LIPID MODIFICATION OF G PROTEIN ALPHA SUBUNITS

拟议研究的目的是确定其性质， 脂类修饰与支抗的相互关系和相关性 和GTP结合调节蛋白(G蛋白)α亚基的功能。 G蛋白是信号转导通路中不可或缺的一部分， 各种各样的荷尔蒙。阿尔法亚单位是与之有关的实体 G蛋白的同一性通常被等同于，并且是一种主要的 决定G蛋白与受体相互作用的决定因素 以酶或通道为靶标。到目前为止各种阿尔法亚基 已鉴定的是N-肉豆蔻酰化和/或棕榈酰化。这个 这些修改的相关性最常用以下方面来描述 锚定到膜上。尽管人们对脂肪酸有很高的兴趣 然而，酰化反应的基本机制和功能相关性问题是 文件记录不佳。 这项提案中要解决的问题具体涉及 生物合成支链亚基背景下的脂类修饰 蛋白质相互作用和亚基稳定性。第一个目标是 项目是定义棕榈酰化所需的结构元素 G蛋白α亚基。要检验的假设是：i)N- α/I-棕榈酰化反应需要肉豆蔻酰化和/或锚定。 例如亚基，以及ii)仍未定义的修饰或多肽 序列，在α/S-1的棕榈酰化中起类似的作用， Alpha/Q-和类Alpha/12亚基。实现这一目标将 强化与膜的相互作用是先决条件的概念 对于棕榈酰化，提供了一个框架来理解为什么至少 存在两个不同的事件来实现棕榈酰化，并提供 识别替代修改和/或 膜接触点。该项目的第二个目标将是 确定激素对G蛋白α亚基的调节 棕榈酰化超越了为阿尔法/S建立的范围，并且可以 因此可作为G蛋白活化的一个指标。要提供的信息 不仅有机械的价值，而且有实用的价值，因为完好无损- G蛋白激活的细胞分析已被证明是难以捉摸的。第三个目标 将对脂肪酸的化学计量和异质性进行量化 用质谱仪进行酰化反应。这将提供定量的洞察力 以前缺乏研究的脂肪酸酰化动力学 包括放射性同位素标记。质谱学将另外 用于探索其他修改。最后，第四个目标将是 研究棕榈酰化与α亚基功能的相关性。 需要检验的假设是：i)棕榈酰化通常是多余的 膜附着的其他机制，但具有如下重要性 不参与，ii)棕榈酰化单独或与N- 肉豆蔻酸化促进了与其他蛋白质的相互作用，例如， 受体、β-伽马和糖基磷脂酰肌醇锚定蛋白， 和iii)棕榈酰化有助于防止亚基在 完整的细胞。

The objective of the proposed research is to determine the nature, interrelationship, and relevance of lipid modifications to the anchorage and function of GTP-binding regulatory protein (G protein) alpha subunits. G proteins are integral to pathways of signal transduction established for a wide variety of hormones. The alpha subunit is the entity with which the identity of a G protein is most often equated, and is a major determinant in stipulating interactions of the G protein with receptors and target enzymes or channels. The various alpha subunits so far identified are subject to N-myristoylation and/or palmitoylation. The relevance of these modification is most often described in terms of anchorage to membranes. Despite the high level of interest in fatty acid acylation, however, basic issues of mechanism and functional relevance are poorly documented. The questions to be addressed in this proposal deal specifically with lipid modifications in the context of biosynthetic anchorage, subunit protein interactions, and subunit stability. The first goal of the project is to define elements of structure required for palmitoylation of G protein alpha subunits. The hypotheses to be tested are i) that N- myristoylation and/or anchorage is required for palmitoylation of alpha/i- like subunits, and ii) that a still undefined modification, or peptide sequence, serves a similar purpose in the palmitoylation of alpha/s-1, alpha/q-, and alpha/12-like subunits. Achievement of this goal will strengthen the concept that interactions with membranes are a prerequisite to palmitoylation, provide the framework for understanding why at least two different events exist to achieve palmitoylation, and provide the information necessary for identifying alternate modifications and/or points of membrane contact. The second goal of the project will be to determine whether hormonal regulation of G protein alpha subunit palmitoylation extends beyond that established for alpha/s, and can therefore serve as an index of G protein activation. The information to be obtained has not only mechanistic, but pragmatic value, since intact- cell assays of G protein activation have proven elusive. The third goal will be to quantitate the stoichiometry and heterogeneity of fatty acid acylation by mass spectrometry. This will provide quantitative insight into the dynamics of fatty acid acylation previously lacking in studies involving radioisotopic labeling. Mass spectrometry will additionally be used to explore other modifications. Finally, the fourth goal will be to investigate the relevance of palmitoylation to alpha subunit function. Hypotheses to be tested are i) that palmitoylation is normally redundant to other mechanisms of membrane attachment but assumes importance as these are disengaged, ii) that palmitoylation alone or together with N- myristoylation facilitates interactions with other proteins, e.g., receptors, beta gamma, and glycosylphosphatidylinositol-anchored proteins, and iii) that palmitoylation helps prevent degradation of the subunit in the intact cell.