Role of intracellular sphingolipids in calcium signaling

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的人类病理学有关。Ca信号的时空特性可以调节转录因子的活性，直接影响基因的表达。Ca通常通过肌醇1，4，5-三磷酸（InsP 3）从细胞内储存动员，InsP 3激活含有内源性Ca通道（InsP 3Rs）的特异性受体并定位于内质网。值得注意的是，许多增加InsP 3的细胞表面受体同时也增加鞘氨醇和鞘氨醇-1磷酸（鞘氨醇-IP）的细胞内产生。已经提出了这两种鞘脂的钙动员作用，但它们的作用机制尚不清楚，它们激活的细胞内钙通道尚未确定。此外，它们与InsP 3的功能相互作用尚未被表征。目前的提议旨在测试细胞内鞘氨醇和鞘氨醇-IP两者通过InsP 3R动员Ca的假设。该提议的具体目的是：1）表征细胞内鞘氨醇和鞘氨醇-IP的Ca动员作用-单独或与InsP 3组合作用-使用将在活细胞内被光活化的笼状前体，并通过单细胞显微荧光测定法和数字成像分析它们的作用。还将研究在几种质膜受体刺激后的Ca信号传导期间InsP 3和两种鞘脂之间的相互作用; 2）确定不同InsP 3R亚型的表达是否可以确定由细胞内鞘氨醇和鞘氨醇-IP诱发的Ca信号的类型。使用基因工程化以表达特定InsP 3R亚型的细胞的实验将与用不同InsP 3R亚型转染的哺乳动物细胞系进行的研究组合; 3）确定鞘氨醇和鞘氨醇-IP是否直接打开InsP 3R通道和/或功能性调节InsP 3的作用。为此，将采用来自在平面脂质双层中重构的天然和重组InsP 3R亚型的单通道记录。该提议的长期目标是鉴定鞘氨醇和鞘氨醇-IP动员细胞内Ca（单独或与InsP 3组合作用）的机制和细胞内通道，并表征它们在质膜受体刺激后的细胞信号传导中的作用。

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.