Identification and characterization of a G protein-coupled for the neuropeptide proctolin in Drosophila melanogaster

Erik C. Johnson*, Stephen F. Garczynski†, Dongkook Park*, Joe W. Crim†, Dick R. Na¨ ssel‡, and Paul H. Taghert*§

*Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110; †Department of Cellular Biology, University of Georgia, Athens, GA 30602; and ‡Department of Zoology, Stockholm University, S-106 91 Stockholm, Sweden

Edited by John H. Law, University of Arizona, Tucson, AZ, and approved March 24, 2003 (received for review January 8, 2003) Proctolin is a bioactive neuropeptide that modulates interneuronal through heterotrimeric G proteins in the cockroach (20), we and neuromuscular synaptic transmission in a wide variety of arthro- included this peptide among other potential ligands with which pods. We present several lines of evidence to propose that the orphan to test functional activation of the orphan peptide GPCR G protein-coupled receptor CG6986 of Drosophila is a proctolin potentially encoded by CG6986. We found that CG6986 expres- receptor. When expressed in mammalian cells, CG6986 confers second sion confers selective proctolin sensitivity to a heterologous cell messenger activation after proctolin application, with an EC50 of 0.3 type. We then extended our studies to further implicate CG6986 nM. In competition-based studies, the CG6986 receptor binds proc- as a strong candidate for a Drosophila proctolin receptor. nM). By microarray analysis, CG6986 4 ؍ tolin with high affinity (IC50 transcript is consistently detected in head mRNA of different geno- Materials and Methods types, and under different environmental conditions. By blot analysis, Molecular Cloning. We constructed a full-length CG6986 coding anti-CG6986 antibodies detect a band in tissue homogenates similar sequence appropriate for expression (25) in HEK cells by to the predicted size of the protein. Proctolin receptor immunosignals stepwise PCR amplification using RACE-generated cDNAs and are found in the hindgut, heart, and in distinct neuronal populations a CG6986 EST as templates. Details of the cloning steps and of the CNS; such patterns correlate with previous demonstrations of reagents are found in Supporting Materials and Methods, which is proctolin biological activity, and in several instances, with areas of published as supporting information on the PNAS web site, proctolin peptide immunosignals. The identification of a bona fide www.pnas.org. proctolin receptor provides the basis for a mechanistic analysis of this critical synaptic modulator. Cell Culture, Transfections, and Functional Assays. HEK-293 cells that contain a FLP recombination target (FRT) site integrated he pentapeptide proctolin (RYLPT) was the first neuropep- within the genome (Invitrogen) were cultured on DMEM sup- Ttide to be isolated and sequenced from (1). It was plemented with 10% FBS and 100 units per ␮g per ml of a purified based on its myotropic actions on the hindgut, and penicillin͞streptomycin solution. Cells were transfected with a was subsequently found to have wide distribution throughout the total of 10 ␮g of plasmid DNA [a 9:1 ratio of pOG44 to CG6986 arthropods (2–4). In the crab Cancer, proctolin has potent effects in pcDNA5͞FRT] by using Lipofectamine (Invitrogen) according on defined neural circuits within the stomatogastric system (5) to the manufacturer’s recommendations. After 48 h, the cells and at the neuromuscular junction (6). In insects, proctolin is a were selected on DMEM containing 100 ␮g͞ml hygromycin B. major peptide cotransmitter and it modulates contractions of Cells were split at a 1:5 ratio every 3 days and maintained under both somatic and visceral muscles (7, 8). Proctolin-like immu- an atmosphere of 5% CO at 37°C in a humidified incubator. nosignals are found within a small number of in the 2 After selection for resistance to antibiotics, cells were assayed for larval CNS of Drosophila and these include efferents to body- wall muscles, heart, and viscera (9). ligand-dependent receptor activation by monitoring changes in The mechanisms of actions of proctolin have also been intracellular calcium after incubation with the dye, Fluo3-AM investigated. Proctolin elevates levels of inositol trisphosphate (Molecular Probes). Details of dye incubation and fluorescence measurements are provided in Supporting Materials and Methods. (IP3) and cAMP, and increases calcium entry through voltage- gated ion channels (10–13). Several studies have suggested the existence of multiple proctolin receptor subtypes based on Binding Assays. Membranes were prepared by differential cen- binding studies and activities of proctolin (14, 15) or its structural trifugation through sucrose. Proctolin was iodinated by a chlo- analogues (16–19). However, more recent studies (15, 20, 21) of ramine-T method (26). Conditions for determination of specific 125 proctolin binding support a model featuring a single proctolin binding of I-labeled proctolin to membranes were as follows: receptor. The identification of a specific proctolin receptor will membranes were added to a 1.5-ml polypropylene microfuge help address issues regarding possibilities of multiple receptors, tube containing 100 pM 125I-labeled proctolin in 50 mM the precise nature of proctolin-dependent signaling pathways, Tris⅐HCl, pH 7.5, 1ϫ Hanks’ balanced salt solution, 1.5% BSA, and phylogenetic differences in proctolin signaling. The signif- and protease inhibitor in the presence of various amounts of icance of such studies is substantial because proctolin exerts unlabeled proctolin or other peptides. For HEK cells transfected such a widespread role as a synaptic modulator in arthropod with CG6986, the ability of unlabeled proctolin to displace physiology. binding of the label to membranes was compared with six other Analysis of the completed Drosophila genomic sequences (22, neuropeptides. Binding reactions were conducted at room tem- 23) identified Ͼ100 genes encoding G protein-coupled receptors perature for 2 h and were terminated by centrifugation and (GPCRs). Further analysis indicated that 44 such receptors are subsequent washing. Pellets were collected for determination of likely to have peptide ligands and that the majority of these are derived from ancestors for mammalian peptide GPCR genes (24). The CG6986 gene encodes a predicted peptide GPCR that This paper was submitted directly (Track II) to the PNAS office. is distantly related to the thyrotropin-releasing factor receptor Abbreviations: GPCR, G protein-coupled receptor; SP, sex peptide. family (24). Because proctolin was previously shown to signal §To whom correspondence should be addressed. E-mail: [email protected].

