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Two Subtypes of Ecdysis-triggering Hormone Receptor inDrosophila melanogaster

2003; Elsevier BV; Volume: 278; Issue: 20 Linguagem: Inglês

10.1074/jbc.m301119200

1083-351X

Yoonseong Park, Young‐Joon Kim, Vincent Dupriez, Michael E. Adams,

Insect and Arachnid Ecology and Behavior

Insect ecdysis is a hormonally programmed physiological sequence that enables insects to escape their old cuticle at the end of each developmental stage. The immediate events leading to ecdysis, which are initiated upon release of ecdysis-triggering hormones (ETH) into the bloodstream, include respiratory inflation and sequential stereotypic behaviors that facilitate shedding of the cuticle. Here we report that the Drosophila geneCG5911 encodes two functionally distinct subtypes of G protein-coupled receptors through alternative splicing (CG5911a and CG5911b) that respond preferentially to ecdysis-triggering hormones of flies and moths. These subtypes show differences in ligand sensitivity and specificity, suggesting that they may play separate roles in ETH signaling. At significantly higher concentrations (>100-fold), certain insect and vertebrate peptides also activate these receptors, providing evidence that CG5911 is evolutionarily related to the thyrotropin-releasing hormone and neuromedin U receptors. The ETH signaling system in insects is a vital system that provides opportunities for the construction of models for the molecular basis of stereotypic animal behavior as well as a target for the design of more sophisticated insect-selective pest control strategies. Insect ecdysis is a hormonally programmed physiological sequence that enables insects to escape their old cuticle at the end of each developmental stage. The immediate events leading to ecdysis, which are initiated upon release of ecdysis-triggering hormones (ETH) into the bloodstream, include respiratory inflation and sequential stereotypic behaviors that facilitate shedding of the cuticle. Here we report that the Drosophila geneCG5911 encodes two functionally distinct subtypes of G protein-coupled receptors through alternative splicing (CG5911a and CG5911b) that respond preferentially to ecdysis-triggering hormones of flies and moths. These subtypes show differences in ligand sensitivity and specificity, suggesting that they may play separate roles in ETH signaling. At significantly higher concentrations (>100-fold), certain insect and vertebrate peptides also activate these receptors, providing evidence that CG5911 is evolutionarily related to the thyrotropin-releasing hormone and neuromedin U receptors. The ETH signaling system in insects is a vital system that provides opportunities for the construction of models for the molecular basis of stereotypic animal behavior as well as a target for the design of more sophisticated insect-selective pest control strategies. ecdysis-triggering hormone pre-ecdysis-triggering hormone G protein-coupled receptor neuromedin U open reading frame reverse transcriptase PCR Chinese hamster ovary thyrotropin-releasing hormone pheromone biosynthesis-activating neuropeptide A defining feature of growth and metamorphosis in insects is the periodic shedding of cuticle known as ecdysis (1Truman J.W. Ewer J. Ball E.E. J. Exp. Biol. 1996; 199: 749-758PubMed Google Scholar). This complex process, by which the insect escapes its outer shell and sheds the cuticular lining of the foregut, hindgut, and respiratory system, is mediated by a peptide hormone-signaling cascade that results in a sequence of precisely timed physiological and behavioral events. Initiation of the ecdysis sequence coincides with the appearance of ecdysis-triggering hormones (ETHs)1 in the bloodstream, which act directly on the central nervous system to elicit patterned motor output characteristic of pre-ecdysis and ecdysis behaviors (2Zitnan D. Ross L.S. Zitnanova I. Hermesman J.L. Gill S.S. Adams M.E. Neuron. 