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First published online May 24, 2005
Journal of Experimental Biology 208, vii-a (2005)
Copyright © 2005 The Company of Biologists Limited
doi: 10.1242/jeb.01649
Outside JEB |
THAT ALL-IMPORTANT TAN...
University of Glasgow
j.a.t.dow{at}bio.gla.ac.uk
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The arthropod exoskeleton is both a blessing and a curse. It provides mechanical protection, a permeability barrier and a lightweight skeleton; but it must be moulted regularly as it becomes constricting when the animal grows. The multi-stage moulting process involves the growth of a new flexible cuticle underneath the old one, the detachment from and shedding of the old one (ecdysis), then the expansion of the new one before it is finally tanned - that is, made rigid by chemical crosslinking. Classic neck-ligation experiments (tying the neck with ligature silk to prevent soluble brain factors from reaching the rest of the body) showed that brain-derived factors are responsible for moulting. Since then, it has become clear that moulting is under multiple, sequential neurohormonal control. In March this year, two research groups simultaneously revealed an important missing link in this pathway - the last step in the process, the tanning hormone bursicon.
Bursicon is released from the insect's head after ecdysis; if an insect's neck is ligated soon enough after moulting, its cuticle does not tan. Painstaking work has shown that bursicon acts through the second messenger cyclic AMP. Second messengers signal the arrival of hormones (like bursicon) at the cell surface to target molecules in the cell. Bursicon's effects can be mimicked in neck-ligated insects by injecting their abdomen with either cyclic AMP analogues or a central nervous system-derived peptide fraction of around 30 kDa in size, too large to be a neuropeptide. That is, bursicon is a protein hormone, explaining why it has proved hard to identify.
To determine bursicon's peptide sequence, one of the groups laboriously purified bursicon from the cockroach Periplaneta. They located partial sequences in bursicon that matched a known class of mammalian hormone, the cystine knot hormones. These hormones are thrown into characteristic 3-D shapes by internal disulphide bridges between cysteine residues, and typically form dimers (complexes made up of two proteins). The sequence of the cockroach hormone also matched closely a single gene in the Drosophila genome, CG13419, marking it as a likely candidate gene that codes for bursicon. A strong candidate receptor for the hormone was also known; another Drosophila gene, rickets, showed a very similar mutant phenotype to the effects of neck ligation, and rickets mutants could be rescued by injection of cyclic AMP. However, the big problem was that the peptide encoded by CG13419 itself had no effect on the receptor encoded by rickets. Something was missing!
The insight that came to both groups was that bursicon might be a heterodimer (a complex of two different proteins) rather than a homodimer (a complex of two of the same protein). That is, the CG13419 gene product needed to associate with a different cystine knot protein before it became active. The two groups came to the same answer by different routes; one noticed that the honeybee homologue of CG13419 contained a second cystine knot sequence, which matched a further Drosophila gene; the other looked for Drosophila genes similar to CG13419. Both identified CG15284 as a candidate to form the heterodimer with the protein encoded by CG13419.
Now, everything fits together. Both groups showed that the two proteins, when co-expressed in vitro, form a 35 kDa protein, close to the original mass predicted from the cockroach bursicon. This heterodimer also potently activated the rickets receptor in vitro to generate cyclic AMP; they had found the missing link. In addition, synthetic bursicon displayed strong tanning activity when the groups injected it into neck-ligated insects. Finally, CG13419, CG15284 and rickets were all expressed at times consistent with their playing a role in ecdysis. The close similarity between the proteins encoded by these three Drosophila genes and those in other insects suggests that this pathway for cuticle hardening is tightly conserved, at least across insects.
References
Luo, C. W., Dewey, E. M., Sudo, S., Ewer, J., Hsu, S. Y.,
Honegger, H. W. and Hsueh, A. J. (2005). Bursicon, the insect
cuticle-hardening hormone, is a heterodimeric cystine knot protein that
activates G protein-coupled receptor LGR2. Proc. Natl. Acad. Sci.
USA 102,2820
-2825.
Mendive, F. M., Van Loy, T., Claeysen, S., Poels, J., Williamson, M., Hauser, F., Grimmelikhuijzen, C. J. P., Vassart, G. and Vanden Broeck, J. (2005). Drosophila molting neurohormone bursicon is a heterodimer and the natural agonist of the orphan receptor DLGR2. FEBS Lett. 579,2171 -2176.[CrossRef][Medline]
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