insect tachykinin receptors as well as mammalian neurokinin receptors [146, 147]. This receptor is also able to recruit βarrestin-GFP in response to Drosophila tachykinin-related peptides [63], and is internalized in response to these peptides [145]. It appears that CG7887 may have preferential affinity for DTK-1, while the other tachykinins display uniformly lower activity levels [145], although further evaluation of this provisional conclusion will be needed. The CG6515 receptor is able to elicit βarrestin redistribution in specific response to tachykinin application [63]. It is currently unclear which second messenger(s) are liberated by this receptor, although early studies that expressed this receptor in NIH-3T3 cells measured changes in IP3 levels in specific response to tachykinin peptides derived from the Locust [18]. The CG7887 receptor is distributed throughout the central nervous system and in discrete peripheral loci. Specifically, within the larval CNS, antisera against the CG7887 receptor labels two descending neuronal projections and soma that are potentially expressing the SIFamide peptide [145,148]. This receptor is also expressed within the tubules [145], which is consistent with previous reports of the tachykinins possessing diuretic activity [143]. Western Blots using receptor antisera detected a single band from fly extracts, the size of which is consistent with predictions of the CG7887 receptor protein. The anatomical organization of the second tachykinin receptor is currently unknown, but given the roles of the tachykinin peptides, the expectation is that this receptor will be expressed in different cellular populations with disparate functional roles.

Leucokinin (LK): A potent diuretic factor Leucokinin peptides function as a potent diuretic factor in Drosophila and other insects and are characterized by a diagnostic FXXWG-amide motif at their C-termini. This peptide was initially identified in the cockroach, Leucopharea [149] and the Drosophila gene encoding this peptide has been cloned [150]. In Drosophila, there is a sole leucokinin peptide member, also referred to as Drosokinin and is the longest known member of this peptide family. Leucokinin is expressed in a pair of lateral horn neurons within the central brain and in eight to ten pairs of neurons along the ventral ganglia [151]. This peptide exerts strong diuretic effects and its site of action has been mapped to the stellate cells of the Malpighian tubule, where it causes increases in intracellular calcium [152].


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A leucokinin receptor has been identified in Drosophila and is encoded by the CG10626 gene [153]. This receptor is a member of the neurokinin receptor subfamily [16] and is also highly related to a leucokinin receptor isolated from the snail, Lymnaea [154]. In agreement of experiments probing the nature of the effects of leucokinin on the stellate cells of the tubule, CG10626 expression in S2 cells leads to leucokinin-evoked calcium increases [153]. The estimated EC50 of such signaling was estimated to be 0.04 nM, as specifically measured by increases of aqueorin luminescence and such values are in general agreement with leucokinin effects and signaling on isolated tubules [153]. An antibody generated against the leucokinin receptor specifically detects a single protein band of the predicted size of the leucokinin receptor. This band is similarly detected in transfected S2 cells, which suggests that this reagent is an authentic reporter for the receptor protein [153]. Within the tubule, expression of this receptor appears to be limited to the stellate cells of the tubule; it is also widely expressed throughout the CNS and other peripheral tissues. While the effects of leucokinin on diuresis are well-documented, the central roles of the leucokinin peptide are currently unknown. However, the receptor is widely expressed in the brain and a specific subset of six neurons express a distinct diuretic peptide called DH44 [155]. Such observations suggest a complex neuroendocrine circuitry controlling the release of diuretic factors which may represent central regulatory elements underlying this physiology. This hypothesis is bolstered by previous observations of extensive coexpression of various diuretic hormones in other Arthropods [10, 155, 156]. A future genetic analysis of leucokinin signaling in Drosophila will likely provide insight into the central and peripheral elements of osmotic regulation.

SIFamide SIFamide was isolated via brute force isolation from the grey flesh fly, Neobelleria on the basis of being active on a Locusta hindgut bioassay [157]. Following its purification, however, it was found not to be myotropic in either Neobelleria or Drosophila [157]. Since its identification, this neuropeptide has been found in a wide variety of Arthropods including the shrimp, Procambrus. The gene encoding this neuropeptide has been isolated in Drosophila, and the distribution of the neuropeptide has been assessed utilizing an antibody against the flesh fly neuropeptide [148]. In Drosophila, two tracts of immunolabeling are seen along the ventral nerve cord [148] and appear to be similar to staining with antisera against one of the Drosophila tachykinin receptors, specifically the CG7887 or DTKR receptor [145]. While this needs to be empirically determined, it may be that tachykinin signaling is a major regulatory feature of SIFamide release, and this may suggest functional properties of SIFamide signaling.

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Recently, a candidate SIFamide receptor has been identified and is encoded by the CG10823 gene [52]. Specifically, introduction of this cDNA to a CHO cell line stably expressing the Gα16 construct leads to dose dependent increases in calcium as measured with the aequorin based luminescence [52]. This receptor is a member of the Drosophila neurokinin group [16] and the estimated sensitivity to the SIFamide peptide is approximated to be 20 nM for an EC50 [52]. Genetic analysis and localization of this receptor might offer insight into the functional roles of SIFamide signaling in insects. In other arthropods, localization of the SIFamide peptide suggests that the function of this neuropeptide is in olfactory information processing [158], and such efforts in insects such as Drosophila will determine if this functional role is conserved as well.