1999; 23: 1-20Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar,3Zitnan D. Adams M.E. J. Exp. Biol. 2000; 203: 1329-1340PubMed Google Scholar). In the moth Manduca sexta, two peptides called MasPETH and MasETH act in concert to trigger successive phases of the ecdysis behavioral sequence. Remarkably, the temporal features of these motor patterns recorded from the isolated nervous system closely reflect the behavior observed in whole animals. Related peptides inDrosophila melanogaster known as ETH1 and ETH2 trigger ecdysis, although their respective roles in the recruitment of different parts of the ecdysis sequence are less clear. As yet the molecular and cellular targets of ETHs have not been described. Additional signaling molecules operating downstream of ETH within the central nervous system include the eclosion hormone and the crustacean cardioactive peptide (2Zitnan D. Ross L.S. Zitnanova I. Hermesman J.L. Gill S.S. Adams M.E. Neuron. 1999; 23: 1-20Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 4Ewer J. Truman J.W. J. Comp. Neurol. 1996; 370: 330-341Crossref PubMed Scopus (94) Google Scholar, 5Zitnan D. Kingan T.G. Hermesman J. Adams M.E. Science. 1996; 271: 88-91Crossref PubMed Scopus (199) Google Scholar, 6Gammie S.C. Truman J.W. J. Exp. Biol. 1999; 202: 343-352PubMed Google Scholar, 7McNabb S.L. Baker J.D. Agapite J. Steller H. Riddiford L.M. Truman J.W. Neuron. 1997; 19: 813-823Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Identification of ETH receptors would be of great help in defining the cellular elements involved in ecdysis behaviors. With this objective, we took advantage of the Drosophila genome project to investigate G protein-coupled receptors (GPCRs) that are likely to respond to ETH (8Hewes R.S. Taghert P.H. Genome Res. 2001; 11: 1126-1142Crossref PubMed Scopus (451) Google Scholar, 9Adams M.D. Celniker S.E. Holt R.A. Evans C.A. Gocayne J.D. Amanatides P.G. Scherer S.E. Li P.W. Hoskins R.A. Galle R.F. George R.A. Lewis S.E. Richards S. Ashburner M. Henderson S.N. Sutton G.G. Wortman J.R. Yandell M.D. Zhang Q. Chen L.X. Brandon R.C. Rogers Y.-H.C. Blazej R.G. Champe M. Pfeiffer B.D. Wan K.H. Doyle C. Baxter E.G. Helt G. Nelson C.R. Gabor Miklos G.L. Abril J.F. Agbayani A. An H.-J. Andrews-Pfannkoch C. Baldwin D. Ballew R.M. Basu A. Baxendale J. Bayraktaroglu L. Beasley E.M. Beeson K.Y. Benos P.V. Berman B.P. Bhandari D. Bolshakov S. Borkova D. Botchan M.R. Bouck J. Brokstein P. Brottier P. Burtis K.C. Busam D.A. Butler H. Cadieu E. Center A. Chandra I. Cherry J.M. Cawley S. Dahlke C. Davenport L.B. Davies P. de Pablos B. Delcher A. Deng Z. Mays A.D. Dew I. Dietz S.M. Dodson K. Doup L.E. Downes M. Dugan-Rocha S. Dunkov B.C. Dunn P. Durbin K.J. Evangelista C.C. Ferraz C. Ferriera S. Fleischmann W. Fosler C. Gabrielian A.E. Garg N.S. Gelbart W.M. Glasser K. Glodek A. Gong F. Gorrell J.H. Gu Z. Guan P. Harris M. Harris N.L. Harvey D. Heiman T.J. Hernandez J.R. Houck J. Hostin D. Houston K.A. Howland T.J. Wei M.-H. Ibegwam C. Science. 2000; 287: 2185-2195Crossref PubMed Scopus (4781) Google Scholar). The search for ETH receptors was narrowed to a small number of candidates on the basis of an assumption of ligand-receptor co-evolution, in which receptors for peptides having a C-terminal amino acid motif consisting of PRX amide would have high sequence homology. An exhaustive analysis of theDrosophila genome yielded two groups of GPCRs, the neuromedin U (NMU) group and the vasopressin group (10Park Y. Kim Y.J. Adams M.