Bursicon, the cuticle-hardening , is a heterodimeric cystine knot protein that activates G protein-coupled receptor LGR2

Ching-Wei Luo*, Elizabeth M. Dewey†, Satoko Sudo*, John Ewer‡, Sheau Yu Hsu*, Hans-Willi Honegger†§, and Aaron J. W. Hsueh*§

*Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305-5317; †Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235; and ‡Department of Entomology, Cornell University, Ithaca, NY 14853

Communicated by J. Woodland Hastings, Harvard University, Cambridge, MA, December 31, 2004 (received for review December 17, 2004) All arthropods periodically molt to replace their exoskeleton (cu- and named as LGRs. The LGR genes are conserved in inver- ticle). Immediately after shedding the old cuticle, the neurohor- tebrates and vertebrates (8) and can be divided into three groups mone bursicon causes the hardening and darkening of the new as follows: group A, vertebrate glycoprotein hormone receptors; cuticle. Here we show that bursicon, to our knowledge the first group B, melanogaster LGR2 (DLGR2) and verte- heterodimeric cystine knot hormone found in , consists of brate orphan receptors LGR4͞5͞6 (9); and group C, mammalian two proteins encoded by the genes burs and pburs (partner of relaxin͞INSL3 receptors (10, 11). Based on genetic analyses in burs). The pburs͞burs heterodimer from D. melanogaster, bursicon was proposed to act through the G binds with high affinity and specificity to activate the G protein- protein-coupled receptor DLGR2, encoded by the rickets coupled receptor DLGR2, leading to the stimulation of cAMP gene (12). signaling in vitro and tanning in neck-ligated blowflies. Native Based on the comparison with three P. americana partial bursicon from Periplaneta americana is also a heterodimer. In D. peptide sequences (13), Dewey et al. (5) reported the identifi- melanogaster the levels of pburs, burs, and DLGR2 transcripts are cation of burs, an Ϸ15-kDa protein encoded by the D. melano- increased before ecdysis, consistent with their role in postecdysial gaster gene CG13419 as a bursicon candidate. Our preliminary cuticle changes. Immunohistochemical analyses in diverse insect data, however, indicated that recombinant burs was not bioac- species revealed the colocalization of pburs- and burs-immunore- tive. Because burs is a cystine knot protein with an uneven activity in some of the neurosecretory neurons that also express number of cysteine residues and because many ligands of this crustacean cardioactive peptide. Forty-three years after its initial family form heterodimers (14), we hypothesized that the burs description, the elucidation of the molecular identity of bursicon gene product could heterodimerize with another cystine knot and the verification of its receptor allow for studies of bursicon protein encoded by the D. melanogaster gene CG15284, which we actions in regulating cuticle tanning, wing expansion, and as yet call pburs (partner of burs). Here, we show that the bursicon unknown functions. Because bursicon subunit genes are homolo- (pburs͞burs) is, to our knowledge, the first heterodimeric cystine gous to the vertebrate bone morphogenetic protein antagonists, knot hormone discovered in insects. Recombinant pburs͞burs is our findings also facilitate investigation on the function of these capable of high-affinity binding to the receptor DLGR2 to proteins during vertebrate development. stimulate cAMP production in vitro and to induce tanning in the neck-ligated bioassay in vivo. BMP antagonist ͉ heterodimeric polypeptide ligand ͉ LGR Materials and Methods nsects comprise Ͼ90% of all animal species and are of great Isolation of D. melanogaster burs and pburs cDNAs and Identification Ieconomical and ecological importance. The evolutionary suc- of Their Orthologs in Other Insect Species. The protein sequence of cess of insects is due partially to their exoskeleton, which pburs was identified by using the burs sequence against the D. provides protection, mechanical support, and an effective barrier melanogaster database with BLAST (15). For the amplification of the to desiccation and infections. During immature stages, contin- pburs cDNA, primers 5Ј-TGGGAGTGGGTCAGCATGCA-3Ј ued growth requires that insects replace their exoskeleton. and 5Ј-TAGTTTATGTTGAGGCATTA-3Ј were used. The re- During this molting process, a new cuticle is synthesized and sulting PCR products were cloned into the pcDNA3.1͞Zeo(ϩ) secreted by underlying epidermal cells and then is hardened after expression vector (Invitrogen) and confirmed by sequencing. Sim- the remains of the old cuticle are shed at ecdysis. Molting and ilarly, primers 5Ј-AAGGACACTCGCAGTCGGGCCGA-3Ј and ecdysis are regulated by at least six different (1). The 5Ј-CTATTGCAGAGCAATGCGCCGGA-3Ј were used to am- steroid 20-hydroxyecdysone directs the synthesis of the new plify the burs cDNA from D. melanogaster. To identify its orthologs, cuticle (2), and then a cascade of peptide hormones orchestrates the pburs protein sequence was used in TBLASTN searches against the events surrounding ecdysis. different insect genomes. The sequences of D. melanogaster burs According to current knowledge, the last uncharacterized and mammalian bone morphogenetic protein (BMP) antagonists hormone in this cascade, bursicon, is an Ϸ30-kDa neurohor- were identified from the GenBank database [accession nos. mone that is released after the completion of ecdysis (3, 4) and AY672905 (burs), AAC39725 (gremlin), BAA92265 (DAN; dif- triggers the tanning (melanization and sclerotization) of the new ferential screening-selected gene aberrative in neuroblastoma), cuticle as well as wing expansion (5). Bursicon of diverse insects, including blowfly (Calliphora erythrocephala), cockroach

