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TRANSMEMBRANE SIGNALING VIA RECEPTORS AND G PROTEINS

通过受体和 G 蛋白的跨膜信号传导

基本信息

批准号：
2176376
负责人：
PAUL C STERNWEIS
金额：
$ 34.31万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-04-01 至 1995-03-31
项目状态：
已结题

项目摘要

Strategies are proposed for the elucidation of mechanisms by which a large family of membrane-associated proteins, G protein, mediate regulation of intracellular processes by extracellular signals (hormones, neurotransmitters, light, etc.). This family includes the Gs and Gt proteins which stimulate adenylyl cyclase and the visual cGMP- phosphodiesterase, respectively. Other G proteins are implicated in the regulation of phospholipase activities and ion channels. They mediate hormonal regulation of inositol polyphosphates, diacylglycerol, arachidonic acid, membrane potential, and Ca2+ influx. Three major questions are addressed. First, how are more that 100 receptors, 15 or more, and several regulated activities linked together? Second, what is the specificity of subunit interaction within the G protein family? Third, how extensive is the spectrum of regulation of the G proteins? The first question is addressed in part by attempts to reconstitute regulation of signalling pathways with purified G proteins. A specific focus will be on the characterization of a new G protein, Gq, and other similar proteins that are not substrates for bacterial toxins. Receptor interaction will be examined in membranes as well as by reconstitution with purified receptors. Functional studies will focus on the regulation of phospholipases. More general techniques will use immobilized alpha and betagamma subunit matrices in attempts to isolate, and thus identify regulated proteins. Interaction among unique G protein subunits will be examined in detail. This will be supported by the preparation of specific complexes of the beta and gamma subunits through expression in a baculovirus system. The interaction of these complexes with individual alpha subunits, their role in receptor interaction, and their potential regulation of effector molecules will be examined. Post-translational modification, which effects the functional interaction of the alpha and betagamma subunits with each other and effectors, will be examined further. This is facilitated by isolation of labeled subunits with affinity matrices. The potential spectrum of regulation by G proteins will be examined be characterizing other proteins with which they interact. Strategies for exploiting subunit affinity columns in this endeavor are proposed. One focus is on an 80 kDa protein that binds to the betagamma subunits in the presence of Ca2+. In summary, this proposal examines several aspects of G protein regulation. It will help define the specificity and action of these common signalling pathways for hormones.

人们提出了一些策略，以阐明大范围的膜相关蛋白家族，G蛋白，介导调节由细胞外信号(激素、神经递质、光等)。该系列包括Gs和GT 刺激腺酰环化酶和视觉cGMP的蛋白质- 磷酸二酯酶。其他G蛋白也参与了磷脂酶活性和离子通道的调节。他们进行调解多聚肌醇、二酰甘油、花生四烯酸的激素调节酸、膜电位和钙离子内流。解决了三个主要问题。首先，怎么会有100多个 15个或更多的受体和几个受调控的活动联系在一起？第二，G蛋白中亚基相互作用的特异性是什么？家人？第三，对G的监管范围有多广蛋白质？第一个问题在一定程度上是通过尝试重组纯化的G蛋白对信号通路的调节。一种特定的重点将集中在一种新的G蛋白，GQ和其他不是细菌毒素底物的相似蛋白质。受体将在膜中检查相互作用，以及通过与纯化的受体。功能研究将集中在调节磷脂酶。更通用的技术将使用固定的阿尔法和 Betagamma亚单位矩阵试图分离，从而识别调节蛋白质。我们将详细研究独特的G蛋白亚基之间的相互作用。这将通过制备Beta的特定络合物来支持和伽马亚基通过在杆状病毒系统中表达。这个这些复合体与单个α亚基的相互作用，它们的作用在受体相互作用中，及其对效应器的电位调节将对分子进行检查。翻译后修饰，会产生什么影响 α亚基和β亚基之间的功能相互作用其他和效应器，将进一步研究。这是通过以下方式促进的用亲和矩阵分离标记的亚基。 G蛋白调节的潜在光谱将被研究确定与之相互作用的其他蛋白质的特征。战略以实现建议在这一努力中利用亚基亲和柱。一焦点是一种80 kDa的蛋白，它与β-淀粉样蛋白中的β亚基结合。钙离子的存在。综上所述，这项建议研究了G蛋白调控的几个方面。这将有助于定义这些常见信令的特异性和作用荷尔蒙的途径。