6198–6203 ͉ PNAS ͉ May 13, 2003 ͉ vol. 100 ͉ no. 10 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1030108100 Downloaded by guest on September 30, 2021 Fig. 1. Effects of peptides on HEK cells stably expressing CG6986 or CG10698. Fluorescence was measured before and immediately after peptide addition. The concentration of all peptides shown was 10Ϫ8 M. Compared with control cells, only proctolin induced significant changes in cells expressing CG6986 and only corazonin induced significant changes in cells expressing CG10698. AstB, allatostatin B; AKH, adipokinetic hormone; CCAP, crustacean cardioacceleratory peptide; ETH, ecdysis-triggering hormone; FMRF, DPKQDFMRFamide; Ast, allatostatin A; AstC, allatostatin C; DMS, dromyosuppressin; PDF, pigment dispersing factor; IFa, IF amide; dTK, Drosophila tachykinin; and CAP-2B1, cardioacceleratory peptide 2B1.

radioactivity on a Packard Gamma II counter. Raw counts at a 1:1000 dilution, and antisera preadsorbed with synthetic obtained from the binding assays were converted to percent total peptide immunogen (100 nmol͞ml). Images were obtained on a binding, and resulting data were analyzed by nonlinear regres- Zeiss Axioplan microscope equipped with a Hamamatsu charge- sion analysis with GRAPHPAD PRISM software (San Diego). coupled device camera (Ichinocho, Japan) using OPENLAB 3.1.2 More complete details are provided in Supporting Materials and software (Improvision, Coventry, U.K.) and PHOTOSHOP 6.0 Methods. software (Adobe Systems, Mountain View, CA).