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11423-11428Crossref PubMed Scopus (299) Google Scholar). Functional analysis revealed that the NMU group likely arose through ligand-receptor co-evolution, because it responds to several groups of peptides with a C-terminal PRX amide motif characteristic of the ETHs. In contrast, the vasopressin group responds to unrelated peptides, and thus the PRX amide in this group appears to have arisen through convergent evolution. The monophyletic group of NMU receptors was found to be activated by all three categories of insect PRX amide peptides (10Park Y. Kim Y.J. Adams M.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11423-11428Crossref PubMed Scopus (299) Google Scholar): pyrokinins or pheromone biosynthesis-activating neuropeptide (PBAN)-like peptides ending with the amino sequence motif -FXPRXa (where final "a" means amide) (11Raina A.K. Jaffe H. Kempe T.G. Keim P. Blacher R.W. Fales H.M. Riley C.T. Klun J.A. Ridgway R.L. Hayes D.K. Science. 1989; 244: 796-798Crossref PubMed Scopus (390) Google Scholar, 12Matsumoto S. Kitamura A. Nagasawa H. Kataoka H. Orikasa C. Mitsui T. Suzuki A. J. Insect Physiol. 1990; 36: 427-432Crossref Scopus (182) Google Scholar), cardioactive CAP2b-like peptides with the -FPRXa motif (13Huesmann G.R. Cheung C.C. Loi P.K. Lee T.D. Swiderek K.M. Tublitz N.J. FEBS Lett. 1995; 371: 311-314Crossref PubMed Scopus (103) Google Scholar, 14Morris H.R. Panico M. Karplus A. Lloyd P.E. Riniker B. Nature. 1982; 300: 643-645Crossref PubMed Scopus (122) Google Scholar), and ecdysis-triggering hormones having a -PRXa motif (2Zitnan D. Ross L.S. Zitnanova I. Hermesman J.L. Gill S.S. Adams M.E. Neuron. 1999; 23: 1-20Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 5Zitnan D. Kingan T.G. Hermesman J. Adams M.E. Science. 1996; 271: 88-91Crossref PubMed Scopus (199) Google Scholar, 10Park Y. Kim Y.J. Adams M.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11423-11428Crossref PubMed Scopus (299) Google Scholar, 15Adams M.E. Zitnan D. Biochem. Biophys. Res. Commun. 1997; 230: 188-191Crossref PubMed Scopus (41) Google Scholar, 16Park Y. Zitnan D. Gill S.S. Adams M.E. FEBS Lett. 1999; 463: 133-138Crossref PubMed Scopus (98) Google Scholar). Surprisingly, some receptors exhibited sensitivity to multiple peptides. In particular, CG8795 was activated by DrmPK2, HUGγ, and ETH1, with sensitivity to DrmPK2 and HUGγ being far greater than to ETH1 (DrmPK2 > HUGγ > ETH1). The responsiveness of CG8795 to only relatively high concentrations of ETH1 suggested that it is unlikely to be the physiological ETH receptor. We therefore expanded the search profile for ETH receptors to include CG5911, which encodes a receptor related to the NMU group. As predicted (8Hewes R.S. Taghert P.H. Genome Res. 2001; 11: 1126-1142Crossref PubMed Scopus (451) Google Scholar), CG5911 gene products occur as two alternative splice variants termed CG5911a and CG5911b. CG5911b responds to ETH1 and the related peptides MasETH and MasPETH from moths at subnanomolar concentrations while showing relative insensitivity to other peptides sharing C-terminal PRX amide motifs. A pattern of cross-reactivity in a second set of peptides provides further support for the hypothesis that CG5911 encodes the authentic ETH receptor. cDNA was synthesized using Superscript (Invitrogen) with mRNA isolated by Dynabeads (Dynal) from whole flies (∼50 individuals of Canton S) in both larval and adult stages by priming at poly(A) sites. PCR reactions conducted in 20-μl volumes contained 20 mm Tris-HCl, pH 8.4, 50 mm KCl, 2 mm MgCl2, 0.2 mm dNTP, 0.5 μm of each primer, 0.5 unit ofTaq polymerase (Invitrogen), and 0.5 unit of Pfupolymerase (Stratagene). The reaction mixture was denatured initially for 5 min at 94 °C and then subjected to 35 cycles of 94 °C for 1 min, 53 °C for 1 min, and 72 °C for 1 min and 30 s. The RT-PCR products were cloned in pGEM-T-EZ vector (Promega) and sequenced. Start and stop codons of the open reading frames (ORF) were confirmed by the primers in the RT-PCR designed on the outside of the in-frame stop codons of the ORF. The primers shown in Fig. 1 are Fa (5′-AAGGACGATGGCGATGCTGA) paired with Ra (5′-GCTTACCCCTTACTGCCAGT) and Fb (5′-CCCGAGGATGTTGTCCCTGT) paired with Rb (5′-GATGGGATTCAGATCTTGCT). The CG5911 ORFs were inserted into the pXOON vector (17Jespersen T. Grunnet M. Angelo K. Klaerke D.A. Olesen S.P. BioTechniques. 