Licensing of IP3 receptors to evoke cytosolic calcium signals

细胞有不同的功能。这种分工要求细胞之间相互交流并感知周围环境。这些对话发生在围绕着每个细胞的质膜（PM）上。PM中的受体是细胞的天线：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。这些信号通路中最广泛的需要IP3受体（IP3Rs），这是一种从细胞内储存（ER）释放钙（Ca）的通道。IP3导致这些通道打开，允许钙从内质网迅速泄漏，有两个重要的后果：钙被递送到它所调节的细胞内目标，钙从内质网的损失刺激PM通道的打开，更多的钙可以通过通道流入细胞。关键是ip3r是细胞外信号触发Ca信号从而产生细胞反应的中枢。ip3r激发的Ca信号的组成部分是“Ca泡”，这是一种短暂的Ca信号（持续约十分之一秒），当少数ip3r聚集在一起打开时发生。一个典型的细胞表达大约10,000个IP3Rs，其中大多数在内质网内快速移动，但是Ca泡只在几个固定的位点重复发生（涉及不超过几百个IP3Rs）。通过标记内源性IP3Rs，使我们能够在显微镜下看到它们，同时观察Ca信号，我们已经证明，唯一引起Ca泡的IP3Rs是固定的，停在PM下面。这些观察结果确立了迄今为止尚未认识到的对IP3R监管的额外水平的需求：即什么“许可”IP3R回应？我们最近的研究表明，在表达人类癌症中常见突变的一种蛋白质（KRas）的细胞中，一种蛋白质被上调，从而激活了IP3Rs。这种蛋白质，KRAP，将IP3Rs系在PM下方的细胞骨架（肌动蛋白）上，并沿着Ca进入的位点。在没有KRAP的细胞中，所有IP3Rs都是可移动的，IP3不能唤起Ca信号。具有额外KRAP的细胞具有更多的固定ip3r， IP3引起更多的Ca泡和更大的Ca信号。因此，KRAP决定了细胞是否可以通过Ca信号对细胞外刺激做出反应。这一发现很有趣，因为它将IP3Rs的亚细胞地理与其活性联系起来，并表明许可可能由Ras蛋白（在许多癌症中发生突变）和肌动蛋白骨架调节。后者很有趣，因为肌动蛋白骨架是动态的，它的活性是由PIP2控制的，PIP2也是制造IP3的分子。因此，PIP2在调节IP3R活性中可能有两个作用：通过组装和锚定肌动蛋白骨架使它们作出反应，并作为IP3的来源，导致被许可的IP3R打开。KRAP对IP3Rs的许可也表明，钙从内质网的损失可能在局部调节钙进入PM的机制。我的提案将使用配备的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IP3Rs做出反应。我们使用基因编辑方法来标记或操纵关键蛋白，使它们在调节Ca信号的天然表达水平上被看到。我们将讨论三个问题：1。KRAP和IP3Rs相互作用的复合物的组成和结构是什么？KRAP对ip3r做了什么来允许他们（“许可”他们）做出回应？PIP2在IP3R调控中是否具有双重作用：通过肌动蛋白调控获得许可，并作为IP3的来源？我们解决细胞生物学中的基本问题，即理解无处不在的细胞内信号通路被调节以传递空间组织信号的机制。我们的工作也有潜力，因为我们开发了破坏KRAP-IP3R相互作用的肽，提供了第一个有用的ip3r拮抗剂，这将在实验上有用，并可能为新疗法提供途径。

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.