Ecdysis triggering hormone (ETH) Ecdysis-triggering hormone (ETH) is a neuropeptide that regulates the progression of one developmental stage to the next. This peptide, when injected in to the hemolymph can cause precocious ecdysis (shedding of the exoskeleton) [124], and so was implicated as part of the regulatory neuroendocrine events that coordinate such developmental milestones. This neuropeptide is paramount in what amounts to a complex and elegant peptidergic circuitry controlling the timing and sequence of events to coordinate this truly amazing developmental feat. The structure of ETH is that two peptides are produced from processing of the ETH precursor molecule, and both of these peptides are characterized by PRXamide at their C-terminus [159]. ETH is specifically expressed along neuroendocrine cells lining the trachea, referred to as Inka cells [160]. Recovery of a null mutant in the eth gene produces lethal defects in the ecdysis program which notably are rescued with injections of exogenous ETH [161]. ETH promotes the release of Eclosion hormone (EH) which in turn stimulates the further release of ETH via a positive feedback loop [162]. EH and ETH promote the release of CCAP [162]. ETH is thought to initiate pre-ecdysis behaviors and coordinate various physiologies through the subsequent release of these other peptide hormones [162]. In Manduca sexta, corazonin promotes the release of ETH, and it remains to be tested if corazonin is a regulatory element in ETH release in Drosophila [106]. Two different research groups have recently identified ETH receptors encoded by the CG5911 gene. This gene is alternatively spliced to give rise to two different isoforms, both of which are activated by ETH peptides. Specifically, Iversen et al. 2003 and Park et al., 2003 demonstrated ETH dependent changes in calcium fluorescence when CG5911 is expressed in CHO cells with a promiscuous G protein [163-164]. Both receptor variants,


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CG5911A and CG5911B are activated by both ETH-1 and ETH-2. However, these two studies disagree with respect to sensitivity of these receptors for the ETH peptides, as well as to the specificity of the response. Iversen et al., found that only the ETH peptides were able to elicit any significant change in calcium fluorescence with the following sensitivities (EC50s for 5911A (ETH1 ~ 200 nM, ETH-2 ~ 1800 nM) and for 5911B (ETH-1 ~ 37 nM, ETH-2 ~ 160 nM) [163]. Park et al., found in agreement with Iversen et al., in that the 5911B receptor was more sensitive than the 5911A variant, but their estimates of EC50s were for 5911A (ETH-1 ~ 414 nM, ETH-2 ~ 4300 nM) and for 5911B (ETH-1 ~ 0.9 nM, ETH-2 ~ 1.4 nM) [164]. These two studies also disagree with respect to cross-reactivity with other peptides sharing a PRX motif at their C-terminus, including hugin γ, and CAP2B-1 peptides. Notably, these receptors are activated by heterologous peptides from insects but also mammalian Neuromedin U [164]. These differences may be reconciled by differing receptor expression levels or peptide purities. ETH receptor transcripts were found in embryonic, larval, pupal, and adult tissues with the 5911A isoforms peaking in larval stages and the 5911B isoforms peaking in pupal stages [163], suggesting differing functional roles for these two ETH receptors. Exact tissue distribution for each isoform is currently unknown and such knowledge will address the possibility of functional redundancy or discrete functions of these ETH receptors. Nonetheless, these ETH receptors and investigation of their roles will advance our understanding of how critical developmental timing events are regulated by neuropeptides. Finally, it remains open for further investigation the amount that these different peptides which share structural feature with ETH contribute (if anything) to ecdysis behavior.

Cardioacceleratory peptide 2b (CAP2b) and pyrokinins A number of different peptide families are characterized by structural similarities specifically occurring at their C-termini. The cardioacceleratory peptides (also referred to as periviscerokinins) originally isolated in Manduca possess a diagnostic –FPRXamide and the pyrokinins/PBAN peptides possess a –FXPRLamide sequence and lastly the ecdysis triggering hormones possess a –PRXamide sequence. The CAP peptides were originally isolated in the tobacco hawkmoth, Manduca sexta, based on their potent cardioacceleratory effects [165]. Pursuant to their identification, related peptides (periviscerokinins) have been identified in many different insects [166]. Three different peptides are produced from cleavage of the capability (capa) gene in Drosophila and all share the FPRXamide at their C-terminus [167]. In Drosophila, CAP2B-1 and CAP2B-2 possess diuretic properties and signal through a calcium and nitric oxide signaling system, as determined through

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examinations of physiological responses on isolated Malpighian tubules [167168]. The third peptide yielded from proteolytic processing of the capa gene product is called Pyrokinin 1 or CAP2B-3 and possesses the FXPRLamide Cterminal sequence motif. Another gene called hugin encodes two peptides, one related to ecdysis triggering hormones called hugin γ and another with a pyrokinin motif called Pyrokinin-2 [169]. The hugin transcript is expressed throughout each developmental stage; however, levels peak in larval and pupal stages. Expression of the hugin peptide is restricted to the subesophageal ganglia in the embryonic and larval CNS [169]. Ectopic expression of the hugin peptide produces lethality associated with failures in ecdysis, and application of both hugin encoded peptides increases heart rate in Drosophila [168]. Additionally, introduction of a tetanus toxin to alter synaptic transmission in hugin neurons alters feeding behaviors in a nutrient-dependent fashion [170]. A number of potential receptor molecules have been identified for the CAP2B and pyrokinin peptides. Notably, all of these receptors are evolutionarily related to each other and are Drosophila members of the Neuromedin U subfamily. A receptor that is specific for the capa peptides is encoded by the CG14575 gene [104, 171]. Introduction of mRNA for the CG14575 gene into Xenopus oocytes leads to CAP2B induced changes in calcium [104] as measured by gating of the chloride conductance present in these cells with an estimated EC50 of 150 nM and 230 nM for the CAP2B-1 and CAP2B-2 peptides, respectively. Likewise, introduction of this receptor cDNA in CHO cells with the promiscuous G protein leads to CAP2B peptideevoked changes in calcium levels as monitored by aequorin luminescence with an estimated EC50 of 69 nM and 110 nM for CAP2B-1 and CAP2B-2. Transcript levels were measured by Iversen et al., 2002 and these investigators found higher levels of mRNA in the larval stages and reduced, yet consistent expression in adult bodies [171]. This agrees with data presented by Wang et al., 2004, in which the transcriptome of the Malpighian tubules was evaluated and this transcript was found to be enriched in adult tubules [172]. Two receptor paralogs, encoded by the CG8784 and CG8795 genes are receptors for the pyrokinins and related peptides. CHO cells expressing either of these receptors along with the promiscuous G protein exhibits calcium increases in response to the hug γ or pyrokinin-2 peptides [173]. In this expression system, these receptors show a preference for the pyrokinin 2 peptide by one order of magnitude and exhibit EC50 values in the nanomolar range [173]. In Xenopus oocytes, expression of these receptors leads to increased amplitude of the calcium-activated chloride conductance in response to a number of these structurally similar peptides. Rapid desensitization in the oocyte expression system made it difficult to estimate exact sensitivity that