Immunocytochemistry and Western Blots. A synthetic peptide cor- Results responding to the C-terminal 20 aa of the predicted CG6986 Functional Assay. We generated six independent HEK 293 lines NEUROSCIENCE product was used to generate rabbit antisera (Multiple Peptide that were stably transfected with a cDNA corresponding to Systems, San Diego). An antiserum raised in rabbit to proctolin CG6986 and one line stably transfected with a cDNA corre- conjugated to thyroglobulin with glutaraldehyde was produced sponding to CG10698. We tested these lines with a library of commercially (Euro Diagnostica, Malmo¨, Sweden). HEK cells were fixed for 60 min at RT in 4% paraformaldehyde in PBS, containing 7% picric acid (vol͞vol). The cells were washed and stained for1hatroom temperature in anti-CG6986; cells were washed, then stained with Cy3-conjugated anti-rabbit antibodies (Jackson Laboratories, West Grove, PA). For Western blots, SDS gels were electrotransferred to poly(vinylidene difluoride) membranes, then incubated with primary antibody at a 1:750 dilution for 1 h, and signals were detected after incubation with an alkaline phosphatase-conjugated anti-rabbit antibody. Immunocytochemistry was performed on tissues of adults and third-instar larvae of the Oregon R strain. Fresh-frozen, 15-␮m cryostat sections were collected from adult heads, thoraces, and abdomens and fixed in 4% paraformaldehyde in sodium phos- phate buffer for 1 h. Sections were incubated for 24 h with diluted antireceptor (1:1,000) and antipeptide (1:2,000) antisera. We used either Cy3-tagged secondary antiserum or the biotin- streptavidine method (with peroxidase detection) using a Fig. 2. Dose responsiveness of HEK cells expressing CG6986 to proctolin and DAKO ABC kit following manufacturer’s recommendations. SP application. The percentage changes due to proctolin (■) and SP (Œ) Whole-mount staining was performed on adult and larval CNS addition were computed as means Ϯ SE from eight wells tested per dose per and intestine by using Cy3 detection. We tested preimmune sera peptide.

Johnson et al. PNAS ͉ May 13, 2003 ͉ vol. 100 ͉ no. 10 ͉ 6199 Downloaded by guest on September 30, 2021 bound 125I-proctolin specifically. For label at 0.1 nM, total specific binding was composed of Ϸ8% of the added 125I- proctolin. Nonspecific binding (addition of 1 ␮M unlabeled proctolin) constituted only 1% of the label bound. The binding of 125I-proctolin was displaced by addition of unlabeled proctolin in a concentration-dependent manner (Fig. 3), with an IC50 of 4 nM (R2 ϭ 0.9941). In contrast, label was not displaced to any similar degree by corazonin, [Leu-5]enkephalin, [Met-5]- enkephalin, FMRFamide, leucomyosupressin (LMS), or leuco- kinin. Lack of such competition by other peptides further supports the specificity of binding exhibited by synthetic proc- tolin. Membranes prepared from untransfected control cells exhibited no specific binding for 125I-proctolin (data not shown).

Expression. We evaluated potential expression of both transcript and protein levels in Drosophila tissues. Measures of CG6986 RNA levels were mined from data collected in previously

125 described microarray experiments that studied adult head RNA Fig. 3. Competition for I-proctolin binding to membranes prepared from (ref. 29; raw data are available at http:͞͞circadian.wustl.edu). HEK cells stably transfected with CG6986. Membranes were incubated with 100 pM 125I-proctolin and various concentrations of unlabeled proctolin (■) That data set includes measurements from two genotypes (yw [Leu-5]enkephalin (Œ), [Met-5]enkephalin (), leucomyosupressin (᭜), FMRF- and period w). Each genotype was also measured in each of two amide (F), leucokinin VIII (ᮀ), or corazonin (‚)for2hat30°C. Values indicate environmental conditions (12 h light, 12 h dark, and constant means Ϯ SE (n ϭ 3). darkness). Finally, the data includes measurements made at 4-h intervals to monitor possible diurnal and͞or circadian variation. CG6986 is expressed in adult head at low, but sustained levels insect bioactive peptides for receptor activation by monitoring (Fig. 4A) that match those of a previously identified peptide changes in calcium-dependent fluorescence. The addition of GPCR [CG1147, neuropeptide F receptor (NFPR); ref. 30]. This individual peptides to untransfected cells resulted in small consistent level of expression is notable given that roughly half responses (Fig. 1). CG6986-transfected cells responded to proc- of the Ϸ14,000 genes represented on the microarray were not Ͻ Ϫ12 tolin at 10 nM (P 10 ), but not to any of 14 other peptides reliably detected with adult head RNA (29). The average tested (Fig. 1). At 1 ␮M, both proctolin and sex peptide (SP) Ϫ Ϫ expression levels for these peptide GPCRs did not change produced responses (P Ͻ 10 8 and P Ͻ 10 4, respectively), significantly as a function of genotype, of environmental con- although the magnitude of the response to proctolin was much dition (Fig. 4A), or of time of day (data not shown). CG6986 larger. Further, CG6986 was sensitive to proctolin at subnano- protein levels were detected with an antibody directed to the C molar concentrations, whereas it did not respond to SP at terminus of the predicted protein. HEK cells that were stably concentrations Ͻ1 ␮M (Fig. 2). CG10698-transfected cells dis- transfected with CG6986, but not with untransfected cells, were played specific responses to corazonin with an EC50 comparable specifically stained (data not shown). By Western blot analysis to values reported (27, 28) and did not respond to proctolin at (n ϭ 5), we observed a band of Ϸ60 kDa (Fig. 4B), which is close doses as high as 1 ␮M. From the dose-response curve (Fig. 2), to the predicted value of 62 kDa for the CG6986 protein we calculated an EC50 value for proctolin on CG6986 cells of 0.3 (Berkeley Drosophila Genome Project). nM (R2 ϭ 0.9662). That value suggested that proctolin is a good candidate to be an endogenous ligand of the receptor encoded Distribution of Immunosignals. In the adult brain, punctate labeling by CG6986. was observed in the neuropil of the superior median and lateral protocerebrum, of the ventral lateral protocerebrum, of the Binding Properties. To test the hypothesis that proctolin might be upper division of central body, and of the subesophageal gan- an endogenous ligand of CG6986, we characterized CG6986 glion (Fig. 5A and C–F). In the optic lobe, labeling was detected binding properties with a RRA. Membranes prepared from HEK in the serpentine layer of the medulla (Fig. 5D) and in a basal cells that were stably transfected with the CG6986 receptor layer of the lamina (Fig. 5E). Labeling was also seen in a number

