ATTACHMENT, TARGETING & LOCALIZATION OF G ALPHA SUBUNIT

附件、瞄准

• 批准号: 6386650
• 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/6386650
• 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（改编自申请人的摘要）：异三个鸟嘌呤 由Galpha和Gbeta/Gamma组成的核苷酸结合蛋白（G蛋白） 亚基，细胞膜跨受体转导信号 包括腺苷循环酶，磷酸二酯酶的各种效应子， 磷脂酶和离子通道。 受体刺激使Galpha达到 将GTP交换为GDP，并与GBETA/GAMMA解离。 两个亚基 与效应子相互作用，直到Galpha将GTP水解为GDP。 激素受体相互作用非常具体，但是许多受体-G蛋白 相互作用不太具体。 用G蛋白进行重构研究， 受体和效应子表明，许多Galpha亚基都可以搭配到 相同的受体和效应子。 由于多个G蛋白偶联信号传导 每个真核细胞中都存在途径，有可能 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的重要机制 特异性。 该建议将使用突变的Galpha亚基（有些已经 特征是根据晶体结构制作的，以解决 关于Galpha如何依恋膜的问题，并在特定的 膜域。 Galpha亚基与膜的相互作用 在Galpha家庭中有所不同。 使用体外测定和瞬态 转染，Galpha-O的N-和C末端的突变，Galpha-S Galpha-Q将用于识别对膜结合重要的区域 这是脂质的已知角色以及与 beta/伽玛。 使用这些技术，Galpha-O的氨基酸11-14已经 发现通过身份不明的膜有助于膜结合 蛋白质。 Galpha靶向特定的膜结构域 在MDCK细胞（偏振上皮）中进行了研究。 在这些中跟随galpha 细胞，表达galpha-O（通常不表达）的稳定细胞系，并且 建立和特征的表位标记为galpha亚基 免疫荧光和共聚焦显微镜。 Galpha-o本地化 外侧膜和与内源性Galpha-i2和ZO-1重叠（紧密 结（TJ）蛋白）。 galpha-o的突变galpha-o亚基和嵌合体 和Galpha-S（顶端和基底外侧定位）将用于 确定对特定膜靶向重要的区域。 路径 将建立针对Galpha-O和内源性Galpha I2的靶向目标 galpha-s和脉冲追逐标签和选择性膜的Galpha-Q 生物素化。 激活的Galpha-O（Q205L）的表达也位于 基底外侧膜并使用该膜加速形成TJ TJ生物发生的CA+2开关模型。 TJ中Galpha亚基的角色 生物发生将通过表达稳定的细胞系进一步表征 野生型和激活的galpha亚基。 准确的信号对于 所有细胞和信号通路经常在缺血组织后破坏 受伤。 TJ形成对于发展上皮组织和 在缺血（急性管状坏死）中恢复期间。 这些研究可能 给出新见解以了解所有细胞中信号特异性的理解， 并可能允许制定防止或纠正异常的策略 在患病或受伤的组织中发出信号。

项目成果

Heterotrimeric G proteins and apoptosis: intersecting signaling pathways leading to context dependent phenotypes.

异三聚体 G 蛋白和细胞凋亡：交叉信号通路导致背景依赖性表型。

• DOI: 10.2174/156652409788488784
• 发表时间: 2009
• 期刊: Current molecular medicine
• 影响因子: 2.5
• 作者: Yanamadala,Vijay;Negoro,Hideyuki;Denker,BradleyM
• 通讯作者: Denker,BradleyM

Domains necessary for Galpha12 binding and stimulation of protein phosphatase-2A (PP2A): Is Galpha12 a novel regulatory subunit of PP2A?

Galpha12 结合和刺激蛋白磷酸酶 2A (PP2A) 所需的结构域：Galpha12 是 PP2A 的新型调节亚基吗？

• DOI: 10.1124/mol.106.033555
• 发表时间: 2007
• 期刊: Molecular pharmacology
• 影响因子: 3.6
• 作者: Zhu,Deguang;Tate,RobertI;Ruediger,Ralf;Meigs,ThomasE;Denker,BradleyM
• 通讯作者: Denker,BradleyM

Identification of polycystin-1 and Gα12 binding regions necessary for regulation of apoptosis.

鉴定调节细胞凋亡所需的多囊蛋白-1 和Gα12 结合区域。

• DOI: 10.1016/j.cellsig.2010.09.005
• 发表时间: 2011
• 期刊: Cellular signalling
• 影响因子: 4.8
• 作者: Yu,Wanfeng;Ritchie,BenjaminJ;Su,Xuefeng;Zhou,Jing;Meigs,ThomasE;Denker,BradleyM
• 通讯作者: Denker,BradleyM

Galpha12 directly interacts with PP2A: evidence FOR Galpha12-stimulated PP2A phosphatase activity and dephosphorylation of microtubule-associated protein, tau.

Galpha12 直接与 PP2A 相互作用：Galpha12 刺激 PP2A 磷酸酶活性和微管相关蛋白 tau 去磷酸化的证据。

• DOI: 10.1074/jbc.c400508200
• 发表时间: 2004
• 期刊: The Journal of biological chemistry
• 作者: Zhu,Deguang;Kosik,KennethS;Meigs,ThomasE;Yanamadala,Vijay;Denker,BradleyM
• 通讯作者: Denker,BradleyM