Molecular mechanisms underlying rapid nongenomic actions of ecysteroids

类固醇是体内天然存在的物质，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，它们可以打开细胞核中一系列不同基因的表达，这些基因对该激素具有特异性。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，类固醇与细胞表面上的受体作用，这些受体在其外表面结合激素，改变其形状，然后将有关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结合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果蝇（Drosophila melanogaster）的新型细胞表面GPCR在动物中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，因为它可以被一类昆虫类固醇激素（称为蜕皮类固醇）和一种肾上腺素样分子（称为多巴胺）打开。这种受体在昆虫中可能相当于一种非常罕见的脊椎动物受体，称为“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样分子阻断。因此，这项研究的结果将直接相关的研究后一种受体在脊椎动物，包括人类。我们计划使用标记分子来观察类固醇和多巴胺附着在果蝇受体上的部位。我们将看到其他具有相似或不同结构的分子如何容易地阻止标记分子附着。这将帮助我们跟踪每种物质与这些位点结合的情况，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。我们还计划确定类固醇和多巴胺通过该受体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，为这种昆虫受体的功能，在一些快速的细胞表面行动的蜕皮甾类化合物，已被描述在神经细胞和肌肉之间的通信研究，并在昆虫神经系统的各个部分的生长和发育的研究。我们将观察是否关闭这些制剂中的受体可以阻断蜕皮类固醇的观察效果。果蝇所有基因的DNA中的碱基序列现在已经确定，但有些基因的功能仍然未知，它们被称为“孤儿”基因。因此，我们将观察是否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来创造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提供关于这种受体在昆虫神经系统中控制生长和信号传导的功能的额外信息。上述研究将为类固醇激素通过细胞表面G蛋白偶联受体发挥快速作用提供基础资料。他们还将提供有关这种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.