(Periplaneta americana), cricket (Gryllus bimaculatus), locust Abbreviations: BMP, bone morphogenetic protein; CCAP, crustacean cardioactive peptide. (Locusta migratoria), and meal beetle (Tenebrio molitor) (4), Data deposition: The sequences for pburs reported in this paper have been deposited in the initiates tanning in neck-ligated (6, 7), suggesting the GenBank database [accession nos. AY823257 (D. melanogaster), AY823258 (A. mellifera), conservation of this signaling system that is essential for insect AY823259 (A. gambiae), and AY823260 (B. mori)]. survival. §To whom correspondence may be addressed. E-mail: [email protected] or Recently, a subfamily of G protein-coupled receptors with a [email protected]. large ectodomain containing leucine-rich repeats was identified © 2005 by The National Academy of Sciences of the USA

2820–2825 ͉ PNAS ͉ February 22, 2005 ͉ vol. 102 ͉ no. 8 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0409916102 Downloaded by guest on October 1, 2021 AAH46632 (PRDC; protein related to DAN and Cerberus); stretch of four residues (boxed) in D. melanogaster pburs is BAC82440 (Coco), AAK92484 (cerberus), AAN45848 (USAG1), identical to a partial peptide of purified bursicon (GT28) found and AAQ88990 (SOST)]. The sequence alignment and phyloge- in P. americana (13). Based on the known cystine knot structures netic tree were constructed by the neighbor-joining method using of TGF-␤ and CG-␤ (14), the structure of pburs could be the MEGALIGN program in the DNASTAR software package (DNA- predicted (Fig. 1B). Both pburs and burs are likely to form a STAR, Madison, WI). cystine knot and contain two intramolecular disulfide bonds between C2–C8 and C5–C11. In addition, a free cysteine at Expression and Purification of D. melanogaster pburs and burs. The position 6 may be involved in the formation of an intermolecular expression vector pcDNA3.1 containing D. melanogaster pburs or bridge. Analyses of the pburs gene structures from the four burs was transfected into mammalian 293T cells by using Lipo- insects indicated that they consist of two or three exons (Fig. 1C). fectamine 2000 (Invitrogen), and transfected cells were selected Of interest, the first exon in D. melanogaster is split into two by the Zeocin-containing medium. Selected cells were allowed to exons in A. mellifera and B. mori, whereas exons 2 and 3 in A. reach confluence and then cultured for 72 h in a serum-free mellifera and B. mori are combined into one exon in A. gambiae. medium (DMEM͞Ham’s F12 medium containing 100 ␮g͞ml Phylogenetic analyses based on the cystine knot region of several penicillin, 100 ␮g͞ml streptomycin, and 2 mM L-glutamate). related proteins further indicated that pburs, together with burs, After filtration, the conditioned media were concentrated. To showed close sequence relationships with cystine knot proteins facilitate purification, D. melanogaster burs and pburs constructs in the human BMP antagonist family (Fig. 1D). Some of these were tagged with His-6- and FLAG-tagged epitopes, respec- proteins have been found to antagonize the actions of BMP tively, at the N terminus by replacing the endogenous signal ligands during embryonic development and organogenesis peptide with a prolactin signal peptide and the epitope tags. To (18, 19). generate recombinant bursicon, cells coexpressing pburs and burs were clonally selected and confirmed based on tagged Bursicon Is a Heterodimer of pburs and burs Capable of Activating epitopes. Conditioned media were further purified by using DLGR2. We generated specific antibodies against pburs and burs. metal chelating Sepharose (Amersham Pharmacia) against His- As shown in Fig. 2A (Upper), in blots of SDS-polyacrylamide 6-burs followed by anti-FLAG M1 affinity gel (Sigma) against gels, a single high-molecular mass band of Ϸ30 kDa could be FLAG-pburs. Protein purity and biochemical characteristics recognized by both anti-pburs and anti-burs antibodies in ganglia