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these receptors have for the pyrokinin 2 and the hugin peptide, yet Park et al., 2002 were able to estimate EC50 values for the responses to ETH-1 and the pyrokinin-1 (CAP2B-3) peptides of the CG8795 receptor [104]. These observations might indicate a greater sensitivity of this receptor for the pyrokinin-2 and hugin γ peptides, as it follows that the degree of desensitization may predict relative ligand sensitivity. Spatial expression of these receptor paralogs is unknown, yet injection of double stranded RNA species of either of these receptors caused heighten embryo lethality, perhaps suggesting developmental roles for these receptors. Further analysis of these receptors is clearly required, as it is still unresolved to what extent these receptors are able to respond in vivo to alternative PRX containing peptides. It is worthy to note that the assignment of these being bona fide pyrokinin receptors may be premature; the response of CG8795 was as robust to ETH-1 as other reported figures for the “ETH receptor” [163-164]. Clearly, localization of each of these receptors will either support the notion of dedicated receptors for specific ligands, or perhaps will indicate that these receptors are able to elicit varying degrees of signaling, dependent upon local concentrations of different peptides. Another receptor belonging to the NMU subgroup displays broad ligand specificities for structurally related peptides. The receptor encoded by CG9918 displays sensitivity to the CAP2B-3 peptide at very large concentrations in Xenopus oocytes [104]. This receptor, when expressed in mammalian cells, is more selective for the CAP2B-3 peptide with an EC50 of ~ 50 nM [174]. It is also responsive to other PRXamide containing peptides including ETH-1, Pyrokinin-2, and the hug γ peptides albeit about ten-fold less responsive [174]. Clearly, these Neuromedin U receptors display differing pharmacological profiles with respect to the PRXamide peptides and much work is required to appreciate the roles of the PRXamide peptides and their Neuromedin U–like receptors.

FMRFamide and dromyosuppressin (DMS) The FMRFamide-related peptides (FaRPs) are a group of peptides with a similar C-terminus, that was first identified in molluscs and include the FMRFamides, myosuppressins, and the sulfakinins (previously discussed). The FMRF precursor undergoes posttranslational processing to yield eight mature peptides [175-177]. Dromyosupressin (DMS) is encoded by a distinct gene and is the sole Drosophila member of the FLRFamide subfamily [178]. Several of the individual peptides produced from the processing of the FMRFamide gene are cardioinhibitory in Drosophila [179, 180]. These same peptides exert excitatory actions at the Drosophila neuromuscular junction [175, 181]. Similarly, DMS significantly lowers heart rate in Drosophila [180];

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contrasting with the effects that this peptide has on the larval neuromuscular junction (NMJ) [177]. The central effects of the FMRFamides are thought to underlie processing of visual information as these peptides are highly expressed near the photoreceptors of adult Drosophila [182, 183]. A number of GPCRs have been identified as candidate receptors for the FMRFamides and supressins. The receptor encoded by the CG2114 gene displays greater sensitivity to the FMRFamide containing peptides but is responsive to DMS albeit at higher concentrations [51, 184]. This receptor is also activated by other RFamide containing peptides including the sulfakinins and sNPFs [51, 184]. This receptor also appears to show differential sensitivity to the different FMRFamides produced from processing of the FMRFamide precursor. In CHO cells, the pharmacological profile of the CG2114 receptor is PDNFMRFa > DPKQDFMRFa > (TPAEDFMRFa = SPKQDFMRFa = MDSNFMRFa) > SVQFMRFa > SDNFMRFa, with no apparent signaling from the SAPQDFMRSa [184]. However, the data reported by Meeusen et al., 2002 with the same receptor in CHO cells disagree with this rank order. Specifically, (PDNFMRF = SDNFMRF = DPKQDFMRF = TPAEDFMRFa), with EC50s ranging from 1.8 nM to 2.1 nM in contrast to Cazzamali et al., who found a range of 0.9 nM to the 100 nM range [51, 184]. Perhaps these differences stem from purity of synthetic peptides used in these different experiments or different absolute levels of receptor expression. In HEK cells, the CG2114 receptor is able to induce the redistribution of the βarrestin-GFP molecule in specific response to either the FMRFamides or DMS [63]. Expression of this receptor appears to be constant throughout development and widespread through different tissues as evaluated with Northern Blots and RTPCR analysis [51, 184]. Additionally, a pair of linked receptor paralogs encoded by CG8985 and CG13803, show greater sensitivity to the DMS peptide but are also activated by the FMRFamides in the nanomolar range. Introduction of either of these cDNAs in CHO cells, co-expressed with Gα16, leads to calcium increases, as monitored by aequorin luminescence, in response to DMS with both receptors displaying an EC50 of ~ 40 nM [185]. In HEK-293 cells, the signaling and desensitization properties of these receptor paralogs were quantified. The CG13803 receptor displayed a sensitivity of 0.17 nM for the DMS peptide and 4.2 nM for the DPKQDFMRFamide and the CG8985 receptor displayed EC50s of 1.8 nM and 13 nM for DMS and DPKQDFMRFamide, respectively, as measured by decrements of forskolin-induced increases in cAMP [63]. Additionally, GTPγS measurements were carried out for the CG13803 receptor and significant effects were measured with 10 nM doses of DMS. Notably, these receptors were also able to recruit a βarrestin-GFP chimeric protein to the membrane, but the details of the response are interesting. Specifically, the


