Homeostatic Regulation of PIP2-Calcium Signaling

项目摘要/摘要 磷脂酰肌醇4，5-二磷酸(PIP2)-钙信号通路在刺激生长后被激活 因子受体、抗原受体和G蛋白偶联受体通过包括神经递质和 组胺。磷脂酶C的激活触发质膜(PM)上PIP2脂类的水解 生成二酰基甘油(DAG)和三磷酸肌醇(IP3)。IP3随后释放存储在 内质网(ER)激活胞浆钙效应器，用于下游信号事件，如 分泌、增殖和迁移。消耗的PM PIP2和ER钙必须迅速恢复以维持 信号反应和维持细胞内环境平衡。PIP2-钙信号的这种动态平衡调节 需要将磷脂酰肌醇(PI)从ER运输到PM以重新合成PIP2并存储操作 激活钙离子内流以重新填充内质网钙离子储存区的钙离子通道(SOCE)。SOCE已被广泛研究； 然而，PIP2-钙信号转导过程中PIP2补充的机制还不是很清楚。 最近，我们和其他人发现了脂转移蛋白Nir2，它定位于ER-PM连接，其中 急诊室与PM保持联系，以调解PM PIP2的补充。这项建议的目标是界定 受体诱导的钙信号转导中Nir2在内质网-质膜连接处调节PIP2补充的机制。 我们的初步研究发现，Nir1是一种缺少脂转移蛋白结构域的Nir蛋白，是一个结合伙伴 Nir2在内质网-质膜连接处的正向调节。此外，我们最近的数据显示，膜- 在所有扩展突触中存在的发夹序列的整形对于调节PIP2是很重要的 急诊室-PM交界处的补给。此外，我们还发现，DAG转化为磷脂酸(PA)通过 DAG激酶(DGK)对Nir2在内质网-PM连接的定位起着关键作用。在哺乳动物的十种DGK中 细胞中，DGK的异构体DGK(DGKε)最近被发现定位于内质网-质膜连接处。我们的中心假设 在受体诱导的钙信号转导过程中，PM PIP2的补充是由Nir1和Nir1的寡聚体介导的 DGKε在由E-Syts塑造的ER-PM结处从DAG产生PA后形成Nir2。我们建议 使用生化分析和先进的成像技术来验证我们的中心假设的三个具体目标 确定Nir1、E-Syts和DGKε调节PIP2补给的机制。我们期待着 成功完成拟议的研究将建立分子和细胞调控机制 受体刺激引起的PIP2水解后的补充。恢复PM PIP2对维持至关重要 CA2信号转导和维持PM PIP2水平对膜运输、细胞骨架动力学和离子至关重要 在受体刺激的细胞中运输。Nir1基因突变与视网膜营养不良和突变患者有关 在DGK，ε患有肾衰竭。我们提出的研究可能会为开发治疗药物提供知识 治疗受这些疾病影响的患者的策略。

PROJECT SUMMARY / ABSTRACT Phosphatidylinositol 4,5-bisphosphate (PIP2)-Ca2+ signaling is activated following stimulation of growth factor receptors, antigen receptors, and G protein-coupled receptors by ligands including neurotransmitters and histamine. The activation of phospholipase C triggers hydrolysis of the PIP2 lipid at the plasma membrane (PM) to generate diacylglycerol (DAG) and inositol trisphosphate (IP3). IP3 subsequently releases Ca2+ stored in the endoplasmic reticulum (ER) to activate cytosolic Ca2+ effectors for downstream signaling events such as secretion, proliferation, and migration. The consumed PM PIP2 and ER Ca2+ must be quickly restored to sustain signaling responses and to maintain cellular homeostasis. This homeostatic regulation of PIP2-Ca2+ signaling requires transport of phosphatidylinositol (PI) from the ER to the PM for PIP2 resynthesis and store-operated Ca2+ entry (SOCE) that activates Ca2+ influx to refill the ER Ca2+ store. SOCE has been studied extensively; however, the mechanisms underlying PIP2 replenishment during PIP2-Ca2+ signaling are not well understood. Recently, we and others discovered the lipid transfer protein Nir2 that localizes at ER-PM junctions, where the ER is in contact with the PM, to mediate PM PIP2 replenishment. The objective of this proposal is to define the mechanisms regulating PIP2 replenishment by Nir2 at ER-PM junctions during receptor-induced Ca2+ signaling. Our preliminary studies identified Nir1, an Nir protein lacking a lipid transfer protein domain, as a binding partner and positive regulator of Nir2 at ER-PM junctions. In addition, our recent data revealed that the membrane- shaping hairpin sequence present in all extended synaptotagmins (E-Syts) is important for regulating PIP2 replenishment at ER-PM junctions. Moreover, we found that conversion of DAG into phosphatidic acid (PA) by DAG kinases (DGKs) is crucial for Nir2 localization at ER-PM junctions. Among the ten DGKs in mammalian cells, the epsilon-isoform of DGK (DGKε) is recently shown to localize at ER-PM junctions. Our central hypothesis is that PM PIP2 replenishment during receptor-induced Ca2+ signaling is mediated by the oligomers of Nir1 and Nir2 formed following PA production from DAG by DGKε at ER-PM junctions shaped by E-Syts. We propose three specific aims to test our central hypothesis using biochemical analysis and advanced imaging techniques to determine the mechanisms by which Nir1, E-Syts and DGKε regulate PIP2 replenishment. We expect that successfully completion of the proposed studies will establish the molecular and cellular mechanisms regulating PIP2 replenishment following hydrolysis induced by receptor stimulation. Restoring PM PIP2 is critical to sustain Ca2+ signaling and maintain PM PIP2 levels critical to membrane trafficking, cytoskeletal dynamics, and ion transport in receptor-stimulated cells. Mutations in Nir1 are linked to retinal dystrophy and patients with mutations in DGKε suffer from renal failure. Our proposed studies may provide knowledge for developing therapeutic strategies to treat patients affected by these diseases.