were analyzed after electrophoresis by using a 12% SDS- extracts of P. americana (lane 1) run under nonreducing condi- BIOLOGY

polyacrylamide gel. tions. In contrast, two smaller bands corresponding to pburs DEVELOPMENTAL (Ϸ14 kDa) and burs (Ϸ16 kDa) monomers were found under In Vitro and in Vivo Bioassays of Bursicon. For assessing the cAMP reducing conditions (the nature of the two bands at Ϸ44 kDa, production mediated through DLGR2 (8, 16), full-length detected by using anti-burs antibodies under reducing condi- DLGR2 cDNA was cloned into the pcDNA3.1͞Zeo(ϩ) expres- tions, is presently unknown). In addition, 293T cells were sion vector (Invitrogen) and confirmed by sequencing. The transfected with plasmids containing pburs and͞or burs with or DLGR2-containing plasmid then was transfected into human without epitope tags to generate recombinant proteins. Immu- 293T cells by using Lipofectamine 2000 (Invitrogen). After noblotting showed that the conditioned medium from trans- 18–24 h of transfection, cells were harvested in the cAMP assay fected cells contained heterodimers or homodimers (lanes 2–5). buffer (DMEM͞F12 supplemented with 0.1% BSA and 0.25 mM When cells were cotransfected with pburs and burs (lane 3), only 3-isobutyl-1-methylxanthine) and plated on 24-well culture the heterodimers were detected. The molecular masses of the plates (2 ϫ 105 cells per ml). After 30 min of culture, cells were recombinant proteins were slightly larger than those found in the treated with ligands for 16 h before measurement of total cAMP ganglia extracts from P. americana. Differential posttransla- in triplicate by a specific RIA (17). tional modifications of native and recombinant proteins may In vivo bursicon activity was assessed by using the neck-ligated account for these differences. fly bioassay (3). The concentration of recombinant pburs, burs, To test the bioactivity of these proteins, 293T cells overex- or bursicon (pburs͞burs heterodimer) was adjusted to 10 ng͞␮l. pressing DLGR2 were treated with medium containing het- A5-␮l test solution of recombinant proteins or ganglia extracts erodimeric pburs͞burs with or without epitope tags. Both het- from P. americana was injected into ligated flies (Sarcophaga erodimeric preparations stimulated dose-dependent increases in bullata) at adult eclosion. The extent of tanning bioactivity was cAMP production, comparable to levels induced by P. americana quantitated by assigning a score between 0 (no tanning) and 6 ganglia extracts (Fig. 2A Lower). In contrast, medium from cells (dark tanning) as described in ref. 4. In each experimental series, transfected with either pburs or burs alone were ineffective. By 6–10 flies were injected and scored, and the mean was calcu- using the neck-ligated fly bioassay, injections with recombinant lated. Phosphate buffer (pH 7.3) was injected as a control. The heterodimer preparations or with P. americana ganglia extracts average of the means of three experiments Ϯ SD is shown to stimulated maximal tanning in a 0- to 6-point scoring system indicate the variance of the data. (Fig. 2B). In contrast, treatment with recombinant pburs or burs Antibody generation, receptor binding assay, quantitative alone induced minimal tanning, similar to that obtained by using real-time PCR, immunohistochemistry, and in situ hybridization phosphate buffer. Protein elutions of P. americana ganglia procedures are described in Supporting Text, which is published extracts from SDS gels run under nonreducing conditions as supporting information on the PNAS web site. showed that only the 28- to 33-kDa fraction (bursicon) was functional in the neck-ligated fly bioassay (4) and activated Results DLGR2 (Fig. 2A Lower). The recombinant heterodimer of Identification of pburs and Its Relationship with Other Cystine Knot pburs and burs therefore corresponds to bursicon. Proteins. Based on GenBank searches for novel cystine knot proteins as potential dimerization partners for burs, we identi- Specific High-Affinity Binding of Labeled Bursicon to DLGR2. To assess fied the CG15284 gene in the fruit fly (D. melanogaster), which receptor-binding ability, we purified recombinant epitope- we have named pburs (partner of burs). As shown in Fig. 1A, the tagged bursicon by using two sequential affinity columns against predicted pburs protein is highly conserved among D. melano- polyHis and the FLAG epitope appended to burs and pburs, gaster, mosquito (Anopheles gambiae), honey bee (Apis mellifera), respectively. The purity of the bursicon heterodimer was con- and silkworm (), and all contained the 11 cysteine firmed by using SDS͞PAGE followed by Coomassie blue stain- residues that are also present in the D. melanogaster burs. A ing (Fig. 3A, lanes 1–3). Under nonreducing conditions, purified

