PALMITOYLATION AND TARGETING OF G PROTEINS

细胞内信号通路依赖于适当的和独特的 其组成蛋白质的亚细胞位置。机制 负责可逆靶向外周膜蛋白， 对不同的细胞膜知之甚少。 这项研究经费 将在异源三聚体的背景下解决这个问题。 （字母γ）G蛋白。 G蛋白作为分子开关， 将信息从激活的受体传递到适当的效应器 蛋白质（例如腺苷酸环化酶、磷脂酶C、cGMP 磷酸二酯酶和离子通道）。 分子机制 G蛋白的GT3循环及其受体调节， 效应器正变得越来越好理解。 更不好 我们理解的是负责将G蛋白靶向其 合适的蜂窝位置，以及独特的蜂窝环境- 和与G蛋白相互作用的蛋白质， 影响G蛋白的功能。 本项目的主要目标是 建议是阐明细胞棕榈酰化的机制， 共价连接到16碳脂肪酸的半胱氨酸，G蛋白 α亚基（Ga α）及其特异性靶向机制 细胞内或质膜。 G蛋白β γ的作用 将通过采用以下方法测试Galpha棕榈酰化中的亚基： 破坏体内α β γ相互作用的策略。的 将通过以下方法解决Ga棕榈酰化的亚细胞位点 检查差异定位的G α; α 12的棕榈酰化 存在于质膜上，而α 13和α 14的剪接变体存在于质膜上， alphai 2（salphai 2）在高尔基体膜上发现。 膜抑制剂 囊泡运输将被用来进一步确定细胞途径 用于棕榈酰化。 了解特定高尔基体的机制 Galalpha的定位，脂质修饰的作用， 肉豆蔻酰化和棕榈酰化，以及β-γ相互作用将是 通过突变体G α的表达进行测试。

Intracellular signaling pathways depend upon appropriate and unique subcellular locations of their constituent proteins. Mechanisms responsible for reversibly targeting peripheral membrane proteins to different cellular membranes are poorly understood. This research grant will address this question in the context of heterotrimeric (alphabetagamma) G proteins. G proteins act as molecular switches to relay information from activated receptors to appropriate effector proteins (e.g. adenylyl cyclase, phospholipase C, cGMP phosphodiesterase, and ion channels). Molecular mechanisms underlying the GTPase cycle of G proteins and its regulation by receptors and effectors are becoming increasingly well understood. Much less well understood are mechanisms responsible for targeting G proteins to their appropriate cellular location, and how a unique cellular environment - and proteins that interact with G proteins to target or retain them there - affect a G protein's function. The major objectives of this proposal are to elucidate mechanisms of cellular palmitoylation, covalent attachment to cysteines of a 16 carbon fatty acid, of G protein alpha subunits (Galpha) and mechanisms of specific targeting of Galpha to intracellular or plasma membranes. The role of G protein betagamma subunits in the palmitoylation of Galpha will be tested by employing strategies to disrupt the alphaybetagamma interaction in vivo. The subcellular site of palmitoylation of Galpha will be addressed by examining the palmitoylation of differentially localized Galpha; alphai2 resides at the plasma membrane, whereas alphai3 and a splice variant of alphai2 (salphai2) are found at Golgi membranes. Inhibitors of membrane vesicle transport will be used to further define the cellular pathway for palmitoylation. To understand mechanisms of specific Golgi localization of Galpha, the role of lipid modifications, both myristoylation and palmitoylation, and betagamma interaction will be tested by expression of mutant Galpha.