2002; 32: 536-538Crossref PubMed Scopus (124) Google Scholar), which contains an enhanced green fluorescent protein marker. Phylogenetic analysis was done in PAUP version 4.0b8a for generation of a distance tree with 1000 bootstrapping samplings. Transient expression ofCG5911 gene products was accomplished in the CHO-WTA11 cell line exhibiting stable expression of the luminescent reporter aequorin (18Rizzuto R. Simpson A.W. Brini M. Pozzan T. Nature. 1992; 358: 325-327Crossref PubMed Scopus (782) Google Scholar) and the G protein Gα16 (19Offermanns S. Simon M.I. J. Biol. Chem. 1995; 270: 15175-15180Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). Cells were grown in complete Ham's F12 medium at 37 °C in 5% CO2. Transfection with pXOON-CG5911a and pXOON-CG5911b was performed using FuGene6 (Roche Molecular Biochemicals) according to the manufacturer's protocol at a DNA to FuGene6 ratio of 3:1. Transfected cells were observed under an epifluorescence microscope 1 day later to determine transfection efficiency as measured by the number of cells expressing enhanced green fluorescent protein. Before functional assays, cell suspensions were incubated in coelanterazine h (Molecular Probes) according to previously defined protocols (20Le Poul E. Hisada S. Mizuguchi Y. Dupriez V.J. Burgeon E. Detheux M. J. Biomol. Screen. 2002; 7: 57-65Crossref PubMed Scopus (107) Google Scholar). Luminescence assays were performed in opaque 96-well microplates (Corning) using a Beckman model LD400 Luminometer. After the addition of cells to a well, luminescence was monitored using an 0.5-s sampling time for 20 s. Each 96-well microplate contained multiple wells for positive controls (100 and 300 μm ETH1) and negative controls (buffer only). Luminescence at each ligand concentration was integrated during the 20-s response interval and normalized to the largest positive control response in each plate after the subtraction of background values obtained from negative controls. Luminescence measured in multiple replica wells (2–4 wells) for one concentration of ligand was averaged for the analysis. Data collected from at least three replica plates were used for analysis using the Origin analysis program (OriginLab Corp., Northampton, MA). The sources of peptides were described previously (10Park Y. Kim Y.J. Adams M.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11423-11428Crossref PubMed Scopus (299) Google Scholar), and additional vertebrate peptides (NMU-8, TRH, and ghrelin) were purchased from the American Peptide Company. MasETHacid was synthesized at the University of California, Riverside, using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry and automated solid state techniques. A list of peptides used in this study is provided in Table I.Table ISequences for signaling peptides used in this studyVertebrate peptidesNeuromedin U8YFLFRPRNaTRHpQHPaNeurotensinpQLYENKPRRPYILGhrelinGSSFLSPEHNRVQQRKGSKKPPAKLNPRDrosophila PRXamideHUGγpQLQSNGEPAYRVRTPRLaDrm-PK-2SVPFKPRLaCAP2B-1GANMGLYAFPRVaCAP2B-2ASGLVAFPRVaCAP2B-3TGPSASSGLWFGPRLaETH1DDSSPGFFLKITKNVPRLaETH2GENFAIKNLKTIPRIaOther invertebrate PRX peptidesHezPMPSLAYDDKSFENVEFTPRLaHezPBANLSDDMPATPADQEMYRQDPEQIDSRTKYFSPRLaMasCAP2BpQLYAFPRVaSCPBMNYLAFPRMaMasETHSNEAISPFDQGMMGYVIKTNKNIPRMaMasETHacidSNEAISPFDQGMMGYVIKTNKNIPRMMasPETHSFIKPNNVPRVaOther insect neuropeptidesCCAPPFCNAFTGCaCorazonin (CRZ)pQTFQYSRGWTNaDrmAKHpQLTFSPDWaMasMIPAWQDLNSAWaFMRFamideFMRFaMyomodulinPMSMLRLaProctolinRYLPTLowercase "a" indicates