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βarrestin-GFP was translocated to the membrane in CG13803 expressing HEK cells in response to DMS, but not to DPKQDFMRFamide [63]. Furthermore, the response of HEK cells expressing the CG8985 receptor and the βarrestinGFP reporter did not change when presented with either DMS or DPKQDFMRFamide. These findings are supported in the CHO cells, were Egerod et al., 2003 note differences in the desensitization kinetics with these receptors [63, 185]. Curiously, incorporation of the G protein-coupled receptor kinase (GRK2) lead to βarrestin-GFP redistribution in CG13803 expressing cells in response to DPKQDFMRFamide and in CG8985 expressing cells in response to DMS [63]. These authors concluded that these peptides differ in their abilities to recruit the arrestin molecule, presumably due to the poor recognition by specific kinase molecules. Whether or not these receptor behaviors are indicative of in vivo receptor properties remains to be addressed. All three of these RFamide receptors (CG2114, CG8985, and CG13803) are members of the Thyrotropin releasing hormone receptor family [16] and given that these receptors do respond to related peptides, the interpretations of the actions of any one peptide is problematic to determine. Studies aimed at determining receptor expression will be required to fully appreciate functional roles of these receptors. It is tempting to invoke the CG2114 receptor as the excitatory FMRF/DMS receptor at the Drosophila NMJ [181] and the CG8985/CG13803 as the inhibitory DMS/FMRF receptor(s) at the heart and crop [179, 180] based on the signaling properties of these three receptors. Whether or not these receptors are responding to related peptides in a native setting will have to be determined and such efforts will furnish a greater understanding of the functions (known and novel) of these signaling systems.

Proctolin: The first insect neuropeptide Proctolin occupies a special place in the field of insect endocrinology, as it was the first insect neuropeptide to be isolated and sequenced [186]. This pentapeptide, RYLPT, was isolated by virtue of its potent myotropic effects on the hindgut from the cockroach, Periplaneta americana. In addition, proctolin has been shown to be a largely ubiquitous peptide with an invariant primary sequence that subsequently has been identified in a very large number of disparate arthropod species [187]. The bioactivity of proctolin includes cardioregulation [188,189], a co-transmitter at the NMJ [188] and a myotropic factor for various somatic and visceral musculatures [186,187]. Given that this was the first insect peptide to be identified, the identification of the gene encoding the proctolin peptide took thirty years of scrutiny. This underscores the technical difficulties associated with identifying neuropeptide genes. This recent development was contingent on the completed annotation of the Drosophila genome. Recently, Taylor et al., 2004 established

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the gene that encodes the proctolin gene to be CG7105. Specifically, antisera against sequences corresponding to the preproelements of the proctolin precursor, labels the same cohort of cells as antibodies specific for the mature proctolin peptide [190]. The proctolin gene encodes a single copy of the RYLPT sequence and is expressed in neurosecretory cells that terminate in the ring gland, and a number of cell bodies within the ventral nerve cord [190]. Overexpression of the proctolin gene leads to increased heart rate, consistent with previous reports of the cardioactivity of this transmitter [188]. The receptor encoded by the CG6986 gene has been shown to be a proctolin receptor (Proc-R). This receptor is a member of the Thyrotropin releasing hormone receptor subfamily [16]. Introduction of the ORF of the CG6986 gene in two different heterologous systems confers sensitivity to proctolin as monitored by increases in calcium concentrations [113, 191], proctolin binding [113] and by recruitment of βarrestin to the plasma membrane [63]. Egerod et al., 2003 utilized the Gα16 subunit in CHO cells and measured calcium changes in response to proctolin application [191]. In HEK cells, addition of the Gα16 subunit proved unnecessary, as direct measures of calcium changes were detectable in response to proctolin [113]. Therefore, these authors propose that the proctolin receptor couples to Gq proteins, which are upstream of calcium and the IP3 pathway [113]. This proposed signaling pathway of Proc-R signaling is consistent with in vivo observations [192]. Both of these studies agree that the peptide-receptor interaction is highly specific as both studies estimated subnanomolar EC50 values. Further, a radioreceptor assay demonstrated specific binding of proctolin to membranes derived from stable HEK cells expressing the receptor, and estimated an IC50 in the nanomolar range. Lastly, the proctolin receptor is able to rapidly and specifically recruit an arrestin-GFP fusion protein and such interactions are typical for this class of receptor molecule [63]. Northern blots on mRNA from the CG6986 gene suggests that this receptor is highly expressed in the central nervous system and weakly expressed in the gut [191]. An antibody specific to the proctolin receptor detects a single protein, the size of which is consistent with the approximate size of the proctolin receptor [113]. This same antibody detects high levels of expression in the hindgut and is found in neurons that innervate the heart [113]. The differing results obtained by RNA versus immunohistochemical techniques presumably stem from differential sensitivities of the relative assays employed, or potentially from transcript levels being poor indicators of protein expression. The Proc-R is also expressed in tissues which suggest novel functional physiological roles of proctolin signaling. Expression of ProcR in the ellipsoid body in the brain suggests a role for proctolin in mediating visual processing; likewise, receptor expression in pericardial cells


