Phosphoinositide-calcium Signaling In Cell Regulation

细胞调节中的磷酸肌醇-钙信号转导

• 批准号: 6991153
• 负责人: TAMAS BALLA
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6991153
• 关键词: B lymphocyte; alcohol phosphotransferase; calcium flux; cell growth regulation; cell surface receptors; chickens; cytoplasmic receptors; endoplasmic reticulum; enzyme mechanism; enzyme structure; green fluorescent proteins; inositol phosphates; intermolecular interaction; lipid biosynthesis; phosphatidylinositols; phospholipase C; protein binding; protein localization; voltage gated channel

The Unit of Molecular Signal Transduction investigates signal transduction pathways that mediate the actions of hormones and growth factors in mammalian cells, with special emphasis on the role of phosphoinositide-derived messengers. Phosphoinositides are a small fraction of the cellular phospholipids, but play a critical role in the regulation of many (if not all) signaling protein complexes that assemble on the surface of cellular membranes. Phosphoinositides regulate protein kinases and GTP-binding proteins, as well as membrane transporters including ion channels, thereby controlling many cellular processes such as proliferation, apoptosis and metabolism. Our group focuses on one family of enzymes, the phosphatidylinositol 4-kinases (PI4Ks) that catalyze the first committed step in phosphoinositide synthesis. Current studies are aimed at (1) understanding the function and regulation of several phosphatidylinositol (PI) 4-kinases in the control of the synthesis of hormone-sensitive phosphoinositide pools; (2) characterizing the structural features that determine the catalytic specificity and inhibitor sensitivity of PI 3- and PI 4-kinases; (3) defining the molecular basis of protein-phosphoinositide interactions via the pleckstrin homology and other domains of selected regulatory proteins; (4) developing tools to analyze inositol lipid dynamics in live cells; (5) determining the importance of the lipid-protein interactions in the activation of cellular responses by G protein-coupled receptors and receptor tyrosine kinases. One of the most important regulators of phosphoinositide levels are the phospholipase C (PLC) enzymes that hydrolyze the lipid, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Of the various isoforms of PLCs, the delta forms are the most evolutionarily conserved. These enzymes are believed to be recruited to membranes and regulated by their lipid substrate, PI(4,5)P2, via interaction with their pleckstrin homology (PH) domain, a protein module that is characterized by high-affinity binding to phosphoinositides. Recent studies have identified a new isoform of PLCdelta, the delta4 enzyme, and indicated that its PH domain selectively binds the lipid PI(4,5)P2, but not the soluble inositol 1,4,5-trisphosphate (IP3). To understand the role of the PLCdelta4 PH domain in the localization and function of PLCdelta4 enzyme, the inositol-lipid and inositol phosphate binding properties of the full-length enzyme, and