Role of intracellular sphingolipids in calcium signaling

细胞内鞘脂在钙信号传导中的作用

• 批准号: 6740241
• 负责人: Alessandro Fatatis
• 金额: $ 25.27万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6740241
• 关键词: biological signal transduction; calcium flux; cell line; cytoplasmic receptors; digital imaging; electrophysiology; fluorimetry; inositol phosphates; intermolecular interaction; lipid bilayer membrane; lipid metabolism; membrane proteins; nuclear receptors; phosphatidylinositols; receptor expression; recombinant proteins; sphingolipids; sphingosine; transfection

DESCRIPTION (provided by applicant): AIterations in calcium (Ca) signaling are implicated in human pathologies ranging from neoplastic to neurodegenerative diseases. The spatio-temporal characteristics of Ca signals can regulate the activity of transcription factors and directly affect gene expression. Ca is generally mobilized from intracellular stores by inositol 1,4,5-trisphosphate (InsP3), which activates specific receptors containing an intrinsic Ca channel (InsP3Rs) and localized to the endoplasmic reticulum. Notably, many cell-surface receptors that increase InsP3 concurrently increase the intracellular production of sphingosine and sphingosine-1 phosphate (sphingosine-lP) as well. A Ca-mobilizing role for these two sphingolipids has been proposed, but their mechanism of action is unclear and the intracellular Ca channel(s) they activate yet to be identified. Furthermore, their functional interactions with InsP3 have not been characterized. The current proposal aims to test the hypothesis that both intracellular sphingosine and sphingosine-lP mobilize Ca through InsP3Rs. The specific aims of this proposal are: 1) To characterize the Ca-mobilizing action of intracellular sphingosine and sphingosine-lP - acting separately or in combination with InsP3 - using caged-precursors that will be photo-activated inside living cells and their effects analyzed by single-cell microfluorimetry and digital imaging. The reciprocal interactions between InsP3 and the two sphingolipids during the Ca signaling following the stimulation of several plasma membrane receptors will also be investigated; 2) To establish whether the expression of different InsP3R-subtypes can determine the type of Ca signals evoked by intracellular sphingosine and sphingosine-lP. Experiments using cells genetically engineered to express specific InsP3R-subtypes will be combined with studies performed with mammalian cell lines transfected with distinct InsP3R-subtypes; 3) To ascertain whether sphingosine and sphingosine-lP directly open the InsP3R-channel and/or functionally modulate the action of InsP3. To this end, single-channel recording from native and recombinant InsP3R subtypes reconstituted in planar lipid bilayers will be employed. The long-term objective of this proposal is to identify the mechanisms and intracellular channels through which sphingosine and sphingosine-lP mobilize intracellular Ca - acting alone or in combination with InsP3 - and characterize their role in cellular signaling following the stimulation of plasma membrane receptors.

描述（由申请人提供）： 钙 (Ca) 信号传导的改变与从肿瘤到神经退行性疾病等人类病理学有关。 Ca信号的时空特征可以调控转录因子的活性，直接影响基因表达。 Ca 通常通过肌醇 1,4,5-三磷酸 (InsP3) 从细胞内储存中调动，它激活包含内在 Ca 通道 (InsP3Rs) 并定位于内质网的特定受体。值得注意的是，许多增加 InsP3 的细胞表面受体同时也会增加细胞内鞘氨醇和 1 磷酸鞘氨醇 (sphingosine-IP) 的产生。已经提出了这两种鞘脂的 Ca 动员作用，但它们的作用机制尚不清楚，并且它们激活的细胞内 Ca 通道尚未确定。此外，它们与 InsP3 的功能相互作用尚未得到表征。目前的提案旨在检验细胞内鞘氨醇和鞘氨醇-LP 均通过 InsP3R 动员 Ca 的假设。该提案的具体目标是： 1) 表征细胞内鞘氨醇和鞘氨醇-LP 的 Ca 动员作用 - 单独作用或与 InsP3 组合 - 使用将在活细胞内光激活的笼状前体并分析其效果通过单细胞微荧光测定法和数字成像。还将研究刺激几种质膜受体后的 Ca 信号传导过程中 InsP3 和两种鞘脂之间的相互作用； 2) 确定不同InsP3R亚型的表达是否可以决定细胞内鞘氨醇和鞘氨醇-LP诱发的Ca信号类型。使用基因工程表达特定 InsP3R 亚型的细胞进行的实验将与用不同 InsP3R 亚型转染的哺乳动物细胞系进行的研究相结合； 3) 确定鞘氨醇和鞘氨醇-LP是否直接打开InsP3R通道和/或功能性调节InsP3的作用。为此，将采用在平面脂质双层中重建的天然和重组 InsP3R 亚型的单通道记录。该提案的长期目标是确定鞘氨醇和鞘氨醇-LP 动员细胞内 Ca 的机制和细胞内通道（单独作用或与 InsP3 联合作用），并表征它们在刺激质膜受体后的细胞信号传导中的作用。