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suggest physiological roles in macrophage activity and lastly, the receptor is expressed in neurosecretory cells within the larval and adult central nervous systems. The identification of the proctolin receptor will prove instrumental in continued mechanistic evaluation of novel and known functional roles of proctolin signaling.

Neuropeptide F (NPF): NPY-like peptide and functions Neuropeptide F is a peptide that shares structural and functional features with the mammalian Neuropeptide Y signaling system, with these similarities evident at both the peptide and receptor level. The NPF neuropeptide contains 36 amino acids and Drosophila NPF shares four of the six residues that are diagnostic characters of vertebrate NPY[193]. Representative members of this peptide are also found throughout different insect orders, including Anopheles, Aedes, Apis, and Tribolium. In Drosophila, the NPF peptide is expressed in four neurons in the brain, and the expression of this peptide is under developmental regulation. Specifically, NPF expression peaks in early third instar larvae and diminishes within twenty four hours, correlating with the onset of food aversion behaviors [194]. Disrupting these neurons using a Diphtheria toxin and overexpression of this peptide both lead to curious alterations in a variety feeding behaviors. Specifically, NPF is thought to promote the active phase of foraging behavior and to inhibit food aversion and social burrowing [194]. A receptor for this peptide has been identified and is encoded by the CG1147 gene [195]. Specifically, expression of this receptor in CHO cells leads to NPF-induced decreases in forskolin-stimulated cAMP levels with an IC50 of 51 nM [195]. Furthermore, Garczynski et al. showed specific displacement of radiolabeled NPF with unlabeled peptide on these mammalian cells expressing the receptor with an IC50 of 65 nM, which is in general accord with the estimates of sensitivity derived from the signaling assays [195]. Confirmation of these results were obtained by Johnson et al. 2003, in which CG1147 transfected HEK cells exhibited NPF dependent βarrestin-GFP translocation to the membrane and not to any other of 16 neuropeptides [63]. Furthermore, the pattern of association of the receptor to the arrestin molecule is consistent with mammalian NPY receptor- arrestin associations [196]. Consistent with the NPF disruption studies, targeted manipulation of the NPFR neurons utilizing the Drosophila binary system to introduce diphtheria toxin to these cells, have indicated novel social aspects to feeding [194] and sensitivity to ethanol [197] that are NPF dependent. Expression of the NPF receptor has been evaluated in third instar larvae utilizing in situ hybridization and immunocytochemistry. NPF-R mRNA is distributed throughout the larval CNS, within a number of cells within the brain lobes and ventral ganglion, and

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within isolated cells within the midgut [195]. Using an antibody against either the 2nd extracellular loop or the 3rd intracellular loop of the NPF-R, Wu et al., found NPF-R immunosignals within the subesophageal and abdominal ganglia [194]. Furthermore, flies deficient in NPF and NPF-R are resistant to ethanol sedation; these phenotypes parallel findings within the mammalian NPY system [197]. Further definition of the NPF circuitry will likely promote a better understanding of peptidergic signaling underlying these complex behaviors.

Short neuropeptide F (sNPF): A feeding regulatory transmitter A number of different peptides are produced from cleavage of the CG13968 protein, some members of which share a common C-terminal sequence. Furthermore, based on sequence similarity to the vertebrate Neuropeptide Y family, and other insect NPF-like peptides; these peptides were referred to as short NPFs (sNPFs) [198,199], although, the homology appears to be limited to C-terminal sequences. Specifically, four peptides are processed from the short NPF precursor; sNPF-1 and sNPF-2 share the Cterminal sequence LRLRFamides, whereas two other peptides, sNPF-3 and sNPF-4 share a RLRWamide at their C-termini [198,199]. The sNPF peptide is apparently expressed at uniform levels throughout the development of the fly, as indicated by Western and Northern Blots [200]. Within the larval nervous system, the peptide is expressed in neurohaemal release sites and in several soma in the brain and ventral ganglion, whereas in the adult nervous system the peptides are expressed in the calyx of the mushroom body and throughout the medulla [200]. Genetic analysis of this peptide has implicated sNPF in an interesting behavioral phenotype. The ORF of the sNPF gene was inserted downstream of a UAS element driven by a heat shock promoter as a means to overexpress the peptide and an RNAi construct was used to reduce sNPF expression. Notably, overexpression leads to an increase in feeding behaviors, and the gene knockdown leads to a decreased number of feeding behaviors [200]. The overexpression construct causes an increased body mass, whereas the RNAi did not decrease body size compared to wild-type controls [200]. Thus, the sNPF peptide is thought to lie at the intersection of feeding and metabolism and at the central and peripheral controls. The receptor encoded by CG7395 (NPFR76F) has been identified as the putative target for the sNPFs, via signaling-based assays [201, 202, 203] and by binding assays [204]. Specifically, each of these four peptides is able to evoke calcium changes when both the receptor and a promiscuous G protein subunit (Gα16) are introduced to CHO cells (201). Likewise, introduction of this receptor and the Gα16 subunit to Xenopus oocytes leads to activation of the inward chloride conductance which is activated by intracellular calcium levels