its isolated PH domain, were analyzed in comparison with the similar features of the PLCdelta1 protein. Our studies showed that the PH domain of PLCdelta4 indeed has a lower affinity to both the lipid, PI(4,5)P2 and the soluble IP3, but still could recognize PI(4,5)P2 in the plasma membrane when expressed as a GFP fusion protein in live cells. In contrast, the full-length PLCdelta4 was found not to be associated with the plasma membrane but primarily with the endoplasmic reticulum, and removal of the PH domain from the enzyme failed to affect its cellular localization. These data suggest that the PH domain of PLCdelta4 is not responsible for the localization of the enzyme, probably because it is not fully exposed in the molecule. Our data suggest that in spite of their similar architecture, the PLCdelta1 and -delta4 enzymes are differentially regulated by their PH domains. As a result of PLC activation, IP3 is generated from membrane PI(4,5)P2 upon stimulation of specific forms of cell-surface receptors. IP3 rapidly binds to its intracellular receptors and releases Ca2+ from non-mitochondrial Ca2+ stores to produce the cytoplasmic Ca2+ increase that triggers specific cellular responses of the target cell. IP3Rs are functional Ca2+ channels that work as homo- or heterotetramers. Each receptor subunit has a channel portion containing six transmembrane helices and a pore-domain located between TM5 and TM6, close to the C-terminus of the protein. The ligand-binding domain (LBD) of the receptor is located near the N-terminus of the molecule, and is separated from the channel domain by a long intervening regulatory region facing the cytoplasm. In spite of intense investigations, little is known about the manner in which the binding of IP3 to the N-terminal LBD affects the channel gating properties of the molecule. To address this issue, we investigated whether the LBD of the InsP3R acts as a tethered adaptor module that regulates the channel activity via InsP3-induced conformational changes. For this, we used a novel molecular approach in which the isolated LBD of the type-I InsP3 receptor or its components were tethered to the cytoplasmic surface of the endoplasmic reticulum (ER), where IP3Rs reside, and the effects of their expression on Ca2+ signaling were compared to those of the same constructs expressed in the cytoplasm. When the IP3R-LBD was expressed with a C-terminal short hydrophobic sequence that targets it to the cytoplasmic surface of the ER, it strongly inhibited agonist-induced Ca2+ signaling. Interestingly, the inhibitory effect of the ER-tethered IP3R-LBD form was not due to IP3 binding but rather was the result of the emptying of the IP3-sensitive Ca2+ stores. Another ER-tethered structurally unrelated InsP3 binding module, the PH domain of the PLC-like p130 protein, did not show such an effect. These data suggested that the IP3R-LBD has structural elements that are capable of releasing Ca2+ from the ER. To determine whether the release of Ca2+ occurs via the IP3Rs, we used