ATTACHMENT, TARGETING & LOCALIZATION OF G ALPHA SUBUNIT

描述(改编自申请者摘要)：杂三聚鸟嘌呤 核苷酸结合蛋白(G蛋白)，由Galpha和Gbeta/Gamma组成 亚基，通过细胞膜将信号从受体传递到 各种效应物，包括腺酰环化酶，磷酸二酯酶， 磷脂酶和离子通道。受体刺激导致Galpha 用GTP换取GDP，并与Gbeta/Gamma解离。两个亚单位 与效应器相互作用，直到Galpha将GTP水解为GDP。 激素-受体的相互作用是非常特殊的，但许多受体-G蛋白 互动不那么具体。G蛋白的重组研究， 受体和效应器显示许多Galpha亚基可以与 同样的受体和效应器。由于多个G蛋白偶联信号 通路存在于每个真核细胞中，有可能 信号通路之间的串扰。一项建议是将G本地化 特定膜结构域中的蛋白质是信号传递的重要机制 专一性。这项提议将使用突变的Galpha亚基(一些已经 基于晶体结构的特征和将被制造的新的一个)来解决 关于Galpha如何附着在膜上并定位于特定区域的问题 膜结构域。Galpha亚基与膜的相互作用 在不同的Galpha家族中有所不同。使用体外试验和瞬变试验 Galpha-o，Galpha-S N-端和C-端的转染和突变 Galpha-Q将用于确定膜结合的重要区域 除了已知的脂类作用外，还有与 贝塔/伽马。利用这些技术，Galpha-o的11-14个氨基酸已经被 被发现有助于通过一种未知的膜结合膜 蛋白质(S)。GALPHA靶向特定的膜结构域 在MDCK细胞(极化上皮)中进行研究。追随Galpha的脚步 细胞，稳定表达Galpha-o的细胞系(不正常表达)和 标记了Galpha亚基的表位正在被建立，并被表征为 免疫荧光和共聚焦显微镜。Galpha-o本地化到 侧膜并与内源性Galpha-I2和ZO-1重叠(紧密 连接(TJ)蛋白)。突变的Galpha-o亚基和Galpha-o嵌合体 和Galpha-S(根尖定位和基底外侧定位)将用于 确定对特定膜靶向重要的区域。《小路》(S) 将为Galpha-0和内源性Galpha I2建立靶向， 脉冲追逐标记选择性膜法测定Galpha-S、Galpha-Q 生物素化作用。活化的Galpha-o(Q205L)的表达也定位于 基底膜和导致加速形成的TJS使用 TJ生物发生的钙交换模型。Galpha亚基在TJ中的作用 生物发生将进一步表征稳定的细胞系表达 野生型和激活的Galpha亚基。准确的信令对于 所有细胞和信号通路在缺血组织后往往被破坏 受伤。TJ的形成对上皮组织的发育和 在缺血恢复期间(急性肾小管坏死)。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， 并可允许制定防止或纠正异常情况的策略 在病变或损伤的组织中发出信号。

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.