Luo et al. PNAS ͉ February 22, 2005 ͉ vol. 102 ͉ no. 8 ͉ 2821 Downloaded by guest on October 1, 2021 Fig. 1. Predicted sequences, gene structure, and phylogenetic analyses of pburs from different insects. (A) Sequence alignment of pburs from different insects and burs from D. melanogaster (D.m.). Conserved residues are shaded, and cysteine residues are numbered. A.g., Anopheles gambiae; A.m., Apis mellifera; B.m., Bombyx mori. A stretch of four residues (boxed) in D.m. pburs is identical to that of a fragment purified from protease-treated bursicon from P. americana.(B) Predicted cystine knot structure of pburs. (C) Intron–exon arrangement of pburs genes in different insects. Exons are represented by boxes. The numbers of amino acid residues for each exon and nucleotides for each intron are shown. (D) Phylogenetic tree of D.m. pburs and burs together with seven human BMP antagonists.

bursicon migrated Ϸ38 kDa (Fig. 3A, lane 3), whereas two lower Colocalization of pburs, burs, and Crustacean Cardioactive Peptide bands were evident under reducing conditions (Fig. 3A, lane 4) (CCAP) in the Nervous System of Different Insects. Because the and confirmed to be pburs and burs monomers by using specific sequences of both pburs and burs are highly conserved in insects, antibodies (Fig. 3A, lanes 5 and 6). Purified bursicon was we used antibodies against D. melanogaster pburs or burs to iodinated and found to bind to cells expressing DLGR2 (Fig. detect neurons expressing these proteins in the central nervous 3B). 125I-bursicon binding was displaced in a dose-dependent systems of three different insect species. Immunohistochemical manner by nonlabeled bursicon but was not affected by pburs or staining for pburs and burs in the third-instar of D. melanogaster (Fig. 4 Top) revealed that pburs was coexpressed burs alone. Likewise, several BMP antagonists, gremlin, DAN, with burs in four bilateral neurons in thoracic and abdominal or PRDC, as well as gonadotropins (human chorionic gonado- neuromeres of the ventral nervous system. However, the somata tropin or equine chorionic gonadotropin), did not compete for in the subesophageal and posterior abdominal neuromeres ex- bursicon binding. Conversion of the displacement curve to the pressed only burs. Likewise, colocalization of pburs and burs saturation and Scatchard plots (Fig. 3C) indicated that bursicon occurred in the large bilateral lateral neurosecretory neurons of Ϫ9 binds to DLGR2 with high affinity (Kd ϭ 2.5 ϫ 10 M). the first three unfused abdominal ganglia and in all anterior bilateral cell pairs in the thoracic ganglia of P. americana and in Developmental Changes in the Expression of pburs, burs, and DLGR2 homologous neurons in the thoracic and abdominal ganglia of in D. melanogaster. To examine the expression of pburs, burs, and the cricket Teleogryllus commodus (Fig. 4 Middle and Bottom). In DLGR2, we