Molecular mechanisms underlying rapid nongenomic actions of ecysteroids

Steroids are naturally occurring substances in the body that act as sex hormones and as hormones that control the growth of tissues such as muscle and brain. Usually steroid hormones work by entering a cell and binding to special molecules called receptors. When these receptors are activated by the steroid, they can then turn on the expression in the nucleus of a range of different genes which is specific for that hormone. However, recent studies have shown that steroid hormones can also produce effects on cells rapidly, by mechanisms that do not involve them entering the cell. In these cases the steroids act with receptors on the surface of the cell which bind the hormone at their outer surface, change their shape and then pass on information about the presence of the steroid hormone to the inside of the cell. The cell then responds in an appropriate way. The protein molecules that make up one class of these cell surface receptors bind other small intercellular signalling proteins, called G-proteins, which are used to pass on messages to the rest of the cell from the receptor. Thus, these receptors are also called G-Protein Coupled Receptors or GPCRs. The research proposed in this application aims to understand what functions a novel cell surface GPCR from the fruitfly, Drosophila melanogaster, carries out in the animal and what processes it controls in the cells of the animal. This cell surface receptor is unusual since it can be turned on by both a class of insect steroid hormones, called the ecdysteroids, and by an adrenaline-like molecule, called dopamine. This receptor may be the insect equivalent of a highly unusual vertebrate receptor called the 'gamma-adrenergic receptor'. We do not yet know the structure of this vertebrate receptor but we do know that it can be activated by steroids and that the actions of these steroids can be blocked by dopamine-like molecules. Thus, the findings of this study will be of direct relevance to studies of this latter receptor in vertebrates, including humans. We plan to look at the sites where the steroids and dopamine attach to this fruitfly receptor using molecules tagged with a marker. We will see how easily other molecules, with a similar or different structure, can stop the tagged molecules attaching. This will help us follow how well each substance binds to these sites, give us information about the architecture of the sites and tell us whether the sites overlap or not. We also plan to determine if the signalling pathways, activated by the steroid and dopamine through this receptor, interact or modulate each other. Further, we plan to seek evidence for a function for this insect receptor in some of the rapid cell surface actions of ecdysteroids that have been described in studies on communication between nerve cells and muscles, and in studies on the growth and development of various parts of the insect nervous system. We will see if turning off the receptor in these preparations can block the observed effects of the ecdysteroids. The sequence of the bases in the DNA of all the genes in Drosophila has now been determined but the functions of some of the genes remains unknown and they have been called 'orphan' genes. Thus, we will look to see if any orphan GPCRs with unusual structural and functional properties may also represent cell surface steroid activated receptors. Finally, we plan to use the power of Drosophila genetics to create strains of flies where the novel steroid receptor has been turned off. The behaviour, and other characteristics, of these flies will provide additional information on the function of this receptor in the control of growth and signalling in the insect nervous system. The above studies will provide basic information on the functions of the rapid actions of steroids through cell surface G-protein coupled receptors. They will also provide detailed information on the potential use of this receptor as a new target site for pesticides.

We plan to carry out a study of the molecular mechanisms underlying the rapid nongenomic actions of ecdysteroids in Drosophila. This study will focus on the determination of the detailed pharmacology and second messenger coupling capabilities of a novel cloned Drosophila G-protein coupled receptor (DmDopEcR)(CG18314) that can be activated by both ecdysteroids and catecholamines. Radioligand binding studies using the plant ecdysteroid, 3[H]-ponasterone, will be used to determine the detailed pharmacology of steroid binding to this receptor and the degree of overlap between the ecdysteroid and catecholamine binding sites on the receptor. Since this receptor can activate the cyclic AMP pathway, when stimulated by dopamine, and the MAPKinase pathway, when activated by ecdysteroids, we will investigate the interactions between these two pathways. Further, we plan to seek evidence for a physiological role for this receptor in some of the rapid nongenomic actions of ecdysteroids that have been described in a range of insect physiological (e.g spotaneous neurotransmitter release at larval neuromuscular junctions) and developmental (e.g. progression of morphological furrow in developing eye optic discs and control of cell proliferation in optic lobe neurogenesis) preparations. In these preparations we will use a combination of detailed pharmacological studies together with RNA interference techniques to knock out the expression of the receptor. We have identified two additional orphan GPCRs that may represent additional candidates for Drosophila steroid activated cell surface receptors. We will carry out pharmacological and second messenger expression studies to identify the cognate ligands for these receptors. Finally, we plan to use the power of Drosophila genetics to knock out, and to selectively misexpress, the gene for DmDopEcR to provide additional evidence for its functional role in the control of insect development and in signalling in the adult nervous system.