Attachment, Targeting & Localization of Galpha Subunits

附件、目标

• 批准号: 6606951
• 负责人: BRADLEY M DENKER
• 金额: $ 34.6万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6606951
• 关键词: G protein; MDCK cell; SDS polyacrylamide gel electrophoresis; biological signal transduction; cell membrane; cellular polarity; cerebellar Purkinje cell; chimeric proteins; cyclic AMP; glutathione transferase; guanosine diphosphate; membrane activity; membrane proteins; nucleic acid sequence; nucleotides; polymerase chain reaction; tight junctions; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): Neurons and epithelial cells are polarized cells that target proteins to both structural and functional compartments within the cell. Because of this compartmentalization, the localization of specific signaling components is necessary to achieve ligand-specific cellular responses. At present, little is known about the compartmentalization of signaling components such as G proteins. In this proposal, we hypothesize that localization of signaling molecules into discrete microdomains prevents signal cross-talk and contributes to specific cellular responses. This renewal application proposes to extend this hypothesis by defining the mechanisms of Ga regulation by two different modulatory proteins important in epithelial cells and neurons. As work in progress, we have demonstrated direct interactions between Galpha12 and Galpha12 with the epithelial cell tight junction (TJ) protein Zona Occludens-1 (ZO-1), and between Galpha-0 and the neuronal protein, Purkinje cell protein-2 (Pcp2). Pcp2 activates Galpha 0 in-vitro by stimulating GDP release. Galpha-12 and Galpha12 are localized within the TJ where they regulate fundamental TJ properties including its assembly and the barrier to paracellular particle movement. Aim 1will focus on ZO-1/Galpha12 and ZO-1/Galpha-12 interactions to determine whether there is direct modulation of Galpha by ZO-1, and/or ZO-1 is a scaffold that organizes signaling complexes important for regulating the junction. Utilizing baculovirus expressed proteins, glutathione-S-transferase (GST) fusion proteins, MDCK cell culture systems, and enzymatic assays, we aim to: (1) define the domains of Galpha12 and Galpha-12 interacting with ZO- 1 (2) determine if ZO- 1 modulates k-cat and k-off of Galpha12 and Galpha-12; (3) test whether phosphorylation of Ga12 or ZO-l affects the interaction, and (4) characterize the Ga12/Src signaling pathway regulating the TJ in MDCK cells. Aim 2 will define the mechanism of Pcp2 activation of Ga0 and the function of this interaction in neurons. Pcp2 contains a Goloco motif, a conserved domain found in several other proteins that regulate Ga subunits. We hypothesize that Pcp2 regulates Ga0 through the Goloco motif to stimulate nucleotide exchange, and this interaction regulates a basic neuronal process such as neurite formation. Here, we will; (1) identify the contact sites and regulatory domains of Ga0 and Pcp2; (2) determine role of serine/threonine phosphorylation (MAP Kinasel) of Pcp2 in regulating the interaction with Ga0, and (3) characterize Ga0/Pcp2 localization in adult and new born cerebellar tissue, and utilize inducible Pcp2 expression in cultured neuronal cells (PC 12) to determine localization, phosphorylation, and the influence on neurite formation. Such experiments will yield insight into the regulation of compartmentalized cellular responses, and could provide the basis for development of novel therapeutics for disorders influencing the epithelial cell TJ as well as those impacting neuronal function.

描述（由申请人提供）：神经元和上皮细胞是极化的 将蛋白靶向结构和功能室的细胞 在细胞内。由于这种隔室化， 特定的信号传导成分对于实现配体特异性细胞是必需的 回答。目前，对 信号传导成分，例如G蛋白。在此提案中，我们假设 信号分子在离散的微域中的定位可防止信号 交叉言论并有助于特定的细胞反应。这个更新 应用建议通过定义GA的机制来扩展这一假设 通过两种不同调节蛋白在上皮细胞中调节 和神经元。随着正在进行的工作，我们展示了直接互动 与上皮细胞紧密连接（TJ）之间的Galpha12和Galpha12之间 蛋白zona occludens-1（ZO-1），在Galpha-0和神经元蛋白之间， Purkinje细胞蛋白2（PCP2）。 pcp2通过刺激GDP激活Galpha 0维特罗 发布。 galpha-12和galpha12位于TJ中，它们调节 基本的TJ属性，包括其组装和障碍 细胞细胞粒子运动。 目标1Will专注于ZO-1/Galpha12和ZO-1/Galpha-12相互作用，以确定是否是否 通过ZO-1对Galpha进行了直接调节，/或ZO-1是一个脚手架 组织信号复合体对于调节交界处的重要性。利用 杆状病毒表达的蛋白质，谷胱甘肽-S-转移酶（GST）融合 蛋白质，MDCK细胞培养系统和酶测定，我们的目标是：（1） 定义与ZO-1（2）相互作用的Galpha12和Galpha-12的域 如果ZO-1调节Galpha12和Galpha-12的K-cat和K-Off； （3）测试是否 GA12或ZO-L的磷酸化影响相互作用，（4）表征 GA12/SRC信号通路调节MDCK细胞中的TJ。 AIM 2意志 定义GA0的PCP2激活的机制及其功能 神经元的相互作用。 PCP2包含一个Goloco图案，发现了一个保守的域 在调节GA亚基的其他几种蛋白质中。我们假设PCP2 通过GOLOCO基序调节GA0以刺激核苷酸交换，并且 这种相互作用调节基本的神经元过程，例如神经突的形成。 在这里，我们会； （1）确定GA0和 pcp2; （2）确定丝氨酸/苏氨酸磷酸化（MAP KINASEL）的作用 PCP2在调节与GA0的相互作用时，（3）表征GA0/PCP2 在成人和新出生的小脑组织中定位，并利用诱导 培养神经元细胞中的PCP2表达（PC 12）确定定位， 磷酸化以及对神经突的影响。这样的实验会 对隔室化细胞反应的调节产生洞察力，以及 可以为疾病开发新型治疗学提供基础 影响上皮细胞TJ以及影响神经元的细胞 功能。