performed real-time PCR analyses by using RNA these somata, the immunostaining was found in large granules preparations from different developmental stages of D. mela- that characterize these endocrine cells. nogaster. As shown in Fig. 5, which is published as supporting Further, by using antibodies against pburs and CCAP in P. information on the PNAS web site, the transcript levels for pburs americana and in situ hybridization of pburs in wild-type and CCAP neuron-ablated fly mutants (Fig. 6, which is published as and burs showed a similar pattern of regulation. They were both supporting information on the PNAS web site), our data suggest low in larva (stages 1 and 2) and gradually increased in pupal that pburs, like burs (5), is colocalized with CCAP in some stages (3 and 4) before reaching the highest levels in pharate CCAP-immunoreactive neurons. Further information can be adults (stage 7), in preparation for tanning after adult eclosion. obtained in Supporting Text. For DLGR2 transcripts, an increase was found in early pupae (stage 4), followed by a decrease in the late pupae (stage 6), Discussion before increasing dramatically in pharate adults. In the adult The present study demonstrates the heterodimeric nature of animals (stage 8), all three transcripts were low. bursicon, a neuroendocrine hormone essential for insect tanning

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Fig. 2. Bursicon, a heterodimer of two cystine-knot-containing polypeptides, is the cognate ligand for DLGR2. (A)(Upper) Immunoblot analyses of bursicon DEVELOPMENTAL from P. americana ganglia extracts and conditioned media from 293T cells transfected with pburs and͞or burs. Samples were run under nonreducing and reducing conditions. Lane 1, ganglia extracts from P. americana; lane 2, recombinant pburs͞burs without epitope tags; lane 3, recombinant epitope-tagged pburs͞burs; lane 4, recombinant pburs without a tag; and lane 5, recombinant burs without a tag. (Lower) Stimulation of DLGR2 by P. americana ganglia extracts or conditioned media from cells cotransfected with pburs͞burs expression constructs (bursicon heterodimer), but not by the individual plasmid (pburs or burs). Both wild-type and epitope-tagged bursicon heterodimers were tested. In addition, proteins eluted from the 28- to 33-kDa region of a SDS gel loaded with P. americana ganglia extracts also were tested (■). Ligand levels were determined from immunoblots by using purified tagged bursicon heterodimers as a standard. (B) Neck-ligated fly bioassay. Recombinant bursicon or P. americana ganglia extracts stimulated complete tanning of flies with a maximum score (5–6 points), whereas flies injected with phosphate buffer (PB), pburs, and burs alone did not tan and received an average score of 0–0.3 points. In three separate experiments, 6–10 flies were injected, and their tanning score was averaged.