C-terminal amidation, "pQ" at the N terminus indicates pyroglutamic acid, and S indicates modification of n-octanoic acid. PRX sequence motifs are underlined as FXPRX, FPRX, andPRX. Prefixes for some peptides indicate species of origin: Drm, Drosophila melanogaster; Mas, Manduca sexta; Hez, Helicoverpa zea. Open table in a new tab Lowercase "a" indicates C-terminal amidation, "pQ" at the N terminus indicates pyroglutamic acid, and S indicates modification of n-octanoic acid. PRX sequence motifs are underlined as FXPRX, FPRX, andPRX. Prefixes for some peptides indicate species of origin: Drm, Drosophila melanogaster; Mas, Manduca sexta; Hez, Helicoverpa zea. TheCG5911 gene has homologous mutually alternative exons 4a and 4b encoding the protein sequence from the middle of transmembrane segment 4 to the C terminus (Figs. 1 and2). The clones used for the expression contain the coding sequence from nucleotide position −24 upstream of the Met site to +368 nucleotides after the 3′ stop codon for CG5911a and from −490 to +8 for CG5911b (see GenBank™ accession numbersAY220741, AY220742, and Celera Genomics sequence GenBankTM accession number AE003734) (Fig.2). The ORF is supported by the in-frame stop codon in the 5′-untranslated region (Fig. 1). Initial BLASTP searches and phylogenetic analyses identified the translation of CG5911 (GenBankTM accession number AAF55872) as a Drosophila GPCR closely related to the monophyletic group of NMU receptors responding to PRX amide peptides (10Park Y. Kim Y.J. Adams M.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11423-11428Crossref PubMed Scopus (299) Google Scholar). Putative transcripts generating CG5911a and -b are grouped with thyrotropin-releasing hormone (TRH) receptor in a distance tree with a bootstrapping value of 83.8% (Fig.3). The prediction of gene structure revealed the presence of homologous mutually alternative exons 4a and 4b from transmembrane segment 4 to the C terminus encoding two splice variants of the receptor (Figs. 1and 2). Stop codons occurring at C-terminal ORFs for CG5911a and CG5911b were identified by RT-PCR using primers located after the stop codon. RT-PCR with the primer covering a stop codon at the 5′ end of the ORF for CG5911b revealed a transcript supporting the ORF prediction. Ecdysis-triggering hormones of both Drosophilaand Manduca elicited robust luminescence responses in the CHO-WTA11 cell line stably expressing Gα16 and aequorin transfected with pXOON-CG5911a and pXOON-CG5911b (Fig.4). The time course of cellular responses was concentration-dependent, with delayed slow responses beginning at 0.1 nm and rapid peak responses occurring at 1 μm for CG5911b (Fig. 4A). At the lowest effective concentrations (0.1 nm), the luminescence response was delayed more than 10 s. Integration of each response shown in Fig. 4A during a 20-s sampling period is shown in Fig. 4B. Both CG5911a and CG5911b showed preferential sensitivity to ETHs, with CG5911b showing a significantly higher apparent affinity for all peptides tested (Fig. 4, C and D).Drosophila ETH1 was the most active ligand for both receptors. For CG5911b, responses were evident at 0.25 nmand reached peak values at 10 nm with an EC50value of 0.9 nm. ETH2 and Manduca PETH were only slightly less active, with EC50 values of ∼2 nm. Interestingly, Manduca ETH was approximately 5 times less active on CG5911b than PETH. CG5911a was markedly less sensitive to all peptides tested compared with CG5911b. The most active peptide again was ETH1, with an EC50 of 414 nm, making it ∼400-fold less active on this receptor than on CG5911b. ETH2, MasPETH, and MasETH had EC50 values greater than 2 μm. CG5911b showed significant sensitivity to a range of ETH-related peptides having the signature PRXa C-terminal amino acid motif (Fig.5). These peptides include SCPB, HUGγ, CAP2b-1, CAP2b-2, PBAN, and