Molecular characterization of Bursicon

The molecular analysis and cellular expression of the insect peptide neurohormone bursicon is described. Bursicon triggers the sclerotization of the soft insect cuticle after ecdysis. Using protein elution analyses from SDS gels, the molecular weight of bursicon from different insects was determined to be approximately 30 kDa. Four partial peptide sequences of Periplaneta americana bursicon were obtained from purified nerve cord homogenates separated on two-dimensional gels. Antibodies produced against one of the sequences identified the cellular location of bursicon in different insects and showed that bursicon is co-produced with crustacean cardioactive peptide (CCAP) in the same neurons in all insects tested so far. Additionally, using the partial peptide sequences, the Drosophila genome project was successfully searched for the gene encoding bursicon. With Drosophila as a tool, the function of the sequence has now been verified using transgenic flies. Sequence comparisons also allowed the verification that bursicon is conserved, corroborating the older data from bioassays and immunohistochemical analyses. The sequence of bursicon will enable further analysis of its function, release, and evolution (Honegger, 2004).

Activation of the cAMP/PKA signaling pathway is required for post-ecdysial cell death in wing epidermal cells of Drosophila melanogaster

At the last step of metamorphosis in Drosophila, the wing epidermal cells are removed by programmed cell death during the wing-spreading behavior after eclosion. The cell death is accompanied by DNA fragmentation demonstrated by the TUNEL assay. Transmission electron microscopy reveals that this cell death exhibits extensive vacuoles, indicative of autophagy. Ectopic expression of an anti-apoptotic gene, p35, inhibits the cell death, indicating the involvement of caspases. Neck ligation and hemolymph injection experiments demonstrate that the cell death is triggered by a hormonal factor secreted just after eclosion. The timing of the hormonal release implies that the hormone to trigger the death might be the insect tanning hormone, bursicon. This is supported by evidence that wing cell death is inhibited by a mutation of rickets, which encodes a G-protein coupled receptor in the glycoprotein hormone family that is a putative bursicon receptor. Furthermore, stimulation of components downstream of bursicon, such as a membrane permeable analog of cAMP, or ectopic expression of constitutively active forms of G proteins or PKA, induces precocious death. Conversely, cell death is inhibited in wing clones lacking G protein or PKA function. Thus, activation of the cAMP/PKA signaling pathway is required for transduction of the hormonal signal that induces wing epidermal cell death after eclosion (Kimura, 2004).

To determine whether a humoral signal coming from the head triggers cell death, the necks of flies were ligated at various times after eclosion and the wings were examined for cell death at 2 hours after the ligation. Ligation just after eclosion suppresses cell death and GFP was still detectable in the nuclei of wing epidermal cells after 2 hours. By contrast, when flies were ligated at 20 minutes after eclosion, the normal pattern of cell death was observed. Ligation at later stages correlates with an increased percentage of flies with wing epidermal cell death. Thus wing epidermal cell death is triggered by a signal emanating from the head shortly after eclosion (Kimura, 2004).

The cellular effects of cAMP are usually mediated by PKA. To determine whether this is also the case for wing epidermal cell death, the effect of reduction or elimination of PKA activity on cell death was examined. A dominant-negative form of the regulatory subunit of PKA (R*), whose ectopic expression is known to reduce the activity of endogenous PKA, was used. When R* was ectopically expressed using the en-Gal4 driver, many cells of the wings remained at 2 hours, or even at 8 hours, after wing spreading, resulting in separation between the ventral and dorsal cuticular sheets in the posterior compartment. Targeted expression of R* caused wavy or curly wings, probably due to the distortion between normal adhesion of dorsoventral cuticles in the anterior compartment and detachment of the cuticle in the posterior compartment (Kimura, 2004).

Next, DC0-dependent PKA activity was eliminated by generating clones of DC0 mutant cells within the developing wings. The DC0 gene in Drosophila encodes a catalytic subunit, one of the components of PKA. Since clones of DC0 mutant cells in the anterior compartment produce anterior duplication of the normal wing pattern, the clones in the posterior compartment were examined to investigate whether the death of wing epidermal cells marked with Histone-GFP is suppressed or not. The cells of the clones remained at 2 hours after wing spreading, although the surrounding cells had already been eliminated by cell death. Thus, reduction or elimination of PKA activity prevents the death of wing epidermal cells (Kimura, 2004).

The effects of constitutive activation of PKA on cell death were examined. A mutationally altered mouse catalytic subunit (mC*) was used that is resistant to inhibition by the regulatory subunit. The mutant catalytic subunit is constitutively active, irrespective of cAMP concentration, and can function in Drosophila cells. Using the en-Gal4 driver, the constitutively active catalytic subunit of mC* was expressed in wing epidermal cells. All eclosing flies had blistered wings. The wing epidermal cells died prior to wing spreading. Thus, constitutive activation of PKA causes the precocious death of wing epidermal cells (Kimura, 2004).