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[202]. Additionally, introduction of CG7395 mRNA in Xenopus oocytes leads to sNPF evoked changes in a potassium conductance via GIRK channel modifications [203]. Mertens et al., found that each of the four sNPFs were approximately equal in their relative efficacy with perhaps heightened sensitivity to the sNPF-3 and sNPF-4 peptides [201]. Feng et al., 2002 and Reahle et al., 2003 found that the sNPF-1 and sNPF-2 peptides were clearly more effective at activating this receptor upon expression in Xenopus oocytes [202, 203]. Garczynski et al., utilized a binding approach and determined that the sNPF receptor is more selective for the longer species of sNPFs (namely the sNPF-1 and -2 peptides) [204]. Expression of the sNPF receptor transcript has been evaluated by PCR and Northern Blot analysis, with differing results. PCR detected receptor transcripts throughout different larval tissues, including Malpighian tubules, brain, and gut. In adult tissues, faint bands were detected in heads and bodies and an intense band in ovaries (201). Northern Blots of adult tissues detected transcript in the heads and appendages, suggesting primary expression in the central and peripheral nervous systems [202]. Future isolation and targeted manipulation of sNPF target cells will illuminate the mechanisms of how feeding behaviors are regulated.

Diuretic hormone 44 (DH44): CRF-like diuretic factor A group of structurally distinct peptide hormones influence the level of fluid secretion by the Malpighian tubules. One of these peptides shares significant sequence similarity to the vertebrate neuropeptide, Corticotropin Releasing Factor (CRF) [205, 206]. Within Drosophila, this CRF homolog functions to increase cAMP within the principal cells of the Malpighian tubule, consequently leading to the activation of a Vo ATPase [207]. In Drosophila, DH44 is expressed by a total of six neurons in the larval brain, and in stark contrast with many other insect orders, this peptide is absent in neuroendocrine cells residing in the gut [155]. Curiously, the entire cohort of neurons also expresses the receptor for an unrelated diuretic hormone, specifically the leucokinin receptor [155]. This observation suggests a complex peptidergic circuit involved in the control of diuresis. In vertebrates, CRF has two different receptor molecules and that appears to be the case in Drosophila as well as for other insect species [47, 48]. In Drosophila, there are two members of the CRF-receptor family, and, as predicted by sequence similarities, one of these receptors has been shown to interact with the DH44 peptide. The CG8422 and CG12370 genes encode members of the CRF receptor subfamily, and CG8422 has been demonstrated as being a receptor for the DH44 peptide [208]. While descriptions for the CG12370 receptor are lacking, it is predicted based in sequence similarities

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that this is also a receptor for the DH44 peptide. Consistent with studies on isolated Malpighian tubules assays, DH44 elicits strong increases in cAMP levels in HEK cells expressing CG8422 cDNA [208]. This receptor also signals though calcium albeit at higher peptide doses and recruits βarrestinGFP in a fashion typical for this family of receptors [208]. Evaluation of the anatomical distribution of this receptor shows expression in a relatively small group of neurons that produce the corazonin neuropeptide [10]. An antibody generated against a portion of the receptor labels the entire cohort of corazonin neurons and fails to label any segment of the Malpighian tubule. This result that suggests that an additional DH44 receptor must be expressed is supported by a recent analysis of genes enriched in tubules [172] and is likely encoded by the CG12370 gene. Surprisingly, these corazonin neurons are targets for both DH44 and a distinct diuretic hormone, DH31 which resembles mammalian calcitonin and CGRP [209]. The Drosophila neural circuit appears to have parallels to circuits within the mammalian hypothalamus as the CRF (DH44) and CGRP (DH31) receptors are expressed by GnRH (CRZ) neurons [210]. Thus, it appears that the neural architecture of these signaling pathways are to some extent conserved, and thus it may be concluded that these Drosophila neurons are filling similar functional roles to the mammalian neural circuit.

Diuretic hormone 31: Insect calcitonin? Another distinct neurohormone with diuretic properties had been identified in the cockroach and this peptide shows sequence similarity with the mammalian calcitonin/CGRP peptides [209]. This peptide, DH31 in Drosophila, mirrors the activity of the CRF related diuretic hormones, in specific regards to signaling via cAMP and its role in diuresis [207, 211]. Members of this peptide are present in a number of different insect orders [e.g., 212]. The distribution of this peptide has not been fully evaluated, although in the Drosophila larval nervous system, this peptide is expressed in a number of cells within the central brain and in distinct populations along the ventral ganglion [10]. A receptor that is highly specific for this neuropeptide has been identified and is encoded by the CG17415 gene [10]. This receptor, as predicted by sequence similarities between the calcitonin and DH31 peptides, belongs to the calcitonin receptor family [16]. Moreover, this receptor interacts with an additional protein, called the receptor component protein (RCP) which is also a feature of mammalian CGRP and adrenomedullin receptors [40]. An additional feature of these mammalian receptors is reliance of the receptor activity modifying proteins (RAMPs) which determine ligand specificity for these receptors [38, 39]. Searches of the Drosophila genome have not yet revealed


33

the existence of any RAMPs, and the lack of RAMP molecules could reflect that these components of receptor signaling are truly absent in Drosophila, or alternatively, the diagnostic characteristics of RAMPs have not been highly conserved, which has obscured the identification of a Drosophila RAMP equivalent. While there are no apparent Drosophila homologs of the RAMP proteins, the CG4875 encodes a Drosophila RCP with homologs present in Apis, Aedes, and Bombyx [10]. The expression patterns of RCP have not been assessed and such information will perhaps indicate RCP association with other receptors (e.g., CG4935 is an orphan receptor which belongs to the Calcitonin family of receptor molecules). The DH31 receptor is expressed in the corazonin neurons along with the DH44 receptor. The receptor is also expressed in the Malpighian tubules and in other neurons along the larval ventral nerve cord and within the adult brain lobes [10]. Recovery of mutants within the DH31 signaling pathway will facilitate a deeper understanding of the neural control of osmotic physiology.

