TRANSMEMBRANE SIGNALING VIA RECEPTORS AND G PROTEINS

通过受体和 G 蛋白进行跨膜信号传导

• 批准号: 3280400
• 负责人: PAUL C STERNWEIS
• 金额: $ 31.93万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/3280400
• 关键词: Baculoviridae; G protein; adenylate cyclase; affinity chromatography; biological signal transduction; calcium binding protein; enzyme induction /repression; enzyme reconstitution; hormone regulation /control mechanism; intracellular transport; laboratory rabbit; membrane activity; membrane permeability; membrane proteins; phosphodiesterases; phospholipase C; posttranslational modifications; protein purification; protein reconstitution; protein structure function; receptor; receptor binding; second messengers; transposon /insertion element; ultracentrifugation

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.

战略提出了阐明的机制，其中大 膜相关蛋白家族，G蛋白，介导 通过细胞外信号（激素， 神经递质、光等）。 该系列包括GS和GT 刺激腺苷酸环化酶和视觉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。 它们介导 多磷酸肌醇、甘油二酯、花生四烯酸的激素调节 酸、膜电位和Ca 2+内流。 三个主要问题得到解决。 第一，100多个人 受体，15个或更多，以及几种相互关联的受调节活动？ 第二，G蛋白内部亚基相互作用的特异性是什么 家人？ 第三，G的监管范围有多广 蛋白质？ 第一个问题部分是通过试图重建 用纯化的G蛋白调节信号通路。 特定 重点将是一个新的G蛋白，Gq的表征，和其他 不是细菌毒素底物的类似蛋白质。 受体 将在膜中以及通过与 纯化的受体 功能研究将集中在调节 磷脂酶 更一般的技术将使用固定的α和 β-γ亚基矩阵，试图分离，从而确定 调节蛋白质。 独特的G蛋白亚基之间的相互作用将被详细检查。 这将通过制备β的特定复合物来支持。 和γ亚基。 的 这些复合物与单个α亚单位的相互作用，它们的作用 在受体相互作用中，以及它们对效应物的潜在调节 分子将被检查。 翻译后修饰， α和β γ亚基与每个 其他和效应，将进一步审查。 这是由以下因素促成的： 用亲和基质分离标记的亚基。 G蛋白调节的潜在谱将被检查， 表征与它们相互作用的其他蛋白质。 战略 提出了在这种奋进中利用亚基亲和柱。 一 重点是一个80 kDa的蛋白质，结合到β γ亚单位在 Ca 2+的存在。 总之，本提案研究了G蛋白调控的几个方面。 这将有助于确定这些共同信号的特异性和作用 激素的途径。