Homeostatic Regulation of PIP2-Calcium Signaling

项目概要/摘要 磷脂酰肌醇4，5-二磷酸（PIP 2）-Ca 2+信号转导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PIP 2脂质在质膜（PM）处的水解。 以产生二酰基甘油（DAG）和三磷酸肌醇（IP 3）。IP 3随后释放储存在细胞中的Ca 2+。 内质网（ER）激活胞质Ca 2+效应子，用于下游信号传导事件， 分泌、增殖和迁移。消耗的PM PIP 2和ER Ca 2+必须迅速恢复以维持 信号传导反应和维持细胞内稳态。这种PIP 2-Ca 2+信号的稳态调节 需要将磷脂酰肌醇（PI）从ER转运到PM以用于PIP 2再合成和储存操作 Ca 2+进入（SOCE），其激活Ca 2+内流以再填充ER Ca 2+库。SOCE已被广泛研究; 然而，在PIP 2-Ca 2+信号传导过程中PIP 2补充的潜在机制还没有很好地理解。 最近，我们和其他人发现了位于ER-PM连接处的脂质转运蛋白Nir 2， ER与PM接触，以调节PM PIP 2补充。本提案的目的是界定 在受体诱导的Ca 2+信号传导过程中，Nir 2在ER-PM连接处调节PIP 2补充的机制。 我们的初步研究确定了Nir 1，一种缺乏脂质转移蛋白结构域的Nir蛋白，作为结合伴侣 和ER-PM连接处的Nir 2的正调节剂。此外，我们最近的数据显示，膜- 在所有延伸的突触结合蛋白（E-Syts）中存在的成形发夹序列对于调节PIP 2是重要的 在ER-PM交界处进行补给。此外，我们发现，DAG转化为磷脂酸（PA）， DAG激酶（DGK）对于Nir 2在ER-PM连接处的定位至关重要。在哺乳动物的10种DGK中， 细胞，DGK的ε-同种型（DGKε）最近显示定位于ER-PM连接处。我们的核心假设 受体诱导的Ca 2+信号传导期间PM PIP 2补充是由Nir 1和Nir 2的寡聚体介导的 Nir 2在E-Syts形成的ER-PM连接处由DGKε从DAG产生PA后形成。我们提出 三个具体的目标是使用生化分析和先进的成像技术来测试我们的中心假设 以确定Nir 1、E-Syts和DGKε调节PIP 2补充的机制。我们预计 成功完成拟议的研究将建立分子和细胞机制， 通过受体刺激诱导水解后的PIP 2补充。恢复PM PIP 2对维持 Ca 2+信号传导和维持PM PIP 2水平对膜运输、细胞骨架动力学和离子转运至关重要。 在受体刺激的细胞中转运。Nir 1突变与视网膜营养不良和突变患者有关 在DGKε中患有肾衰竭。我们提出的研究可能为开发治疗性药物提供知识。 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.