MasETH acid. The most active of these peptides were SCPB, MasETH acid, HUGγ, and CAP2b-1; the EC50 values of these peptides were in the range of 300 nm to 3 μm. Furthermore, the vertebrate peptides NMU and TRH caused low but significant receptor activation when applied at 10 μm as did the molluscan peptide myomodulin (p < 0.001 in Student'st test; Fig. 5B). No activity was observed for a number of other peptide ligands, including some with the C-terminal PRXa motif. These ligands include Drm-PK-2, CAP2b-3, and Hez PMP (Fig. 5B). Ecdysis-triggering hormones are vital regulatory signals that govern the stereotypic physiological sequence leading to cuticle shedding in insects. In previous work we identified ETHs in two moth species, Manduca sexta and Bombyx mori, and in the fruit fly Drosophila melanogaster (2Zitnan D. Ross L.S. Zitnanova I. Hermesman J.L. Gill S.S. Adams M.E. Neuron. 1999; 23: 1-20Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 15Adams M.E. Zitnan D. Biochem. Biophys. Res. Commun. 1997; 230: 188-191Crossref PubMed Scopus (41) Google Scholar, 16Park Y. Zitnan D. Gill S.S. Adams M.E. FEBS Lett. 1999; 463: 133-138Crossref PubMed Scopus (98) Google Scholar). Using genetic tools in Drosophila to excise the ethgene, lethal ecdysis deficits were demonstrated at the end of the first larval instar (21Park Y. Filippov V. Gill S.S. Adams M.E. Development. 2002; 129: 493-503PubMed Google Scholar). Lethality was reversed by the injection of ETH peptides (21Park Y. Filippov V. Gill S.S. Adams M.E. Development. 2002; 129: 493-503PubMed Google Scholar). The ETH signaling system therefore has been demonstrated to be both necessary and sufficient for insects to survive the earliest ecdysis. To expand our understanding of the molecular basis for ETH action, we conducted a search for its likely receptor inDrosophila. In this paper we report on the cloning and functional expression of theDrosophila gene CG5911, predicted by Hewes and Taghert (8Hewes R.S. Taghert P.H. Genome Res. 2001; 11: 1126-1142Crossref PubMed Scopus (451) Google Scholar) to encode splice variants called CG5911a and CG5911b. Our results show that both splice variants of CG5911 are highly selective for ETH1 and related peptides from both fly and moth, with CG5911b responding to subnanomolar concentrations ofDrosophila ETH1. For each receptor subtype the highest sensitivity was observed to Drosophila ETH1 and ETH2, but comparable sensitivity also was observed for MasPETH and MasETH. Other insect and vertebrate peptides, particularly those having C-terminal PRX amide motifs, also activated CG5911b, but only at ∼100–1000-fold higher concentrations. The selectivity and high sensitivity of CG5911 to ETHs alone strongly supports the hypothesis that this gene encodes the authentic ETH receptor. The gene structure of CG5911 with the results of RT-PCR revealed two homologous exons presenting the mutually alternative splice variants CG5911a and CG5911b. Phylogenetic analysis suggests that divergent exons 4a and 4b are the product of duplication. The portion of the receptor protein predicted by exon 4 extends from the middle of transmembrane segment 4 (T4) to the C terminus and therefore includes two extracellular loops (T4-T5 and T6-T7), one intracellular loop (T5-T6), and the C-terminal intracellular tail. Clearly, alternative splicing of exons 4a and 4b to create these receptor isoforms could affect both their ligand and G protein-coupling specificities. Functional differences in CG5911a and CG5911b are suggested by several observations. CG5911b is activated by subnanomolar concentrations of ETH1 (EC50 = 0.9 nm), whereas the sensitivity of CG5911a is more than 400-fold lower (EC50 = 410 nm). The lower sensitivity of CG5911a to all ligands may be a consequence of poor coupling efficiency to the Gα16used in our