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

Abstract

All arthropods periodically molt to replace their exoskeleton (cuticle). Immediately after shedding the old cuticle, the neurohormone bursicon causes the hardening and darkening of the new cuticle. Here we show that bursicon, to our knowledge the first heterodimeric cystine knot hormone found in insects, consists of two proteins encoded by the genes burs and pburs (partner of burs). The pburs͞burs heterodimer from Drosophila melanogaster binds with high affinity and specificity to activate the G proteincoupled receptor DLGR2, leading to the stimulation of cAMP signaling in vitro and tanning in neck-ligated blowflies. Native bursicon from Periplaneta americana is also a heterodimer. In D. melanogaster the levels of pburs, burs, and DLGR2 transcripts are increased before ecdysis, consistent with their role in postecdysial cuticle changes. Immunohistochemical analyses in diverse insect species revealed the colocalization of pburs-and burs-immunoreactivity in some of the neurosecretory neurons that also express crustacean cardioactive peptide. Forty-three years after its initial description, the elucidation of the molecular identity of bursicon and the verification of its receptor allow for studies of bursicon actions in regulating cuticle tanning, wing expansion, and as yet unknown functions. Because bursicon subunit genes are homologous to the vertebrate bone morphogenetic protein antagonists, our findings also facilitate investigation on the function of these proteins during vertebrate development.BMP antagonist ͉ heterodimeric polypeptide ligand ͉ LGR I nsects comprise Ͼ90% of all animal species and are of great economical and ecological importance. The evolutionary success of insects is due partially to their exoskeleton, which provides protection, mechanical support, and an effective barrier to desiccation and infections. During immature stages, continued growth requires that insects replace their exoskeleton. During this molting process, a new cuticle is synthesized and secreted by underlying epidermal cells and then is hardened after the remains of the old cuticle are shed at ecdysis. Molting and ecdysis are regulated by at least six different hormones (1). The steroid 20-hydroxyecdysone directs the synthesis of the new cuticle (2), and then a cascade of peptide hormones orchestrates the events surrounding ecdysis.According to current knowledge, the last uncharacterized hormone in this cascade, bursicon, is an Ϸ30-kDa neurohormone that is released after the completion of ecdysis (3, 4) and triggers the tanning (melanization and sclerotization) of the new cuticle as well as wing expansion (5). Bursicon of diverse insects, including blowf ly (Calliphora erythrocephala), cockroach (Periplaneta americana), cricket (Gryllus bimaculatus), locust (Locusta migratoria), and meal beetle (Tenebrio molitor) (4), initiates tanning in neck-ligated flies (6, 7), suggesting the conservation of this signaling system that is essential for insect survival.Recently, a subfamily of G protein-coupled receptors with a large ectodo...

Transcriptional expression of bursicon and novel bursicon‐regulated genes in the house fly Musca domestica

“…RT-PCR analysis of the house fly bursicon α and β genes in the CNS reveals that both bursicon α and β genes were transcribed as early as in the first instar larvae, reached the maximum levels in pharate adult, and declined sharply after adult emergence. The sharp decline of the bursicon α and β transcripts after adult emergence observed in the house fly CNS is in agreement with the reports in Drosophila (Luo et al, 2005;Mendive et al, 2005) and in B. mori (Huang et al, 2007), indicating active translation and release of bursicon into hemolymph for cuticle sclerotization of the newly emerged fly. The developmental profiles of bursicon α and β transcripts at the larval and pupal stages of the house fly show a relatively steady increase with a little fluctuation while a big fluctuation of bursicon α and β transcripts has been reported at the larval and pupal stages of Drosophila (Luo et al, 2005;Mendive et al, 2005).…”

“…The discrepancy in the levels of bursicon α and β transcripts at the larval and pupal stages between Musca and Drosophila appears to be the source of RNA used in RT-PCR analysis of bursicon α and β transcripts. In Drosophila, RNA used for RT-PCR was extracted from whole fly body (Luo et al, 2005;Mendive et al, 2005) while in Musca RNA was from the CNS (Fig. 1).…”

Genomic regulatory blocks underlie extensive microsynteny conservation in insects

“…Notably, dpp, teashirt, engrailed, and a gene desert The paralogous zinc finger genes elB and noc, implicated in tracheal and appendage development, have different, but partially overlapping, spatial expression patterns during embryonic development (Dorfman et al 2002) and are coexpressed in larval leg and wing discs (Weihe et al 2004). Among the five flies, elB and noc are in conserved microsynteny with a tRNA gene and at least three protein-coding genes (underlined), which have no evidence of being functionally related to elB or noc: pburs encodes a subunit of the hormone bursicon required for wing expansion and associated cuticle changes after flies emerge from pupae (Luo et al 2005); CG3474 is predicted to encode a cuticle component; CG4218 is predicted to encode a protein of unknown function. (B) The paralogous T-box genes H15 and mid are involved in regulation of heart development and have similar spatial expression patterns during embryonic development (Miskolczi-McCallum et al 2005;Reim et al 2005).…”

Functional Dissection of a Neuronal Network Required for Cuticle Tanning and Wing Expansion inDrosophila

“…This persistent juvenile phenotype is likely to result from loss of the hormone bursicon (for review, see Reynolds, 1983;Ewer and Reynolds, 2002), which is normally released into the hemolymph shortly after eclosion and is known from genetic studies to be required for both wing expansion and tanning. Bursicon has been shown to be expressed exclusively in N CCAP during larval development Luo et al, 2005), but its distribution in adults has not been investigated.…”