The induction of cell death was examined at various stages of pharate adults. As seen in the cases of cAMP injection and of ectopic expression of Gs{alpha}*, precocious cell death was induced at G stage and later. This indicates that wing cells acquire competence to respond to PKA activity by G stage, about 3 hours before eclosion (Kimura, 2004).

A mutation in the G-protein coupled receptor gene rickets inhibits wing epidermal cell death. In Drosophila, the rickets gene is a member of the glycoprotein hormone receptor family of the G-protein-coupled receptors and has been suggested to encode a bursicon receptor. Wing epidermal cell death, marked by Histone-GFP, was examined in rk mutants. In the mutants, wing epidermal cells remained at 2 hours, or even at 8 hours, after eclosion. To determine whether the inhibition of cell death is caused by a failure in the reception of a hormonal signal inducing death, the effects were examined of 8-Br-cAMP and hemolymph injection into rk mutants that were neck-ligated at eclosion. In wild-type flies, injection of hemolymph from wild-type flies at 30 minutes after eclosion and injection of 8-Br-cAMP induces cell death. However, in rk mutants, cell death was induced by injection of 8-Br-cAMP but not by injection of hemolymph. This indicates that the mutant cells could not receive the hormonal signal in the hemolymph, although the activation of cell death by cAMP/PKA signaling was normal in the mutant cells (Kimura, 2004).

The peptide hormone, Bursicon, is known to play a role in the post-ecdysial phase of development. Bursicon has been shown to be released before wing expansion and to hasten the tanning reaction, serving to harden the newly expanded cuticle. The results of this study suggest that the hormone that induces cell death of the wing epidermis could be bursicon. (1) Neck ligation and hemolymph injection experiments have demonstrated that the triggering signal to induce death is a humoral factor released after eclosion. This temporal pattern of death-inducing activity in the hemolymph corresponds to that of bursicon. (2) Injection of cAMP induced cell death, implicating cAMP as the second messenger in the cell death pathway. Studies in blowflies have shown that bursicon also acts through cAMP. Recently, in Drosophila, cAMP was shown to induce cuticular melanization in a fashion similar to bursicon. (3) Reception of the hormonal signal inducing cell death is mediated by a probable bursicon receptor, Rickets (DLGR2), which also acts through cAMP. (4) In Lucilia cuprina, it has been proposed that bursicon is the same as fragment disaggregating hormone, which increases the circulating filamentous cellular fragments derived from post-ecdysial death of the wing epidermal cells. Taken together, it is likely that bursicon coordinates events such as the cell death of wing epidermis and the subsequent tanning and hardening of the cuticle. However, another possibility cannot be ruled out -- namely, that several humoral factors could signal through the pathway. Identification of a bursicon gene as CG13419 in Drosophila will facilitate genetic approaches to understand the role of bursicon in wing epidermal cell death (Kimura, 2004).

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

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. Bursicon, the first heterodimeric cystine knot hormone found in insects, consists of two proteins encoded by the genes burs and pburs (partner of burs). The Pburs/Burs heterodimer from Drosophila melanogaster binds with high affinity and specificity to activate the G protein-coupled receptor DLGR2 (Rickets), 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 roles in postecdysial cuticle changes. Immunohistochemical analyses in diverse insect species have 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, these findings also facilitate investigation on the function of these proteins during vertebrate development (Luo, 2005).

Based on GenBank searches for novel cystine knot proteins as potential dimerization partners for burs, the CG15284 gene was identified and named partner of burs (pburs). The predicted Pbursprotein is highly conserved among D. melanogaster, mosquito (Anopheles gambiae), honey bee (Apis mellifera), and silkworm (Bombyx mori), and all contain the 11 cysteine residues that are also present in the D. melanogaster burs. A stretch of four residues in D. melanogaster Pburs is identical to a partial peptide of purified bursicon found in P. americana. Based on the known cystine knot structures of TGF-beta and CG-beta, the structure of Pburs could be predicted. Both Pburs and Burs are likely to form a cystine knot and contain two intramolecular disulfide bonds between C2-C8 and C5-C11. In addition, a free cysteine at position 6 may be involved in the formation of an intermolecular bridge. Analyses of the pburs gene structures from the four insects indicate that they consist of two or three exons. Of interest, the first exon in D. melanogaster is split into two exons in A. mellifera and B. mori, whereas exons 2 and 3 in A. mellifera and B. mori are combined into one exon in A. gambiae. Phylogenetic analyses based on the cystine knot region of several related proteins further indicate that Pburs, together with Burs, show close sequence relationships with cystine knot proteins in the human BMP antagonist family. Some of these proteins have been found to antagonize the actions of BMP ligands during embryonic development and organogenesis (Luo, 2005).

