A novel ryanodine receptor in the hormonal regulation of hepatic metabolism

描述(申请人提供)：儿茶酚胺和加压素等激素对肝脏代谢和分泌的影响是通过细胞内游离钙浓度([钙]c)的变化来调节的，主要是由于第二信使三磷酸肌醇(IP3)从细胞内储存的钙动员起来。这些激素激发的[Ca~(2+)]_c信号以[Ca~(2+)]_c振荡和波动的形式组织，其频率受激素剂量的控制。频率调制的[Ca~(2+)]c振荡可以起到许多调节功能，包括提高保真度、敏感性和对特定过程的靶向调节。虽然IP3受体在这一途径中的作用已被证实，但最近有证据表明，其他钙释放和钙反馈成分有助于扩大[钙]c振荡的时间和空间范围。实现这一点的一种方式是通过IP3R和另一类主要的细胞内钙释放通道-兰尼定受体(RyRs)之间的相互作用。RyRs通常是通过钙离子诱导的钙释放(CICR)机制被钙激活的，但它们也可以被另一种信使-环腺苷二磷酸(CADPR)激活。尽管RyRs在兴奋性组织(主要是肌肉和脑)中的特性已经很好地描述了，但对于非兴奋性细胞中RyRs的识别和特性却知之甚少。我们最近从大鼠肝细胞中鉴定并克隆了一种独特的RyR亚型(RyR1b)，它源于RyR1基因，但有一个替代的起始点，其蛋白质大小仅为全长肌肉RyR的40%。我们假设RyR1b具有不同的功能特性，这是它在肝细胞和其他不可兴奋的分泌细胞中对[Ca~(2+)]_c信号的贡献，在这些细胞中，RyR可能是主要的RyR亚型。具体地说，我们认为RyR1b在促进和维持由IP3R激活启动的内质网钙离子释放中起关键作用。我们将研究RyR1b在两个特定目标中的潜在作用：在目标1中，我们将进行异源表达研究，以探讨RyR1b的功能和调节特性，包括[Ca~(2+)]c、cADPR及其与IP3和IP3R的相互作用。在目标2中，我们将研究RyR1b在原代肝细胞中的亚细胞分布和调控，并确定在激素刺激下，RyR1b对[Ca~(2+)]c信号的时空模式的影响。RyR1b似乎是细胞内钙释放通道超家族的一个重要新成员，具有独特的性质。它可能具有特定的功能，根据不可兴奋的分泌上皮细胞和内分泌细胞的信号要求进行调节。因此，拟议的工作是创新的，有可能产生很大的影响，具有重大的生物医学健康相关性。 与公共健康相关：拟议的工作将调查一种新发现的钙信号蛋白(RyR1b)的作用和调节，该蛋白似乎在调节激素对肝功能的影响方面发挥作用。它还被认为参与了其他组织的信号传递，包括胰腺和消化道细胞。由于这些组织中信号的错乱经常与疾病状态有关(例如，如肝炎、胆汁淤积症、胰腺炎)，表征RyR1b的性质对公众健康具有重大潜在影响。其独特的分子结构可能会产生一个新的治疗靶点。

DESCRIPTION (provided by applicant): The effects of hormones such as catecholamines and vasopressin on hepatic metabolism and secretion are mediated by alterations in the concentration of cytosolic free Ca2+ ([Ca2+]c), largely as a result of Ca2+ mobilization from intracellular stores by the second messenger inositol 1,4,5-trisphosphate (IP3). The [Ca2+]c signals elicited by these hormones are organized in the form of [Ca2+]c oscillations and waves, whose frequency is controlled by hormone dose. Frequency-modulated [Ca2+]c oscillations may serve a number of regulatory functions, including improved fidelity, sensitivity and targeted regulation of specific processes. While the role of IP3-receptors in this pathway is well established, it has recently become evident that other Ca2+ release and Ca2+-feedback components contribute to extend the temporal and spatial range of [Ca2+]c oscillations. One way in which this might be achieved is through interplay between IP3R and the other major class of intracellular Ca2+ release channels, the ryanodine receptors (RyRs). RyRs are typically activated by Ca2+ through a Ca2+-induced Ca2+ release (CICR) mechanism, but they can also be activated by another second messenger, cyclic-ADPribose (cADPR). Although the RyRs have been well-characterized in excitable tissues (primarily muscle and brain), relatively little is known about the identity and properties of RyRs in nonexcitable cells. We have recently identified and cloned a unique RyR isoform from rat hepatocytes (RyR1b), which is derived from the RyR1 gene, but with an alternative start site that gives rise to a protein of only 40% the size of full length muscle RyRs. We hypothesize that RyR1b has distinct functional properties that underlie its contribution to [Ca2+]c signaling in hepatocytes and other nonexcitable secretory cells, where it may be the principal RyR isoform. Specifically, we propose that RyR1b plays a key role in enhancing and sustaining ER Ca2+ release initiated by IP3R activation. We will examine the potential role of RyR1b in two specific aims: In Aim 1 we will carry out heterologous expression studies to investigate the function and regulatory properties of RyR1b, including regulation by [Ca2+]c, cADPR and its interactions with IP3 and the IP3R. In Aim 2 we will investigate the subcellular distribution and regulation of RyR1b in primary hepatocytes, and determine how it contributes to the temporal and spatial pattern of [Ca2+]c signaling during hormonal stimulation of these cells. RyR1b appears to be a major new addition to the superfamily of intracellular Ca2+ release channels, with distinct properties. It may have specific functions that are tuned to the signaling requirements of nonexcitable secretory epithelia and endocrine cells. Thus, the proposed work is innovative and has the potential for high impact, with significant biomedical health relevance. PUBLIC HEALTH RELEVANCE: The proposed work will investigate the role and regulation of a newly discovered calcium signaling protein (RyR1b) that appears to play a role in mediating the effects of hormones on liver function. It is also postulated to participate in signaling in other tissues, including the pancreas and cells of the digestive tract. Since derangements of signaling in these tissues are frequently associated with disease states (eg. hepatitis, cholestasis, pancreatitis), characterization of the properties of RyR1b has significant potential public health impact. Its unique molecular structure may yield a novel therapeutic target.