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）：神经元和上皮细胞极化 将蛋白质定位于结构和功能区室的细胞 在细胞内。由于这种划分， 特异性信号传导组分是实现配体特异性细胞免疫所必需的。 应答目前，人们对药物的分类知之甚少。 信号成分如G蛋白。在本提案中，我们假设， 将信号分子定位到离散的微区中阻止了信号传导。 串扰，并有助于特定的细胞反应。此续订 应用程序提出通过定义Ga的机制来扩展这一假设。 由两种不同的调节蛋白调节，在上皮细胞中很重要 和神经元。随着工作的进行，我们已经证明了直接的互动 Galpha 12和Galpha 12与上皮细胞紧密连接（TJ）之间的关系 蛋白质封闭带-1（ZO-1），以及在Ga-0和神经元蛋白质之间， 浦肯野细胞蛋白-2（Pcp 2）。Pcp 2通过刺激GDP在体外激活Galalpha 0 release. Galpha 12和Galpha 12位于TJ内，它们调节 基本的TJ特性，包括其组装和对 细胞旁颗粒运动。 目标1将重点关注ZO-1/Galpha 12和ZO-1/Galpha 12相互作用，以确定 ZO-1直接调节Ga α，和/或ZO-1是支架， 组织对调节连接重要的信号复合物。利用 杆状病毒表达蛋白，谷胱甘肽-S-转移酶融合 蛋白质，MDCK细胞培养系统和酶测定，我们的目标是：（1） 定义Galpha 12和Galpha 12与ZO- 1相互作用的结构域（2）确定 ZO- 1是否调节Galpha 12和Galpha 12的k-cat和k-off;（3）测试是否 Ga 12或ZO-1的磷酸化影响相互作用，和（4）表征 Ga 12/Src信号通路调控MDCK细胞TJ。目标2将 明确Pcp 2激活Ga 0的机制及其功能， 神经元的相互作用Pcp 2含有Goloco基序，这是一个保守的结构域， 在其他几种调节Ga亚基的蛋白质中。我们假设Pcp 2 通过Goloco基序调节Ga 0以刺激核苷酸交换，以及 这种相互作用调节基本的神经元过程，例如神经突形成。 在这里，我们将：（1）确定Ga 0的接触点和监管域， Pcp 2;（2）确定丝氨酸/苏氨酸磷酸化（MAP Kinasel）的作用 Pcp 2在调节与Ga 0相互作用中的作用，以及（3）表征Ga 0/Pcp 2 定位于成人和新生儿小脑组织，并利用诱导 培养的神经元细胞（PC 12）中的Pcp 2表达以确定定位， 磷酸化以及对神经突形成的影响。这些实验将 深入了解区室化细胞反应的调节， 可以为开发用于疾病的新疗法提供基础， 影响上皮细胞TJ以及影响神经元TJ的那些 功能