Signaling And Pharmacology Of Orphan Receptors CG12796and CG3579in Drosophila Melanogaster

In the nervous system of insects, acetylcholine and dopamine is a major neurotransmitter and neuromodulator. They regulate a wide variety of behaviors and physiological processes. All of them are independent neurotransmitters, exert their effects by binding to specific ion channel or G-protein-coupled receptors. Most currently used insecticides are neurotoxic chemicals that target a limited number of sites. G-protein-coupled receptors have been recognized as very suitable and specific insecticide targets, especially the studies about their receptors, have been paid more attentions in recent years. In the study, we chose model insect fruit fly, D. melanogaster (Diptera:Drosophilidae). Using modern molecular biology methods, we investigated signaling and pharmacology of muscarinic acetylcholine receptors and dopamine receptors in fruit fly.1. Signaling and pharmacology of a novel muscarinic acetylcholine receptor (CG12796) in the fruit fly, D. melanogasterA full-length cDNA of CG12796has been obtained from fruit fly, D. melanogaster. The mRNA of CG12796is present in the developmental stages and various tissues, including larvae, pupae, adult fly and different body parts of adult male and female, head, thorax, abdomen. The highest expression level of CG12796was in the head of adult fly male and female. We generated a CHO-K1cell line that stably expresses pcDN A3-CGI2796in order to examine signaling and pharmacological properties of this receptor. The result show that none of the amines and acetylcholine were found to increase intracellular cAMP levels or decrease the forskolin-stimulated intracellular cAMP levels. The results show CG12796neither coupling Gs nor Gj. Agonists acetylcholine chloride and oxotremorine M can increase intracellular Ca2+level in a dose-dependent way. The EC50values respectively are73.02nM,48.23nM. In addition, the classical antagonists100μM atropine and100uM scopolamine can inhibit100nM acetylcholine-induced intracellular Ca2+ increases. So we named CG12796as the C-type mAChR which is different from the A-type and B-type mAChRs.2. Signaling and pharmacology of a novel dopamine receptor CG13579in the fruit fly, D. melanogasterWe identified a full-length cDNA of putative dopamine receptor CG13579from fruit fly, D. melanogaster. CG13579was predicted as sequence similarity of previously characterized dopamine/ecdysteroid receptor (CG18314) in the fruit fly D. melanogaster. The mRNA of CG13579is present in the developmental stages and various tissues.The highest expression level of CG13579was in the head of adult fly male and female. CG13579encode one transcript. We generated HEK-293cell line stably transfected with cloned cDNA for examining the second messenger coupling and pharmacological properties of CG13579. CG13579can not increase of intracellular Ca2+concentration in response to high concentrations of dopamine. But they can increases the forskolin-stimulated intracellular cAMP levels. To our knowledge, CG13579may be a new family of insect dopamine receptors.