To assess receptor-binding ability, recombinant epitope-tagged Bursicon was prepared by using two sequential affinity columns against polyHis and the FLAG epitope appended to Burs and Pburs, respectively. The purity of the Bursicon heterodimer was confirmed by using SDS/PAGE followed by Coomassie blue staining. Under nonreducing conditions, purified Bursicon migrates ~38 kDa, whereas two lower bands are evident under reducing conditions and confirmed to be Pburs and Burs monomers by using specific antibodies. Purified Bursicon was iodinated and found to bind to cells expressing DLGR2. ¹²⁵I-bursicon binding was displaced in a dose-dependent manner by nonlabeled bursicon but was not affected by Pburs or Burs alone. Conversion of the displacement curve to the saturation and Scatchard plots indicated that Bursicon binds to DLGR2 with high affinity (Luo, 2005).

To examine the expression of pburs, burs, and DLGR2, real-time PCR analyses was performed by using RNA preparations from different developmental stages of D. melanogaster. The transcript levels for pburs and burs show a similar pattern of regulation. They were both low in larva (stages 1 and 2) and gradually increase in pupal stages (3 and 4) before reaching the highest levels in pharate adults (stage 7), in preparation for tanning after adult eclosion. For DLGR2 transcripts, an increase was found in early pupae (stage 4), followed by a decrease in the late pupae (stage 6), before increasing dramatically in pharate adults. In the adult animals (stage 8), all three transcripts are low (Luo, 2005).

Because the sequences of both Pburs and Burs are highly conserved in insects, antibodies against D. melanogaster Pburs or Burs were used to detect neurons expressing these proteins in the central nervous systems of three different insect species. Immunohistochemical staining for Pburs and Burs in the third-instar larva of D. melanogaster revealed that Pburs was coexpressed with Burs in four bilateral neurons in thoracic and abdominal neuromeres of the ventral nervous system. However, the somata in the subesophageal and posterior abdominal neuromeres expressed only Burs. Likewise, colocalization of Pburs and Burs occurred in the large bilateral lateral neurosecretory neurons of the first three unfused abdominal ganglia and in all anterior bilateral cell pairs in the thoracic ganglia of P. americana and in homologous neurons in the thoracic and abdominal ganglia of the cricket Teleogryllus commodus. In these somata, the immunostaining was found in large granules that characterize these endocrine cells (Luo, 2005).

Further, by using antibodies against Pburs and CCAP in P. americana and in situ hybridization of pburs in wild-type and CCAP neuron-ablated fly mutants, the data suggest that Pburs, like Burs, is colocalized with CCAP in some CCAP-immunoreactive neurons (Luo, 2005).

In D. melanogaster, mutations in the rickets gene show crossed postscutellar bristles and kinked femurs (Edmondson (1948). These mutant flies carry lesions in the DLGR2 gene and fail to initiate tanning and wing expansion after adult emergence. Although rickets mutants do not melanize when injected with ganglia extracts containing bursicon, they do than in response to injection of an analog of cAMP (Baker, 2002), consistent with the findings that in cells transfected with DLGR2 bursicon stimulates cAMP production. Because flies with burs mutations show the same phenotypes as rickets mutants, burs was proposed to encode a ligand for DLGR2. The present findings demonstrate, however, that Burs needs Pburs as a partner for DLGR2 activation (Luo, 2005).

In addition to the induction of cuticle tanning, bursicon also has been implicated in a stereotyped behavioral program for wing expansion and in the postecdysial cell death of wing epidermal cells. Real-time PCR results showing increases of pburs, burs, and DLGR2 transcripts at pupariation and eclosion stages of D. melanogaster are consistent with the importance of these genes during insect metamorphosis (Luo, 2005).

In D. melanogaster, three LGR genes have been identified. They encode seven transmembrane proteins with a large ectodomain containing leucine-rich repeats. These three fly LGRs show sequence homology with the three subgroups of mammalian LGRs, including the group A glycoprotein hormone receptors, group B LGR4/5/6, and group C LGR7 and -8. Fly DLGR2, like mammalian LGR4/5/6, has 17-18 leucine-rich repeats, likely involved in ligand binding. A recent study identified a group B LGR ortholog in the gastropod Crassostrea gigas (oyster), suggesting that these receptors have ancient origins. Although the ligands for LGR7 and LGR8 recently have been identified, the ligands for the group B vertebrate LGRs are unknown. The insect pburs and burs genes are homologous to the vertebrate BMP antagonist family of genes found to be important during embryonic development and organogenesis. Only some members of the BMP antagonist family are competitive antagonists capable of direct binding to BMPs, and the exact mechanisms of actions of most proteins in this family are still unknown. Future studies will reveal whether these cystine knot proteins are ligands for vertebrate orphans LGR4/5/6. Of interest, recent studies have demonstrated that mutations in the LGR4 and LGR5 genes in mice are associated with perinatal lethality, suggesting important roles during embryonic development (Luo, 2005 and references therein).

The identification of two cystine knot polypeptides as subunits for the heterodimeric bursicon in insects, together with the demonstration of specific binding and activation of a G protein-coupled receptor with leucine-rich repeats, finally allow for an investigation of the diverse aspects of the physiological functions of this important neuroendocrine hormone (Luo, 2005).

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