Pigment dispersing factor (PDF): A circadian hormone Pigment dispersing factor (PDF) was first isolated in crustacea as a factor able to induce pigment migration in isolated eyestalks [213]. In Drosophila, this hormone is co-localized with markers of the circadian clock, specifically a subset of neurons expressing the period gene [214-215]. This observation may be specific to Drosophila, as this co-localization with clock markers is apparently absent in many different insect orders including the Hymenoptera, Lepidoptera, and even related Diptera [216, 217]. Functional analysis of the action of this peptide in Drosophila has revealed a complex neural circuitry underlying temporal organization of behavior. Ablation of these PDF neurons as well as mutations within the PDF gene produce flies with abnormal circadian behavior [73]. Evaluation of the effects of the PDF mutation on the molecular cycles of the expression and distribution of the PER protein indicates that this neuropeptide is essential for coordination of these activities in different pacemaker populations [218]. Additionally, evaluation of PDF binding sites within the brain implies that one function associated with PDF signaling is to synchronize different oscillators. Another behavior downstream of PDF signaling is geotaxis behavior, initially identified through microarray analysis of selected lines. Notably, a PDF mutation results in increased geotaxis and this behavior is sensitive to the absolute dosage of the PDF gene [219]. Recently, three independent groups identified a GPCR that is responsible mediating downstream signaling of PDF [45, 73, 74]. This receptor is a member of the secretin family of receptors [16], and by sequence analysis is most closely related to other members of the Drosophila calcitonin receptor

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subfamily. These reports all conclude that the CG13758 gene encodes a functional PDF receptor, and notably, genetic analysis implicates this receptor in mediating PDF-dependent behaviors, namely organization of circadian and geotaxis behaviors. Specifically, expression of CG13758 in HEK-293 cells or a Drosophila cell line (S2 cells) leads to PDF evoked accumulation of cAMP [43, 71]. Also, these reports generally agree on the level of PDF sensitivity by the PDF-R, each estimating an EC50 in the nanomolar range. Of interest is the demonstration that the inclusion of Drosophila homolog of Neurofimbratosis 1 (dNF1) potentiated such receptor signaling [43]. This feature is consistent with NF1 action on the mammalian PACAP (PAC-1) receptor [41]. Furthermore, Drosophila NF1 has been implicated as an integral clock output factor [220]. In S2 cells, binding of labeled PDF was exhibited only in CG13758 transfected cells, and was competed away with unlabeled PDF, but not proctolin [71]. Genetic analysis of PDF-R mutant alleles phenocopy the circadian defects seen in PDF mutants [43, 71, 72] as well as the geotaxis deficits seen in peptide mutants [43, 219]. Collectively, these three reports establish the CG13758 gene as encoding the PDF receptor which mediates circadian and geotaxis behaviors. Expression of the PDF receptor has been evaluated utilizing in situ hybridization and immunocytochemistry. Consistent with previous reports on the roles of PDF within the circadian system [218, 221], the PDF receptor is expressed within subsets of clock cells [43, 71]. Thus, it is thought that PDF acts to synchronize different oscillators within the fly brain and coordinate locomotor and other circadian output behaviors temporally. Given that Taghert et al., 2001 found that other neuropeptides are important for governing the temporal organization of behavior [222], knowledge of the identity of these peptide hormones and their connections to PDF are imperative to appreciate the roles of peptide hormones in the circadian system. Undoubtedly, continued and concentrated efforts of these peptidergic circuits will reveal the mechanics of circadian clock output and its relationship to behavior.

Bursicon: The post-eclosion hormone Bursicon is a bioactive peptide that has been implicated in the process of cuticular tanning and hardening and other post-eclosion behaviors [223]. This hormone had been initially described nearly forty years ago, but the structure of the peptide (and consequently, the identification of its receptor) had been elusive until recently, when both the structure had been resolved and its receptor functionally characterized. This important and significant discovery was made by two independent groups working along different lines. In newly enclosed blowflies (Sarcophaga bullata) that have been decapitated or neckligated the cuticle fails to tan, indicating a neural origin of the factor that