Fig. 4. In vivo expression of CG6986. (A) Histograms indicating means (ϮSE) for average fluorescence of CG6986 and CG1147 on each of several genome-wide microarrays (ref. 29; http:͞͞circadian.wustl.edu). For the different conditions listed: WT LD, n ϭ 18; WT DD, n ϭ 12; per LD, n ϭ 12; and for per DD, n ϭ 12. (B) Western blot analysis of whole adult bodies stained with antisera to CG6986-predicted protein. MW standards are indicated in kDa.

6200 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1030108100 Johnson et al. Downloaded by guest on September 30, 2021 NEUROSCIENCE

Fig. 5. Immunolabeling in Drosophila tissues with antiserum to proctolin receptor (A and C–J) and to proctolin peptide (B). (A) Tracing of receptor distribution in adult Drosophila brain (compressed frontal view). A few clusters of cell bodies are seen dorsal to the medulla (Me). Posterior to the central body (CB) and in the median neurosecretory cell group (arrow). Punctate labeling is seen in the Me, dorsally in the CB, adjacent to the median neurosecretory cells and in dorsal (DLP) and ventral (VLP) lateral protocerebrum. (B) Tracing of antiproctolin labeling. Cell bodies are seen in lateral neurosecretory cell group (LNC), dorsal to the medulla and in vertrolateral protocerebrum. Faint punctate processes were also seen in the medulla, but were not drawn. (C–F) Proctolin receptor labeling in adult brain. (C) Anterior brain section with punctate labeling in the CB and protocerebral neuropil. Cell bodies are seen in the MNC group and lateral protocerebrum (arrow). (D) Labeling in the serpentine layer of medulla (optic lobe) and in a pair of dorsal cell bodies. (E) Labeling of neural processes in basal neuropil of lamina (optic lobe). (F) Labeling in the VLP and subesophageal ganglion (SEG). (G) Labeled neurons in larval brain; some of these are neurosecretory cells (NS) with processes arborizing on the wall of the anterior aorta adjacent to the ring gland. (H) Labeling of the adult intestine, in the muscle layer of the hindgut (HG) and basal portion of Malpighian tubules (arrow), but not in the midgut (MG). (I) In the abdominal aorta (AA) of the adult, motor nerve terminals on the heart muscle and adjacent pericardial cells are labeled. (J) Detail of labeled neuronal terminals on heart.

of neuronal cell bodies in the brain and optic lobes (Fig. 5A). Two are two pairs of labeled cell bodies in the lateral dorsal proto- prominently labeled cell bodies are located posteriorly in the cerebrum and a pair in the medulla. superior median protocerebrum and eight others are located in Fig. 5B shows the proctolin immunoreactive cell bodies that the median neurosecretory cell group of protocerebrum. There are present in adult brains. Mostly, there appears good corre-

