LIPID MODIFICATION OF G PROTEIN ALPHA SUBUNITS

拟议研究的目的是确定性质， 脂质修饰与锚定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蛋白是建立的信号转导通路的组成部分， 各种各样的荷尔蒙 α亚基是一种实体， G蛋白的同一性最常被等同，并且是一个主要的 G蛋白与受体相互作用的决定因子 和靶向酶或通道。 到目前为止， 所鉴定化合物进行N-肉豆蔻酰化和/或棕榈酰化。 的 这些修改相关性最常被描述为 锚定膜。 尽管人们对脂肪酸有很高的兴趣， 然而，酰化的机理和功能相关性的基本问题是 记录不良。 本提案中要解决的问题具体涉及 生物合成锚定中的脂质修饰，亚基 蛋白质相互作用和亚基稳定性。 的第一个进球 项目是定义棕榈酰化所需的结构元素， G蛋白α亚单位。 待检验的假设是i）N- 肉豆蔻酰化和/或锚定是α/β-棕榈酰化所必需的。 类似亚基，和ii）仍然未定义的修饰，或肽 序列，在alpha/s-1的棕榈酰化中起到类似的作用， alpha/q-和alpha/12样亚基。 实现这一目标将 加强与膜的相互作用是先决条件的概念 棕榈酰化，为理解为什么至少 存在两种不同的事件来实现棕榈酰化，并提供 识别替代修改所需的信息和/或 膜接触点。 该项目的第二个目标是 确定G蛋白α亚基的激素调节 棕榈酰化超出了α/s的范围， 因此可作为G蛋白活化的指标。 这些信息来 它不仅具有机械性，而且具有实用价值，因为完整- G蛋白活化的细胞测定已被证明是难以实现的。 第三个目标 将定量脂肪酸的化学计量和异质性 通过质谱进行酰化。 这将提供定量的洞察力 脂肪酸酰化的动力学，以前缺乏研究 涉及放射性同位素标记。 质谱分析将另外 用于探索其他修改。 第四个目标是 研究棕榈酰化与α亚基功能的相关性。 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，但假设重要性，因为这些 ii）棕榈酰化单独或与N- 豆蔻酰化促进与其它蛋白质的相互作用，例如， 受体、β-γ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的降解， 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.