Molecular mechanisms underlying rapid nongenomic actions of ecysteroids

类固醇是一种天然存在于体内的物质，作为性激素和控制肌肉和大脑等组织生长的激素。通常，类固醇激素通过进入细胞并与称为受体的特殊分子结合而起作用。当这些受体被类固醇激活时，它们就会开启一系列不同基因的细胞核表达，这些基因是针对这种激素的。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一起作用，受体与细胞外表面的激素结合，改变它们的形状，然后将类固醇激素存在的信息传递给细胞内部。然后细胞以适当的方式作出反应。构成一类细胞表面受体的蛋白质分子结合了其他小的细胞间信号蛋白，称为g蛋白，用于将信息从受体传递到细胞的其余部分。因此，这些受体也被称为g蛋白偶联受体或gpcr。本申请中提出的研究旨在了解来自果蝇（Drosophila melanogaster）的一种新型细胞表面GPCR在动物体内发挥的功能以及它在动物细胞中控制的过程。这种细胞表面受体是不寻常的，因为它可以被一种昆虫类固醇激素（称为表皮类固醇）和一种类似肾上腺素的分子（称为多巴胺）激活。这种受体可能相当于一种非常不寻常的脊椎动物受体，叫做“γ -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多巴胺类分子阻断。因此，这项研究的发现将直接关系到研究后一种受体在脊椎动物，包括人类。我们计划用带有标记的分子来观察类固醇和多巴胺附着在果蝇受体上的位置。我们将看到其他分子，具有相似或不同的结构，可以很容易地阻止标记分子的附着。这将帮助我们了解每种物质与这些位点的结合情况，为我们提供有关位点结构的信息，并告诉我们这些位点是否重叠。我们还计划确定通过这种受体被类固醇和多巴胺激活的信号通路是否相互作用或相互调节。此外，我们计划寻找证据，证明这种昆虫受体在体外类固醇的一些快速细胞表面作用中的作用，这些作用已经在神经细胞和肌肉之间的交流研究中被描述，以及在昆虫神经系统各部分的生长和发育研究中被描述。我们将会看到在这些制剂中关闭受体是否可以阻断所观察到的皮质类固醇的作用。果蝇所有基因的DNA碱基序列现在已经确定，但一些基因的功能仍然未知，它们被称为“孤儿”基因。因此，我们将看看是否有任何具有不寻常结构和功能特性的孤儿gpcr也可能代表细胞表面类固醇激活受体。最后，我们计划利用果蝇遗传学的力量，创造出新的类固醇受体被关闭的果蝇品系。这些果蝇的行为和其他特征将为这种受体在控制昆虫神经系统生长和信号传递方面的功能提供额外的信息。以上研究将为类固醇通过细胞表面g蛋白偶联受体的快速作用功能提供基础信息。它们还将提供有关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.