Johnson et al. PNAS ͉ May 13, 2003 ͉ vol. 100 ͉ no. 10 ͉ 6201 Downloaded by guest on September 30, 2021 spondence between the distributions of peptide and receptor Microarray analysis of the CG6986 transcript confirmed that immunosignals. For example, peptide immunoreactive processes it is expressed consistently in the head of adult . Although were seen in the upper division of the central body, in the lateral absolute levels of CG6986 mRNA appeared low, they were and superior median protocerebrum, and in the medulla (not comparable to those of the neuropeptide F GPCR, CG1147 (30). shown). Proctolin binding sites from locust hindgut and oviducal muscles The adult thoracic abdominal ganglia displayed very little display an estimated size Ϸ50 kDa after partial purification (40). proctolin receptor immunolabeling. In the larval brain, at least We estimated the size of the CG6986 protein at Ϸ60 kDa by two pairs of median neurosecretory cells were immunolabeled Western Blot analysis, a size consistent with the computational together with three more pairs of protocerebral neurons (Fig. prediction. There are several possible reasons for the apparent 5G). In the adult and larval hindguts, intense immunolabeling size difference, including species variation, and͞or the possibility could be seen in the muscle layers (Fig. 5H) and Malpighian of additional genes that encode alternative functional proctolin tubules (especially their basal portions). In adult heart, we receptors. observed distinct labeling of neuronal terminals (Fig. 5 I and J), By immunolocalization, this receptor is highly expressed in as well as strong labeling of the adjacent pericardial cells (Fig. tissues that were previously shown to be physiologically respon- 5I). Preimmune serum and preabsorbed receptor antiserum sive to proctolin. Such data represent still another line of produced no labeling in the nervous system or muscle. evidence to implicate CG6986 as a proctolin receptor. Specifi- cally, marked CG6986 receptor expression in the hindgut is Discussion consistent with the potent myostimulatory effects of proctolin on We demonstrated that the GPCR encoded by CG6986 is likely that tissue in cockroaches (1). A similar physiological action of an integral component of proctolin signaling in Drosophila. proctolin was confirmed for the case of the Drosophila hindgut, Our evidence stems from results of three independent experi- which is also innervated by proctolin-immunoreactive neurons mental approaches. We showed (i) a functional and highly originating in the abdominal ganglia (9). Expression of the specific response to proctolin, (ii) high-affinity binding of the CG6986 receptor in Drosophila Malpighian tubules is consistent pentapeptide to the receptor, and (iii) anatomical distribution with reports that, in the locust, proctolin induces tubule secretion of the receptor consistent with previously described proctolin and stimulates tubule writhing (41). Finally, there appears an sensitivity. excellent correspondence in several brain regions between the We showed that the expression of CG6986 in the heterologous distribution of proctolin receptor immunosignals and those of HEK system specifically conferred sensitivity for the neuropep- proctolin-expressing neurites. There are no previous descrip- tide proctolin. The high sensitivity of this functional assay to tions of the spatial distribution of proctolin in the adult brain of Drosophila Calliphora proctolin is consistent with EC values previously estimated . However, studies in the larger (42) 50 revealed a number of neurons similar to the ones shown herein from in vivo physiological studies of proctolin action (20). for Drosophila. However, expression in a heterologous cell line is not strictly Information regarding locations and times of proctolin recep- comparable to normal receptor expression in vivo; although a tor expression can be of value in the interpretation of proctolin similarity in EC values is suggestive, we do not consider that 50 signaling. For example, several sites where proctolin was as- evidence alone to constitute compelling proof. Therefore, to sumed to act directly, such as hindgut and heart, were immu- establish independent evidence, we evaluated the binding prop- nolabeled here. Several groups have postulated a role for erties of this receptor for proctolin. Our estimation of the IC50 proctolin in regulating heart rate of various insects (16, 43). We value of 4 nM for proctolin competition is in accord with IC50 did not observe staining in the heart proper, but did observe values for other peptide GPCRs (30), and suggests a high-affinity minor staining in the anterior aorta and in some nerve terminals interaction. Significantly, that value is within an order of mag- apposed to cardiac tissue. There are conflicting reports on nitude of the estimated proctolin EC50 value. We also note that proctolin action on heart rate in Drosophila (9, 44). Given the the IC50 estimation represents 10–100 times greater sensitivity mechanism of cardiac pacemaking and its modulation (45), any than was seen for proctolin binding to tissue membrane prepa- direct action of proctolin on heart muscle is likely to be excita- rations from larger insects (20, 21). That discrepancy may derive tory, whereas indirect action onto neurons that innervate the from several cellular differences between the mammalian cell heart could have a net excitatory or inhibitory action (46). line and Drosophila tissues. Alternatively, variation in IC50 Proctolin receptor immunosignals on neurosecretory cells sug- estimations may derive from inherent differences in proctolin gest the peptide may act as a releasing (or inhibiting) factor of receptors among different arthropod species, or differences other, specific peptide hormones. Identifying those cells, and because of receptor interactions with other signaling molecules. relating their secretory products to proctolin actions, may be a ␣ The ‘‘promiscuous’’ G protein G 16 (31) has been used in useful step in providing detail to neural circuits that control several recent studies of Drosophila peptide GPCRs to improve discrete physiological functions. An unexpected role for proc- the chances of generating a measurable response (e.g., refs. 28, tolin in visual processing was indicated by the presence of both 32–33). Addition of this subunit proved unnecessary for study of peptide and receptor immunostaining in the medulla and lamina. ␣ the CG6986 receptor: Coexpression of G 16 did not alter Likewise, the expression of receptor immunosignals in pericar- proctolin sensitivity in HEK cells (data not shown), but did dial cells suggests that proctolin may have unpredicted roles in increase responses to other peptides (E.C.J., unpublished work). cardiac physiology. We assume that the increase in calcium-dependent fluorescence In its transmembrane domains, CG6986 shares only limited after receptor activation in HEK cells is the result of Gq coupling identity with other peptide GPCRs (24). It is distantly related to and concomitant activation of the phospholipase C pathway (34). the candidate dFMRFamide receptor CG2114 (32, 33) and to There is substantial evidence implicating phosphoinositol me- the orphan CG16726 receptor. The primary significance of the tabolism as a component of proctolin signaling (11, 12, 18, 20), present work lies in its potential to use a genetic analysis in but other pathways have also been proposed (35–37). Proctolin Drosophila to explore the functions and mechanisms of proctolin signaling by means of CG6986 could be responsible for all of signaling in vivo. However, the discovery of a candidate proctolin these divergent observations (see refs. 38 and 39 for examples of receptor will also promote further studies of proctolin-signaling GPCRs coupling to multiple signaling pathways). Alternatively, mechanisms underlying synaptic modulation in other arthro- it is possible that additional proctolin receptors may be necessary pods. For example, three different proctolin-expressing neurons to explain all of the proctolin actions so far demonstrated. produce robust modifications of the pyloric circuit in Cancer