DT40 cells that lack all three isoforms of the receptor. Expression of the ER-tethered IP3R-LBD had no effect on the intracellular Ca2+ stores in the triple-knockout cells, while it depleted the stores in the wild-type DT40 cells, indicating that the LBD interacts with the endogenous IP3Rs and leads to their opening. In further experiments we determined that the all-helical domain of the LBD was sufficient to open the channel even though it alone fails to bind IP3, and that the addition of an inhibitory N-terminal segment to the LBD (residues 1-224) greatly decreased its efficacy to release Ca2+. Based on these data, we propose that the LBD of IP3Rs is positioned in close proximity to the channel domain, and that exposure of the all-helical domain from a cryptic state is part of the mechamism by which IP3 binding leads to channel activation. These data provide an important clue to the regulation of IP3R channel function, and open new directions to clarify the molecular details of this process.

分子信号转导的单位研究了介导哺乳动物细胞中激素和生长因子的作用的信号转导途径，并特别强调了磷酸辛辅导层衍生的使者的作用。磷酸肌醇是细胞磷脂的一小部分，但在调节许多（如果不是全部）信号蛋白复合物的调节中起着至关重要的作用，这些信号蛋白复合物在细胞膜表面组装。磷酸肌醇调节蛋白激酶和GTP结合蛋白，以及包括离子通道在内的膜转运蛋白，从而控制了许多细胞过程，例如增殖，凋亡和代谢。我们的小组专注于一种酶家族，即磷脂酰肌醇4-蛋白酶（PI4K），该酶催化了磷酸肌醇合成的第一步。当前的研究针对（1）了解几种磷脂酰肌醇（PI）4-激酶在控制激素敏感磷酸固醇池的功能和调节中； （2）表征确定PI 3-和PI 4-激酶的催化特异性和抑制剂敏感性的结构特征； （3）通过Pleckstrin同源性和选定的调节蛋白的其他结构域定义蛋白磷酸肌醇相互作用的分子基础； （4）开发工具来分析活细胞中的肌醇脂质动力学； （5）确定G蛋白偶联受体和受体酪氨酸激酶在细胞反应激活中脂质蛋白相互作用的重要性。 磷酸肌醇水平最重要的调节剂之一是磷脂酶C（PLC）酶，可水解脂质，磷脂酰肌醇4,5-双磷酸[PI（4,5）P2]。在PLC的各种同工型中，三角洲形式是最保守的。这些酶被认为是通过与其Pleckstrin同源性（PH）结构域相互作用的脂质底物PI（4,5）P2调节的，并由其脂质底物调节，这是一种蛋白质模块，该蛋白质模块的特征在于高亲和力与磷酸肌醇的高亲和力结合。最近的研究已经确定了Delta4酶的一种新的同工型，并表明其pH结构域有选择地结合脂质Pi（4,5）P2，但没有可溶性肌醇1,4,5-三磷酸（IP3）。为了了解PLCDELTA4 pH结构域在PLCDELTA4酶的定位和功能中的作用，与PLCDELTA1蛋白的相似特征相比，分析了全长酶的全长酶以及其分离的pH结构域的全长酶及其分离pH结构域的作用。我们的研究表明，PLCDELTA4的pH结构域确实与脂质，Pi（4,5）P2和可溶性IP3具有较低的亲和力，但是当活细胞中的GFP融合蛋白表示为质膜中的PI（4,5）P2。相比之下，发现全长PLCDELTA4与质膜无关，而主要与内质网络相关，并且从酶中除去pH结构域不影响其细胞定位。这些数据表明，PLCDELTA4的pH结构域对酶的定位概不负责，这可能是因为它在分子中没有完全暴露。我们的数据表明，尽管它们具有相似的结构，但Plcdelta1和-delta4酶还是由其pH结构域差异调节。 由于PLC激活的结果，在刺激特定形式的细胞表面受体时，由膜PI（4,5）P2产生IP3。 IP3迅速与其细胞内受体结合，并从非单位软骨Ca2+储存中释放Ca2+，以产生细胞质Ca2+增加，从而触发靶细胞的特定细胞反应。 IP3RS是功能性CA2+通道，可作为同型或异光学器。每个受体亚基都有一个通道部分，其中包含六个跨膜螺旋和一个位于TM5和TM6之间的孔域，靠近蛋白质的C末端。受体的配体结合结构域（LBD）位于分子的N末端附近，并通过面向细胞质的长长的中间调节区与通道结构域分离。尽管进行了激烈的研究，但对IP3与N末端LBD的结合影响分子的通道门控特性的方式知之甚少。为了解决这个问题，我们研究了INSP3R的LBD是否充当束缚的适配器模块，该模块通过INSP3诱导的构象变化来调节通道活动。为此，我们使用了一种新型的分子方法，在这种方法中，I type-I INSP3受体的分离LBD或其成分被束缚在内质网（ER）的细胞质表面上，IP3RS驻留，其中将其表达对Ca2+信号的影响与Ca2+信号的效果进行比较。当IP3R-LBD用C末端短疏水序列表达，该序列将其靶向ER的细胞质表面时，它极大地抑制了激动剂诱导的Ca2+信号传导。有趣的是，ER连接的IP3R-LBD形式的抑制作用不是由于IP3的结合，而是IP3敏感Ca2+储存的结果。另一个ER连接的结构无关的INSP3结合模块，即PLC样P130蛋白的pH结构域，并未显示出这种作用。这些数据表明，IP3R-LBD具有能够从ER释放Ca2+的结构元素。为了确定Ca2+是否通过IP3RS释放，我们使用了缺少受体所有三种同工型的DT40细胞。 ER系列IP3R-LBD的表达对三重敲除细胞中细胞内Ca2+储存没有影响，而它耗尽了野生型DT40细胞中的存储库，表明LBD与内源性IP3RS和铅与开放的内源性IP3RS相互作用。在进一步的实验中，我们确定LBD的全螺旋结构域足以打开该通道，即使单独的通道无法结合IP3，并且将抑制性的N末端段添加到LBD（残基1-224）大大降低了其释放Ca2+的效果。基于这些数据，我们建议将IP3R的LBD与通道域紧密相近，并且从隐秘状态下全螺旋域的暴露是IP3结合导致通道激活的机制的一部分。这些数据为IP3R通道函数的调节提供了重要的线索，并开放了新的方向，以阐明此过程的分子细节。