and wing expansion. Bursicon consists of two cystine knot epidermal cells (22, 23). Our real-time PCR results showing polypeptides, pburs and burs, capable of activating a G protein- increases of pburs, burs, and DLGR2 transcripts at pupariation coupled receptor, DLGR2. Under reducing conditions, purified and eclosion stages of D. melanogaster are consistent with the recombinant bursicon heterodimers could be separated into importance of these genes during insect . pburs and burs monomers. Although the size of native cockroach Immunohistochemical analyses of pburs and burs demon- bursicon recognized by both anti-pburs and -burs antibodies is strated that both polypeptides are colocalized in four bilateral consistent with previous findings (4, 20), the recombinant D. neurons of the D. melanogaster ventral nervous system and in melanogaster bursicon expressed in mammalian cells showed a select neurons of P. americana and T. commodus. However, in D. higher molecular mass, suggesting posttranslational modifica- melanogaster and T. commodus, some neurons express only burs, tions such as phosphorylation. Both recombinant and native but not pburs. The neurons containing solely burs may serve bursicon induced cAMP production in DLGR2-expressing cells different, yet-unknown functions. Sequence conservation of and stimulated tanning in the in vivo neck-ligated fly bioassay. both pburs (Fig. 1) and burs in diverse insects (5) along with the Specific, high-affinity interactions between bursicon and immunohistochemical results indicate the evolutionary conser- DLGR2 were confirmed by using a radioligand receptor assay. vation of both genes. In addition to insects, bursicon activity has In D. melanogaster, mutations in the rickets gene show crossed also been found in the abdominal nerve cord of the crustacean postscutellar bristles and kinked femurs (21). These mutant flies Homarus americanus (4), suggesting that both pburs and burs are carry lesions in the DLGR2 gene and fail to initiate tanning and likely conserved throughout arthropods. wing expansion after adult emergence. Although rickets mutants Our real-time PCR analyses of burs, pburs, and DLGR2 do not melanize when injected with ganglia extracts containing showed that these transcripts were present before pupal and bursicon, they do tan in response to injection of an analog of adult ecdysis. Maximal levels were observed just before adult cAMP (12), consistent with our findings of the stimulation of emergence, consistent with the known role of bursicon in cAMP production by bursicon in cells transfected with DLGR2. posteclosion tanning and sclerotization of the cuticle, wing Because flies with burs mutations showed the same phenotypes inflation, and epidermal cell death (22). Earlier studies in moth as rickets mutants, burs was proposed to encode a ligand for (Manduca sexta) (24), locust (Schistocerca gregaria) (25), and DLGR2 (5, 12). The present findings demonstrate, however, that Drosophila (26) demonstrated that most CCAP-expressing neu- burs needs pburs as a partner for DLGR2 activation. rons undergo after adult ecdysis, consistent with our In addition to the induction of cuticle tanning, bursicon also findings of low transcript levels for pburs and burs in mature adult has been implicated in a stereotyped behavioral program for D. melanogaster. This low level of pburs and burs expression was wing expansion (12) and in the postecdysial cell death of wing accompanied by a reduction in the levels of DLGR2 message.