assay system. Indeed, the smaller size of peak luminescence responses recorded for CG5911a as compared with CG5911b (data not shown) supports this idea. Further investigation of CG5911a sensitivity using different G proteins will be necessary to resolve this question. Nevertheless, differences in G protein coupling and downstream signal transduction steps could provide a basis for functionally distinct roles for these two receptor subtypes in ETH signaling. In addition to these differences, the ligand sensitivity for the two receptor subtypes is significantly different. The relative potency of ligands on CG5911a (ETH1 > MasETH > ETH2 > MasPETH; see Fig. 4) reflects the same order of biological activity observed for the induction of ecdysis behavior (16Park Y. Zitnan D. Gill S.S. Adams M.E. FEBS Lett. 1999; 463: 133-138Crossref PubMed Scopus (98) Google Scholar). In the case of CG5911b, we observed a somewhat different order of potency (ETH1 > ETH2 > MasPETH > MasETH). The reversal in receptor sensitivity to MasPETH and MasETH suggests that these receptor subtypes discriminate between MasPETH and MasETH and raises the intriguing possibility thatCG5911 orthologs in the Manduca system encode receptors that are ligand-specific. Indeed, preliminary findings involving the Manduca ortholog of CG5911b confirm the preference for MasPETH. 2Y. J. Kim and M. E. Adams, unpublished data. The possible ligand specificity of the two receptor subtypes may account at least in part for divergent activities of these two ligands in the induction of natural pre-ecdysis behavior (2Zitnan D. Ross L.S. Zitnanova I. Hermesman J.L. Gill S.S. Adams M.E. Neuron. 1999; 23: 1-20Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 3Zitnan D. Adams M.E. J. Exp. Biol. 2000; 203: 1329-1340PubMed Google Scholar). Further study ofManduca orthologous isoforms of CG5911 is under way to test this hypothesis. Responses of CG5911b to a second tier of ligands that are active only at >100-fold-higher concentrations reveal further insights into its properties and the likely physiological role of CG5911 as the ETH receptor. All ETHs are C-terminally amidated, and this post-translational modification is essential for biological activity in the silkworm, B. mori (15Adams M.E. Zitnan D. Biochem. Biophys. Res. Commun. 1997; 230: 188-191Crossref PubMed Scopus (41) Google Scholar). Although the injection of synthetic Bombyx ETH elicits the entire ecdysis behavioral sequence in silkworm larvae, the free acid form proved to be largely inactive (15Adams M.E. Zitnan D. Biochem. Biophys. Res. Commun. 1997; 230: 188-191Crossref PubMed Scopus (41) Google Scholar). In the present study, the free acid form of MasETH (MasETHacid, Fig. 5) was found to be more than 100 times less active than native MasETH. CG5911b therefore discriminates between these subtly different forms of the peptide just as we observed previously in behavioral assays (2Zitnan D. Ross L.S. Zitnanova I. Hermesman J.L. Gill S.S. Adams M.E. Neuron. 1999; 23: 1-20Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). We tested a variety of both invertebrate and vertebrate peptides on CG5911b, and most were inactive. Nevertheless, some interesting patterns of activity are apparent among the few peptides that showed some biological activity. Of the invertebrate peptides tested, the peptide HUGγ, encoded by the ortholog of the moth PBANgene as well as PBAN itself, were active at several hundred-fold higher concentrations than authentic ETHs. The low affinity response of CG5911b to HUGγ and its high affinity response to ETH1 and ETH2 complement our previous results, which showed that the related GPCR encoded by CG8795 is highly sensitive to HUGγ but considerably less sensitive to ETHs (10Park Y. Kim Y.J. Adams M.