6202 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1030108100 Johnson et al. Downloaded by guest on September 30, 2021 borealis (5). However, the precise details of the regulatory effects sitivity (49). Likewise, the moth Manduca displays proctolin- depend on which proctolin is activated, and the mech- immunoreactive neurons, but there is no reported evidence of anisms that pattern such fundamental synaptic modulation are functional responsiveness to the peptide (50). Given these unknown. Clearly, information about sites of proctolin receptor suggestions of evolutionary variation, the ability to assay for expression will present a useful means with which to pursue such proctolin receptor expression will strengthen efforts to interpret issues. Comparative studies may also reveal evolutionary diver- the occurrence and significance of proctolin modulation in gences, as proctolin signaling may not be a uniform feature of arthropods. arthropod phylogeny. BLASTP searches of the Anopheles (mos- quito) genome reveals an absence of candidate CG6986 ortho- We thank Anne Karlsson for technical assistance, Cornelis Grimme- logues (47). The mosquito genome contains a candidate proctolin likhuijzen and Michael Adams for sharing unpublished information, Erik gene (48), but its organization is dissimilar to the best candidate Kubli and Jan Veenstra for samples of synthetic peptides, and Randy found in the Drosophila genome (CG7105). Notably, CG7105 Hewes for comments on the manuscript. We also thank the Berkeley Drosophila Genome Project for genomic and EST sequence information. has an expression pattern similar to that of proctolin and The work was supported by a Keck Fellowship (to E.C.J.), a grant from proctolin-precursor immunolabeling (D.R.N., A. M. E. Winther, the Human Frontier Science Program (to D.R.N. and P.H.T.), grants R. J. Siviter, M. Dushay, A. D. Shirras, and R. E. Isaac, from the McDonnell Center (to P.H.T.), and National Institutes of unpublished work). The mosquito hindgut lacks proctolin sen- Health Grant NS21749 (to P.H.T.).

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