Licensing of IP3 receptors to evoke cytosolic calcium signals

IP3 受体许可激发细胞质钙信号

• 批准号: BB/T012986/1
• 负责人: Colin Taylor
• 金额: $ 78.08万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FT012986%2F1
• 关键词: Licensing; IP3; receptors; evoke; cytosolic

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.

细胞专用于不同的角色。这种分工要求细胞之间相互交谈，并感知周围环境。这些对话发生在每个细胞周围的一道屏障--质膜(PM)。PM中的受体是细胞的触角：每个触角探测特定的细胞外信号，改变形状，然后调节细胞内的信号。这些信号通路中最广泛的需要IP3受体(IP3R)，这是从细胞内储存(ER)释放钙(Ca)的通道。IP3导致这些通道开放，允许钙从内质网迅速泄漏，产生两个重要的后果：钙被输送到它调节的细胞内靶点；内质网中钙的损失刺激PM通道的打开，使更多的钙可以流入细胞。关键的一点是，IP3R是细胞外信号的中枢，细胞外信号通过它来激发钙信号，从而产生细胞反应。由IP3R激发的钙信号的基础是“钙喷”，这是当几个聚集的IP3R一起开放时发生的短暂的钙信号(持续约十分之一秒)。一个典型的细胞表达大约10,000个IP3受体，其中大部分在内质网内快速移动，但钙离子喷发只在几个固定的位置重复发生(涉及不超过数百个IP3受体)。通过用标签标记内源性IP3R，允许我们在观察钙信号的同时用显微镜看到它们，我们已经表明，唯一能唤起钙喷出的IP3R是固定的，停在PM的下面这些观察确立了迄今为止尚未认识到的对IP3R额外水平的监管的必要性：即是什么“许可”IP3R做出反应？我们最近的工作表明，在表达在人类癌症中通常突变的蛋白质(KRAS)的细胞中上调的一种蛋白质允许IP3R。这种名为KRAP的蛋白质将IP3R与PM下和钙进入部位旁边的细胞骨架(肌动蛋白)捆绑在一起。在没有KRAP的细胞中，所有的IP3R都是可移动的，IP3不能引起钙信号。有额外的KAP的细胞有更多的不活动的IP3R，IP3能激发更多的钙喷雾和更大的钙信号。因此，KRAP决定了细胞是否可以通过钙信号对细胞外刺激做出反应。这一发现很有趣，因为它将IP3R的亚细胞地理与其活性联系起来，并表明许可可能受到RAS蛋白(在许多癌症中突变)和肌动蛋白骨架的调节。后者很耐人寻味，因为肌动蛋白骨架是动态的，其活性受PIP2控制，PIP2也是制造IP3的分子。因此，PIP2在调节IP3R活性方面可能有两个作用：允许它们通过组装和锚定肌动蛋白骨架来做出反应，以及作为IP3的来源，导致获得许可的IP3R开放。CRAP对IP3R的许可也提出了一种机制，通过这种机制，内质网中钙的损失可能会局部调节跨PM的钙进入。我的提议将使用配备有显微镜的显微镜来观察活细胞中的单个蛋白质和微小的钙信号，以确定CRAP如何许可IP3R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，以便在它们调节钙信号的同时，能够在自然表达水平上看到它们。我们将解决三个问题：1.KRAP和IP3R相互作用的复合体的组成和架构是什么？2.KRAP对IP3R做了什么以允许它们(‘许可’它们)做出反应？3.PIP2在IP3R调节中是否具有双重作用：通过调节肌动蛋白进行许可和作为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控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相互作用的多肽，以提供第一批有用的IP3R拮抗剂，这将在实验上有用，并可能为新的治疗方法提供途径。

项目成果

KRAP tethers IP

KRAP 系留 IP

• DOI: 10.17863/cam.74679
• 发表时间: 2021
• 作者: Thillaiappan N

IP3R at ER-mitochondrial contact sites: beyond the IP3R-GRP75-VDAC1 Ca2+ funnel

ER-线粒体接触位点的 IP3R：超出 IP3R-GRP75-VDAC1 Ca2 漏斗

• DOI: 10.17863/cam.96930
• 发表时间: 2023
• 作者: Atakba P