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11423-11428Crossref PubMed Scopus (299) Google Scholar). These results confirm our phylogenetic analyses, which indicate the close evolutionary relationship betweenCG8795 and CG5911. The question remains as to whether the cross-reactivities of these two ligands (ETH and HUGγ) against CG8795 and CG5911b reflect genuine biological phenomena. Evidence in support of this comes from Roos and colleagues (22Meng X. Wahlstrom G. Immonen T. Kolmer M. Tirronen M. Predel R. Kalkkinen N. Heino T. Sariola H. Roos C. Mech. Dev. 2002; 117: 5Crossref PubMed Scopus (95) Google Scholar), who showed that ectopic expression of the hugingene produced lethal ecdysis defects. The mammalian peptides neuromedin and TRH, the receptors of which are related phylogenetically to CG5911, also activate CG5911b, albeit at relatively high concentrations. The significant bioactivity of these peptides, with the complete absence of responses to neurotensin and ghrelin (assayed at 10 μm), strongly support our phylogenetic analysis, which indicates that CG5911 evolved from a GPCR ancestral to the NMU/TRH receptors. Myomodulin (PMSMLRLamide), a neuropeptide first identified in molluscs, also was found to activate CG5911b. We were unable to find myomodulin-like peptides in the Drosophila genomic data base, but a similar sequence occurs in the Caenorhabditis elegans data base (Y45F10A.5). 3Y. Park and M. E. Adams, unpublished data. Myomodulin could play an analogous role to ETH in nematodes, which with the arthropods have been newly classified in the Ecdysozoa (23Aguinaldo A.M. Turbeville J.M. Linford L.S. Rivera M.C. Garey J.R. Raff R.A. Lake J.A. Nature. 1997; 387: 489-493Crossref PubMed Scopus (1279) Google Scholar). Indeed, antibodies raised against myomodulin cross-react with ETHs in endocrine Inka cells (24O'Brien M.A. Taghert P.H. J. Exp. Biol. 1998; 201: 193-209PubMed Google Scholar), indicating structural similarities between these peptides. During preparation of this manuscript, independent evidence appeared to support the hypothesis that CG5911 functions as the ETH receptor (25Iversen A. Cazzamali G. Williamson M. Hauser F. Grimmelikhuijzen C.J.P. Biochem. Biophys. Res. Commun. 2002; 299: 924-931Crossref PubMed Scopus (63) Google Scholar). These findings confirm that two functional subtypes of CG5911 are produced by alternative splicing and that ETH1 is a more potent agonist against both receptors. These authors (25Iversen A. Cazzamali G. Williamson M. Hauser F. Grimmelikhuijzen C.J.P. Biochem. Biophys. Res. Commun. 2002; 299: 924-931Crossref PubMed Scopus (63) Google Scholar) also demonstrated the presence of an additional 5′-untranslated exon. However, our results demonstrate a higher sensitivity of CG5911 to both ligands and a considerably greater divergence in receptor sensitivity between the two subtypes. These findings could indicate that CG5911a and CG5911b interact with different G proteins and may activate different signal transduction pathways. Our pharmacological and phylogenetic analyses also indicate that ETH and the pyrokinin/CAP2b groups of insect neuropeptides constitute a set of ligands that have evolved with CG5911 and the neuromedin U group of vertebrate receptors and their respective ligands. In summary, we have demonstrated that alternative splice variants ofCG5911 encode two functional receptor subtypes of ETH receptor in Drosophila. Although further work is necessary to confirm their physiological roles in natural ETH signaling, the pronounced ligand sensitivity and specificity of CG5911 gene products along with the phylogenetic relationships of CG5911to both Drosophila and mammalian receptors for PRX amide peptides provide strong evidence for their roles as the physiological ETH receptors. We thank Dr. Tiesheng Shi for the synthesis of MasETHacid and Beckmann Coulter, Inc., for the use of the LD400 luminometer.