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Title: Isolation, expression analysis and functional characterization of the first anti-diuretic hormone receptor in insects

Author
item PALUZZI, JEAN-PAUL - University Of Toronto
item PARK, YOONSEONG - Kansas State University
item Nachman, Ronald
item ORCHARD, IAN - University Of Toronto

Submitted to: Proceedings of the National Academy of Sciences (PNAS)
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/28/2010
Publication Date: 6/1/2010
Citation: Paluzzi, J., Park, Y., Nachman, R.J., Orchard, I. 2010. Isolation, expression analysis and functional characterization of the first anti-diuretic hormone receptor in insects. Proceedings of the National Academy of Sciences. 107:10290-10295.

Interpretive Summary: Because of problems with the development of resistance to conventional pesticides, there is a critical need for new concepts and alternative approaches in controlling insect pests. The basic premise of this research is that "CAPA" neuropeptides (short chains of amino acids) serve as potent messengers in insects to regulate water balance, a vital function in many insects that transmit disease. Nevertheless, these neuropeptides in and of themselves hold little promise as pest control agents because of susceptibility to being degraded in the target pest, and inability to pass through the outside skin and/or digestive tract. We must design neuropeptide mimics that resist degradation by enzymes in the digestive tract and blood of pest invertebrates and interact with the active site within an agricultural or medical pest in such a way as to either over-activate or block critical, neuropeptide-regulated life functions. We report on the isolation, identification and characterization of the first active site that regulates prevention of water loss in insects. This active site interacts specifically with "CAPA" neuropeptide hormones to prevent water loss in the assassin bug, an insect that transmits Chagas disease to humans. Key structural features of the neuropeptides have been identified that allow them to turn this active site on. This discovery will aid in the design of neuropeptide-like compounds capable of disrupting the critical life function of water balance in insect pests. This work brings us one step closer to the development of practical neuropeptide-like substances that will be effective in controlling insect pests that transmit deadly diseases in an environmentally friendly fashion.

Technical Abstract: Diuresis following blood-gorging in Rhodnius prolixus is the major process leading to the transmission of Chagas' disease. We have cloned the cDNA of the first receptor known to be involved in an antidiuretic strategy in insects; a strategy that prevents diuresis. This receptor belongs to the insect CAPA receptor family known in other insects to be activated by peptides encoded within the "capability" gene. We characterize the expression profile in fifth instars and find expression is localized to the alimentary canal. Highest transcript levels are found in Malpighian tubules and the anterior midgut, which are known targets of the anti-diuretic hormone RhoprCAPA-alpha2. Two transcripts were identified, capa-r1 and capa-r2; however the latter encodes an atypical GPCR lacking a region ranging between the first and second transmembrane domain. Our heterologous expression assay revealed the expressed capa-r1 receptor is activated by RhoprCAPA-alpha2 (EC50 = 385nM) but not by RhoprCAPA-alpha1. Structural analogs of the inactive RhoprCAPA-alpha1 were capable of activating the expressed capa-r1 receptor, confirming the importance of the C-terminal consensus sequence common to CAPA-related peptides. In addition, this receptor has some sensitivity to the pyrokinin-related peptide RhoprCAPA-alphaPK1, but with an efficacy approximately 40-fold less than RhoprCAPA-alpha2. Other peptides belonging to the PRXamide superfamily were inactive on the capa-r1 receptor. Taken together, the neuroendocrinological relevance of this receptor in facilitating the anti-diuretic strategy in R. prolixus may make this receptor a useful target for development of agonists or antagonists that could help influence the transmission of Chagas disease that occurs during diuresis in this medically important insect disease-vector.