Licensing of IP3 receptors to evoke cytosolic calcium signals

细胞专门用于不同的角色。这种劳动分工要求细胞相互交谈并感知周围环境。这些对话发生在一个屏障上，即围绕着每个细胞的质膜（PM）。PM中的受体是细胞的触角：每个受体检测特定的细胞外信号，改变形状，然后调节细胞内信号。这些信号传导途径中最广泛的需要IP 3受体（IP 3R），其是从细胞内储存（ER）释放钙（Ca）的通道。IP 3导致这些通道打开，允许Ca从ER快速泄漏，具有两个重要的后果：Ca被递送到其调节的细胞内靶点，并且Ca从ER的损失刺激PM中的通道打开，更多的Ca可以通过该通道流入细胞。关键是IP 3R是细胞外信号引发Ca信号从而引起细胞反应的枢纽。IP 3R引发的Ca信号的构建块是“Ca puffs”，这是一种短暂的Ca信号（持续约十分之一秒），当少数聚集的IP 3R一起打开时发生。一个典型的细胞表达大约10，000个IP 3R，其中大部分在ER内快速移动，但Ca的喷发只在少数固定位点重复发生（涉及不超过几百个IP 3R）。通过用标签标记内源性IP 3R，使我们能够用显微镜看到它们，同时观察Ca信号，我们已经表明，引起Ca喷流的唯一IP 3R是不动的，并停在PM下面。这些观察建立了一个迄今未被认识到的需要额外的IP 3R监管水平：即什么“许可证”IP 3R响应？我们最近的工作表明，在表达蛋白质（KRas）的细胞中上调的蛋白质通常在人类癌症中突变许可IP 3R。这种蛋白质KRAP将IP 3Rs束缚在PM下方的细胞骨架（肌动蛋白）上，并与Ca进入发生的位点一起。在没有KRAP的细胞中，所有的IP 3 R都是移动的，并且IP 3不能引起Ca信号。具有额外KRAP的细胞具有更多不动的IP 3R，并且IP 3引起更多的Ca喷流和更大的Ca信号。因此，KRAP决定细胞是否可以用Ca信号对细胞外刺激做出反应。这一发现很有趣，因为它将IP 3R的亚细胞地理与它们的活性联系起来，它表明许可可能受到Ras蛋白（在许多癌症中突变）和肌动蛋白骨架的调节。后者是有趣的，因为肌动蛋白骨架是动态的，其活性由PIP 2控制，PIP 2也是制造IP 3的分子。因此，PIP 2在调节IP 3R活性方面可能有两个作用：通过组装和锚定肌动蛋白骨架来授权它们做出反应，以及作为导致授权IP 3R打开的IP 3的来源。KRAP对IP 3R的许可也表明了从ER中丢失Ca可能局部调节Ca进入PM的机制。我的建议将使用显微镜来观察活细胞中的单个蛋白质和微小的Ca信号，以确定KRAP如何许可IP 3R做出反应。我们使用基因编辑方法来标记或操纵关键蛋白质，使它们在调节Ca信号时能够在天然表达水平上被观察到。我们将解决三个问题：1。KRAP和IP 3R相互作用的复合体的组成和结构是什么？2. KRAP对IP 3R做了什么来允许他们（“许可”他们）做出响应？3. PIP 2在IP 3R调节中是否具有双重作用：通过肌动蛋白调节许可和作为IP 3的来源？我们解决细胞生物学中的基本问题-即，了解无处不在的细胞内信号传导途径的调节机制，以提供空间组织的信号。我们的工作也有潜力，因为我们开发了破坏KRAP-IP 3R相互作用的肽，以提供第一个有用的IP 3R拮抗剂，这将是实验上有用的，并可能提供新的治疗方法。

Cells are specialized for different roles. This division of labour requires that cells talk to each other and sense their surroundings. These conversations occur across a barrier, the plasma membrane (PM), that surrounds every cell. Receptors in the PM are the cell's antennae: each detects a specific extracellular signal, changes shape, and then regulates intracellular signalling. The most widespread of these signalling pathways requires IP3 receptors (IP3Rs), which are channels that release calcium (Ca) from intracellular stores (ER). IP3 causes these channels to open, allowing Ca to leak rapidly from the ER, with two important consequences: Ca is delivered to the intracellular targets that it regulates and the loss of Ca from the ER stimulates opening of channels in the PM through which more Ca can flow into the cell. The key point is that IP3Rs are hubs through which extracellular signals evoke Ca signals and thereby cellular responses.The building blocks of Ca signals evoked by IP3Rs are 'Ca puffs', which are brief Ca signals (lasting about a tenth of a second) that occur when a handful of clustered IP3Rs open together. A typical cell expresses about 10,000 IP3Rs, most of which move rapidly within the ER, but Ca puffs occur repeatedly at just a few fixed sites (involving no more than few hundred IP3Rs). By labelling endogenous IP3Rs with a tag that allows us to see them with a microscope while simultaneously observing Ca signals, we have shown that the only IP3Rs that evoke Ca puffs are immobile and parked beneath the PM. These observations established a hitherto unrecognised need for an additional level of IP3R regulation: namely what 'licenses' IP3Rs to respond?Our recent work showed that a protein that is up-regulated in cells expressing a protein (KRas) commonly mutated in human cancer licenses IP3Rs. This protein, KRAP, tethers IP3Rs to the cellular skeleton (actin) beneath the PM and alongside the sites where Ca entry occurs. In cells without KRAP, all IP3Rs are mobile and IP3 fails to evoke Ca signals. Cells with extra KRAP have more immobile IP3Rs and IP3 evokes more Ca puffs and larger Ca signals. Hence, KRAP determines whether a cell can respond to extracellular stimuli with a Ca signal. This discovery is interesting because it links the subcellular geography of IP3Rs to their activity, and it suggests that licensing may be regulated by Ras proteins (mutated in many cancers) and by the actin-skeleton. The latter is intriguing because the actin-skeleton is dynamic and its activity is controlled by PIP2, which is also the molecule from which IP3 is made. Hence, PIP2 may have two roles in regulating IP3R activity: licensing them to respond by assembling and anchoring the actin-skeleton, and as the source of the IP3 that causes licensed IP3Rs to open. Licensing of IP3Rs by KRAP also suggests mechanisms by which loss of Ca from the ER may locally regulate Ca entry across the PM.My proposal will use microscopes equipped to look at single proteins and tiny Ca signals in living cells to establish how KRAP licenses IP3Rs to respond. We use gene-editing methods to tag or manipulate key proteins to allow them to be seen at native expression levels as they regulate Ca signals. We will address three questions:1. What is the composition and architecture of the complex where KRAP and IP3Rs interact?2. What does KRAP do to IP3Rs to allow them ('license' them) to respond?3. Does PIP2 have a dual role in IP3R regulation: licensing via regulation of actin and as a source of IP3?We address fundamental issues in cell biology - namely, understanding the mechanisms by which a ubiquitous intracellular signalling pathway is regulated to deliver spatially organised signals. Our work also has potential, as we develop peptides to disrupt KRAP-IP3R interactions, to provide the first useful antagonists of IP3Rs that will be experimentally useful and may provide routes to new therapies.