ATTACHMENT, TARGETING & LOCALIZATION OF G ALPHA SUBUNIT

附件、瞄准

• 批准号: 2734825
• 负责人: BRADLEY M DENKER
• 金额: $ 11.87万
• 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 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734825
• 关键词: G protein; biological signal transduction; cell membrane; cellular polarity; chimeric proteins; confocal scanning microscopy; cyclic AMP; gene mutation; immunofluorescence technique; inositol phosphates; membrane activity; membrane proteins; nucleic acid sequence; polymerase chain reaction; tight junctions; tissue /cell culture; transfection

DESCRIPTION (Adapted from applicant's abstract): Heterotrimeric guanine nucleotide binding proteins (G proteins), composed of Galpha and Gbeta/gamma subunits, transduce signals across cell membranes from receptors to a variety of effectors that include adenylyl cyclases, phosphodiesterases, phospholipases and ion channels. Receptor-stimulation causes Galpha to exchange GTP for GDP and dissociation from Gbeta/gamma. Both subunits interact with effectors until Galpha hydrolyzes GTP to GDP. Hormone-receptor interactions are very specific, but many receptor-G protein interactions are less specific. Reconstitution studies with G proteins, receptors and effectors show that many Galpha subunits can couple to the same receptor and effector. Since multiple G-protein-coupled signaling pathways exist in every eukaryotic cell, there is the potential for crosstalk between signaling pathways. A proposal is that localizing G proteins in specific membrane domains is an important mechanism for signal specificity. This proposal will use mutant Galpha subunits (some already characterized and new one to be made based on crystal structures) to address questions of how Galpha attaches to the membrane, and localizes in specific membrane domains. The interactions of Galpha subunits with the membrane vary among Galpha families. Using in vitro assays and transient transfections, mutations at both the N- and C-termini of Galpha-o, Galpha-s and Galpha-q will be used to identify regions important to membrane binding that are in addition to known roles from lipids and interactions with beta/gamma. With these techniques, amino acids 11-14 of Galpha-o have been found to contribute to membrane binding through an unidentified membrane protein(s). The targeting of Galpha to specific membrane domains is being studied in MDCK cells (polarized epithelia). To follow Galpha in these cells, stable cell lines expressing Galpha-o (not normally expressed) and epitope tagged Galpha subunits are being established and characterized by immunofluorescence and confocal microscopy. Galpha-o localizes to the lateral membrane and overlaps with the endogenous Galpha-i2 and ZO-1 (tight junction (TJ) protein). Mutant Galpha-o subunits and chimeras of Galpha-o and Galpha-s (both apical and basolateral localization) will be used to identify regions important for specific membrane targeting. The pathway(s) of targeting will be established for Galpha-o and endogenous Galpha i2, Galpha-s, and Galpha-q by pulse chase labeling and selective membrane biotinylation. Expression of activated Galpha-o (Q205L) also localizes to the basolateral membrane and causes accelerated formation of TJs using the Ca+2 switch model of TJ biogenesis. A role for Galpha subunits in TJ biogenesis will be further characterized with stable cell lines expressing wildtype and activated Galpha subunits. Accurate signaling is critical for all cells, and signaling pathways are often disrupted after ischemic tissue injury. TJ formation is critical in developing epithelial tissues and during recovery from ischemia (acute tubular necrosis). These studies may give new insights to an understanding of signal specificity in all cells, and may permit the development of strategies to prevent or correct aberrant signaling in diseased or injured tissues.

描述（改编自申请人摘要）：异三聚鸟嘌呤 核苷酸结合蛋白（G蛋白），由G α和G β/γ组成 亚基，使信号穿过细胞膜，从受体到受体， 包括腺苷酸环化酶，磷酸二酯酶， 磷脂酶和离子通道。 受体刺激导致G α 将GTP交换为GDP并从G β/γ解离。 两个亚基 与效应物相互作用，直到Galpha将GTP水解为GDP。 激素-受体相互作用非常特异，但许多受体-G蛋白 交互作用不太具体。 用G蛋白的重构研究， 受体和效应器表明，许多G α亚单位可以耦合到 相同的受体和效应子。 因为多重G蛋白偶联信号 每个真核细胞中都存在这种途径， 信号通路之间的串扰。 一个建议是，本地化G 特定膜结构域中的蛋白质是信号传导的重要机制。 的特异性 这项提议将使用突变的Ga α亚基（一些已经 特征和新的一个要基于晶体结构），以解决 问题是Galalpha如何附着在细胞膜上，并定位在特定的细胞中， 膜结构域 G α亚基与细胞膜的相互作用 不同的阿尔法家族。 使用体外试验和瞬时 转染，Galpha-o、Galpha-s的N-和C-末端突变 和Galpha-q将被用于识别对膜结合重要的区域 除了已知的脂质作用和与 β/γ 利用这些技术，已经将Galpha-o的氨基酸11 - 14 发现通过未识别的膜促进膜结合 蛋白质。 目前正在研究将G α靶向于特定的膜结构域， 在MDCK细胞（极化上皮细胞）中研究。 跟随阿尔法在这些 细胞、表达Galpha-o（正常情况下不表达）的稳定细胞系， 表位标记的G α亚基正在建立并表征为： 免疫荧光和共聚焦显微镜。 galpha-o定位于 侧膜和重叠的内源性Galpha-i2和ZO-1（紧 连接（TJ）蛋白）。 突变的Galpha-o亚基和Galpha-o的嵌合体 和Galpha-s（顶侧和基底侧定位）将用于 鉴定对特异性膜靶向重要的区域。 途径 将为Galpha-o和内源性Galphai2建立靶向， Galpha-s和Galpha-q通过脉冲追踪标记和选择性膜 生物素化 活化的Galpha-o（Q205L）的表达也定位于 基底外侧膜，并导致加速形成TJ使用 TJ生物发生的Ca +2开关模型 Ga α亚基在TJ中的作用 生物发生将进一步表征为稳定的细胞系表达 野生型和活化的G α亚基。 准确的信号传输对于 缺血组织后，所有细胞和信号通路通常都会被破坏 损伤 TJ的形成在上皮组织的发育中至关重要， 在从局部缺血（急性肾小管坏死）恢复期间。 这些研究可能 为理解所有细胞中的信号特异性提供了新的见解， 并且可以允许发展策略以防止或纠正异常的 在患病或受伤的组织中发出信号。