ATTACHMENT, TARGETING & LOCALIZATION OF G ALPHA SUBUNIT

附件、瞄准

基本信息

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
项目类别：
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

项目摘要

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）：异三聚鸟嘌呤核苷酸结合蛋白（G 蛋白），由 Galpha 和 Gbeta/gamma 组成亚基，将信号从受体跨细胞膜转导到多种效应子，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 Galpha 将 GTP 交换为 GDP 并与 Gbeta/gamma 分离。两个亚基与效应器相互作用，直到 Galpha 将 GTP 水解为 GDP。激素-受体相互作用非常具体，但许多受体-G 蛋白交互作用不太具体。 G 蛋白的重构研究，受体和效应器表明许多 Galpha 亚基可以与相同的受体和效应器。由于多个 G 蛋白偶联信号传导每个真核细胞中都存在通路，因此有潜力信号通路之间的串扰。一个建议是本地化 G 特定膜域中的蛋白质是信号的重要机制特异性。该提案将使用突变的 Galpha 亚基（有些已经根据晶体结构制造的特征和新材料）来解决关于 Galpha 如何附着在膜上并定位于特定区域的问题膜域。 Galpha亚基与膜的相互作用 Galpha 家族之间存在差异。使用体外测定和瞬态转染、Galpha-o、Galpha-s N 端和 C 端突变 Galpha-q 将用于识别对膜结合重要的区域除了已知的脂质作用和与贝塔/伽玛。通过这些技术，Galpha-o 的氨基酸 11-14 已被发现有助于通过未识别的膜进行膜结合蛋白质。 Galpha 靶向特定膜域的研究正在在 MDCK 细胞（极化上皮细胞）中进行研究。跟随 Galpha 在这些方面细胞、表达 Galpha-o（通常不表达）的稳定细胞系和表位标记的 Galpha 亚基正在建立和表征免疫荧光和共聚焦显微镜。 Galpha-o 定位于侧膜并与内源性 Galpha-i2 和 ZO-1 重叠（紧密连接（TJ）蛋白）。突变型 Galpha-o 亚基和 Galpha-o 嵌合体 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。途径将为 Galpha-o 和内源性 Galpha i2 建立靶向，通过脉冲追踪标记和选择性膜的 Galpha-s 和 Galpha-q 生物素化。激活的 Galpha-o (Q205L) 的表达也定位于基底外侧膜并导致 TJ 的加速形成 TJ 生物发生的 Ca+2 转换模型。 Galpha 亚基在 TJ 中的作用生物发生将通过表达稳定的细胞系进一步表征野生型和活化的 Galpha 亚基。准确的信号传递对于组织缺血后，所有细胞和信号通路常常被破坏受伤。 TJ 的形成对于上皮组织的发育至关重要从缺血（急性肾小管坏死）恢复期间。这些研究可能为理解所有细胞的信号特异性提供新的见解，并可能允许制定策略来防止或纠正异常情况患病或受伤组织中的信号传导。

项目成果

期刊论文数量（17）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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。