Molecular mechanisms underlying rapid nongenomic actions of ecysteroids

类固醇是体内自然存在的物质，充当性激素和控制肌肉和大脑等组织生长的激素。通常情况下，类固醇激素通过进入细胞并与称为受体的特殊分子结合来发挥作用。当这些受体被类固醇激活时，它们就可以启动一系列针对该激素的不同基因在细胞核中的表达。然而，最近的研究表明，类固醇激素也可以通过不涉及它们进入细胞的机制，迅速对细胞产生影响。在这些情况下，类固醇与细胞表面的受体作用，后者将激素结合在细胞外表面，改变其形状，然后将关于类固醇激素存在的信息传递到细胞内部。然后，细胞以适当的方式做出反应。构成一类细胞表面受体的蛋白质分子与其他被称为G蛋白的小细胞间信号蛋白结合，G蛋白被用来将信息从受体传递到细胞的其他部分。因此，这些受体也被称为G蛋白偶联受体或GPCRs。在这项申请中提出的研究旨在了解果蝇黑腹果蝇的新细胞表面GPCR在动物体内执行什么功能，以及它在动物细胞中控制什么过程。这种细胞表面受体是不寻常的，因为它可以被一类昆虫类固醇激素(称为蜕皮激素)和一种类似肾上腺素的分子(称为多巴胺)激活。这种受体可能是昆虫上的一种非常不寻常的脊椎动物受体，称为“伽马肾上腺素能受体”。我们还不知道这种脊椎动物受体的结构，但我们知道它可以被类固醇激活，这些类固醇的作用可以被类似多巴胺的分子阻断。因此，这项研究的发现将与脊椎动物(包括人类)中的后一种受体的研究直接相关。我们计划利用标记分子观察类固醇和多巴胺与这种果蝇受体结合的部位。我们将看到具有相似或不同结构的其他分子如何容易地阻止标记的分子附着。这将帮助我们跟踪每种物质与这些站点的绑定情况，给我们提供关于站点架构的信息，并告诉我们站点是否重叠。我们还计划确定由类固醇和多巴胺通过这种受体激活的信号通路是否相互作用或调节。此外，我们计划在蜕皮激素的一些快速细胞表面作用中寻找这种昆虫受体的功能的证据，这些作用已在神经细胞和肌肉之间的通讯研究中描述，并在昆虫神经系统不同部分的生长和发育研究中得到描述。我们将看看关闭这些制剂中的受体是否可以阻断蜕皮类固醇的观察到的效果。果蝇所有基因的DNA碱基序列已经确定，但其中一些基因的功能尚不清楚，这些基因被称为“孤儿”基因。因此，我们将寻找是否有任何具有不寻常结构和功能特性的孤儿GPCRs也可能代表细胞表面类固醇激活的受体。最后，我们计划利用果蝇遗传学的力量创造出一种新的类固醇受体已经关闭的果蝇品系。这些苍蝇的行为和其他特征将为这种受体在昆虫神经系统中控制生长和信号传递的功能提供更多信息。上述研究将为类固醇通过细胞表面G蛋白偶联受体发挥快速作用的功能提供基础信息。他们还将提供关于该受体作为杀虫剂新靶点的潜在用途的详细信息。

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.