Luo et al. PNAS ͉ February 22, 2005 ͉ vol. 102 ͉ no. 8 ͉ 2823 Downloaded by guest on October 1, 2021 Fig. 4. Colocalization of pburs and burs in different insect nervous systems. Immunohistochemical detection of pburs and burs immunoreactivities in the ventral nervous system of D. melanogaster, P. americana, and T. commodus. (Top) Double labeling experiments in the third-instar larva of D. melano- gaster.(Middle) pburs and burs colocalized in the large bilateral lateral somata of the second unfused abdominal ganglion of P. americana.(Bottom) Fig. 3. Purification of bursicon and high-affinity binding of labeled bursicon pburs and burs colocalized in the large anterior bilateral neuron pair in the to recombinant DLGR2 receptors. (A) Purification of tagged recombinant second thoracic ganglion of T. commodus. The structure at right is a large ␮ bursicon heterodimers. SDS͞PAGE analyses of conditioned media containing trachea. (Confocal imaging; scale bars, 100 m.) tagged bursicon (lane 1) followed by sequential affinity purification using metal chelating (lane 2) and anti-FLAG M1 affinity chromatography (lane 3, nonreducing conditions; lane 4, reducing conditions). Samples were detected In D. melanogaster, three LGR genes have been identified (8, by using Coomassie blue staining. Purified bursicon proteins were further 16). They encode seven transmembrane proteins with a large confirmed as a heterodimer by using antibodies against pburs (lane 5) or burs ectodomain containing leucine-rich repeats. These three fly (lane 6) under reducing conditions. (B) Binding of 125I-labeled recombinant LGRs showed sequence homology with the three subgroups of bursicon to DLGR2 was competed by nonlabeled bursicon, whereas coincu- mammalian LGRs, including the group A glycoprotein hormone bation with conditioned media containing only pburs, burs, or other proteins receptors, group B LGR4͞5͞6, and group C LGR7 and -8 (31). such as BMP antagonists (DAN, gremlin, or PRDC) or glycoprotein hormones ͞ ͞ (human chorionic gonadotropin, hCG, or equine chorionic gonadotropin, Fly DLGR2, like mammalian LGR4 5 6, has 17–18 leucine-rich eCG) were not effective. (C) Saturation and Scatchard plot (Inset) analyses of repeats, likely involved in ligand binding. A recent study iden- bursicon binding to DLGR2. tified a group B LGR ortholog in the gastropod Crassostrea gigas (oyster) (32), suggesting that these receptors have ancient ori- gins. Although the ligands for LGR7 and LGR8 recently have Although the exact identity of bursicon targets is unclear, this been identified (10, 11), the ligands for the group B vertebrate change could reflect the down-regulation of bursicon receptor LGRs are unknown. The insect pburs and burs genes are expression and͞or the death of bursicon target cells. In other homologous to the vertebrate BMP antagonist family of genes insects, the CCAP- and bursicon-expressing neurons survive into found to be important during embryonic development and adulthood (13, 27). This finding may indicate that bursicon has organogenesis (18, 19, 33). Only some members of the BMP additional unknown functions that can now be investigated. antagonist family are competitive antagonists capable of direct D. melanogaster bearing targeted ablations of CCAP neurons binding to BMPs (18, 33), and the exact mechanisms of actions showed defects in wing expansion and tanning (28), as well as of most proteins in this family are still unknown. Future studies lower bursicon bioactivity, indicating that bursicon expression is will reveal whether these cystine knot proteins are ligands for also disrupted. We have found that both pburs and burs are vertebrate orphans LGR4͞5͞6. Of interest, recent studies dem- expressed in CCAP neurons, consistent with previous work onstrated that mutations in the LGR4 and LGR5 genes in mice showing that CCAP neurons express bursicon bioactivity in are associated with perinatal lethality, suggesting important crickets (27) and cockroaches (13), and some neurons containing roles during embryonic development (34, 35). bursicon bioactivity in Manduca (29) also express CCAP (30). The present identification of two cystine knot polypeptides as Further discussions of the potential relationship between bur- subunits for the heterodimeric bursicon in insects, together with sicon and CCAP can be obtained in Supporting Text. the demonstration of specific binding and activation of a G

2824 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0409916102 Luo et al. Downloaded by guest on October 1, 2021 protein-coupled receptor with leucine-rich repeats, finally allow supported by National Institutes of Health Grant HD23273 (to for an investigation of the diverse aspects of the physiological A.J.W.H.), National Science Foundation Grants 0217637 (to H.-W.H.) functions of this important neuroendocrine hormone. and 0343699 (to J.E.), and by the Bioinformatic Core of U54-HD-31398 as part of the Specialized Cooperative Centers Program in Reproduction Research. Confocal images were performed through the use of the We thank C. Spencer for editorial assistance. The idea that bursicon Vanderbilt University Medical Center Cell Imaging Core Resource might be a heterodimer of two cystine knot proteins was originally (supported by National Institutes of Health Grants CA68485, DK20593, mentioned to one of us (H.-W.H.) by Dr. J. W. Truman. This work was and DK58404).

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