35

promotes tanning [68]. Efforts to isolate the neurohormone underlying this behavior resulted in the identification of a 30 kDa peptide and a partial sequence of the cockroach buriscon was used to identify a Drosophila gene, CG13419 as the gene that encodes bursicon; notably flies bearing mutations in this gene display tanning and wing expansion defects [68, 224]. Dewey et al., 2004 proposed that a homodimer of CG13419 formed a structure called a cystine knot (which is present in mammalian glycoproteins) and that this was the active bursicon [68]. This neuropeptide is co-expressed with CCAP in Drosophila, as well as in other insect orders [225]. This co-expression is an interesting finding as the ecdysis promoting factors EH and ETH promote the release of CCAP; the accompanying release of bursicon would then act to promote post-eclosion behaviors. Baker and Truman proposed that the actions of bursicon were through the DLGR-2 receptor, as mutations in rickets, the gene that encodes the DLGR-2 (CG8930) receptor have defects in cuticular tanning, which cannot be rescued with applications of bursicon-containing extracts [67]. Yet, recombinant bursicon is inactive at this receptor, prompting Luo et al., 2005 to suggest that the bioactive peptide was a heterodimer consisting of another cystine knot protein [68] based on structural observations of the peptide. Luo et al., 2005 found that CG15284, the only other cysteine knot protein in the Drosophila genome, and found that this formed heterodimers with buriscon, and thus named it pBurs (partner of Bursicon). The heterodimeric peptide was then able to elevate cAMP levels in the DLGR-2 receptor as well as induce tanning in neck-ligated blowflies [69]. Furthermore, binding of iodinated heterodimeric bursicon to DLGR-2 membranes was displaced by Burs/pBurs heterodimers and these experiments showed that the receptor bound this peptide complex with high affinity (Kd ~ 2.5 nM) [69]. Finally, analysis of the expression patterns of pBurs indicated that this peptide is co-localized with bursicon in many of the CCAP neurons [69]. Working along other lines, Mendive et al., 2005 found the cysteine knot protein encoded by CG15284, thought to be a candidate for the bursicon peptide based on the lack of bioactivity of the peptide encoded by CG13419 [70]. Searches through the genome of the honeybee, Apis melliferia, identified an ortholog, the ORF of which was considerably larger. Looking for the corresponding peptide in Drosophila led these researchers back to CG13419, and so Mendive et al. 2005, tested the possibility that active bursicon was a dimer of these peptides. Indeed co-expression of both α-burs and β-burs in COS-7 cells led to the formation of a heterodimeric peptide that could stimulate cAMP production in CG8930 expressing cells and induce cuticular tanning in neck-ligated blowflies [70]. The discovery of the bursicon receptor is a major step towards an understanding of how neuropeptides organize these

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critical post-eclosion behaviors and further research on these molecules will make strides towards these ends.

GPA/GPB: A novel glycoprotein hormone Consistent with general descriptions of active glycoprotein hormones as obligatory dimers [226], Sudo et al., have recently identified a new glycoprotein dimer as a ligand for the CG7665 or DLGR1 receptor in Drosophila [228]. Specifically, a heterodimer of Glycoprotein A and Glycoprotein B produces significant increases in cAMP signaling from HEK cells transfected with CG7665 cDNA with an EC50 in the nanomolar range. The expression of the heterodimeric peptide is found throughout development of Drosophila. The functions of this peptide are currently unknown but notably, this receptor was also activated by a dimeric complex consisting of fly GPA-2/human GPB-5. Likewise, this dimeric complex can also activate the human TSH receptor [227]. These remarkable demonstrations of evolutionary conservation may suggest that the functional roles of this novel signaling system may be related to TSH signaling in mammals. Clearly, the identification of this peptide hormone and its receptor will facilitate answering such questions.

Orphan receptors and barren ligands While the field has progressed rapidly since the genome sequence of Drosophila was completed, there are still several orphan receptors which remain unpartnered with a ligand. Currently, there are fifteen orphan receptors that are predicted to have peptide ligands and this number will likely decrease in the near future (Table II). Based on sequence similarities with known receptor molecules, some of these orphans are likely to be additional receptors for known peptides. For example, the receptor encoded by CG12370 is a predicted DH44 receptor, these predictions are based upon strong sequence similarities to the DH44-R1 encoded by CG8422 and that the CG8422 receptor is not expressed in tubules [172]. Thus, a DH44 sensitive tissue expresses a related receptor to an established DH44 receptor. Likewise, the receptors encoded by CG14593 and CG6881 are likely to be AstB and DSK receptors respectively. However, these predictions will need experimental verification. The remaining orphan receptors are likely to be partnered with any one of the following peptide hormones: amnesiac, ion transport peptide, eclosion hormone, NPLP1, NPLP2, NPLP3, PTTH, as well as other potentially novel or undescribed peptides. Given the functional significance of these peptides, the identification of specific receptors will further our understanding of leaning and memory formation, physiology, developmental processes, and a variety of different behaviors. Undoubtedly, the identification of the remaining ligands


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for these receptor molecules will add to our base of knowledge concerning peptide and receptor signaling.

Conclusions and future directions The field has rapidly made progress with the initial descriptions of these important signaling molecules and their cellular targets. Within the next few years, we might project that the entire complement of peptide GPCRs in Drosophila will be partnered with remaining ligands. Inevitably as a consequence of such research endeavors, we will likely learn new fundamental features of peptide GPCR signaling. It is likely that we will have formulated specific hypothesis concerning the intracellular signals that these peptide transmitters liberate, and begin testing these predictions in the natural environs that express their respective receptors. Additionally, the systematic identification of receptor molecules will facilitate the anatomical mapping of target tissues, which represents an important next step, as the neuronal circuitry that underlies various behavioral and physiological programs will be identified. Lastly, the promise of genetic manipulations of these important signaling molecules in amenable systems such as Drosophila, will likely offer insight into the functional aspects of how signaling in individual cells and the alterations that they produce control and regulate diverse behaviors. With more insect genomes currently being annotated, the information collected in Drosophila will provide a solid foundation to those working in other insects and offer insight into the evolutionary significance of these ancient signaling systems.

Acknowledgements I would like to express my gratitude to my colleagues for helpful discussions and constructive criticism on this review: Joe Crim, John Ewer, Susan Fahrbach, Ashley Johnson, Dan Johnson, Orie Shafer, and Paul Taghert.

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