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Bursicon, the Insect Cuticle-Hardening Hormone, Is a Heterodimeric Cystine Knot Protein That Activates G Protein-Coupled Receptor LGR2

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Bursicon, the Insect Cuticle-Hardening Hormone, Is a Heterodimeric Cystine Knot Protein That Activates G Protein-Coupled Receptor LGR2 Bursicon, the insect cuticle-hardening hormone, 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, Drosophila melanogaster LGR2 (DLGR2) and verte- heterodimeric cystine knot hormone found in insects, 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 Drosophila melanogaster 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 fly 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 hormones (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 flies (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
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