A novel ryanodine receptor in the hormonal regulation of hepatic metabolism

描述（由申请人提供）：儿茶酚胺和抗利尿激素等激素对肝脏代谢和分泌的影响是由胞质游离Ca2+ ([Ca2+]c)浓度的改变介导的，主要是由于第二信使肌醇1,4,5-三磷酸（IP3）从细胞内储存的Ca2+动员的结果。这些激素引发的[Ca2+]c信号以[Ca2+]c振荡和波的形式组织，其频率由激素剂量控制。频率调制[Ca2+]c振荡可以服务于许多调节功能，包括提高保真度，灵敏度和特定过程的靶向调节。虽然ip3受体在这一途径中的作用已经确立，但最近发现其他Ca2+释放和Ca2+反馈成分有助于延长[Ca2+]c振荡的时间和空间范围。实现这一目标的一种方法是通过IP3R与细胞内其他主要类型的Ca2+释放通道，即ryanodine受体（RyRs）之间的相互作用。RyRs通常通过Ca2+诱导的Ca2+释放（CICR）机制被Ca2+激活，但它们也可以被另一种第二信使环磷酸腺苷（cADPR）激活。虽然ryr在可兴奋组织（主要是肌肉和大脑）中已经被很好地表征，但对于不可兴奋细胞中ryr的身份和特性知之甚少。我们最近从大鼠肝细胞中鉴定并克隆了一种独特的RyR异构体（RyR1b），它来源于RyR1基因，但具有替代起始位点，产生的蛋白质只有全长肌肉RyR的40%大小。我们假设RyR1b具有独特的功能特性，这是其在肝细胞和其他不可兴奋的分泌细胞中对[Ca2+]c信号传导的贡献的基础，在这些细胞中，它可能是主要的RyR亚型。具体来说，我们提出RyR1b在增强和维持IP3R激活引发的ER Ca2+释放中起关键作用。我们将在两个特定目标中研究RyR1b的潜在作用：在Aim 1中，我们将进行异源表达研究，以研究RyR1b的功能和调控特性，包括[Ca2+]c、cADPR及其与IP3和IP3R的相互作用的调控。在Aim 2中，我们将研究RyR1b在原代肝细胞中的亚细胞分布和调控，并确定它如何在激素刺激这些细胞时影响[Ca2+]c信号的时空模式。RyR1b似乎是细胞内Ca2+释放通道超家族的一个主要新成员，具有不同的特性。它可能具有特定的功能，被调谐到不可兴奋的分泌上皮和内分泌细胞的信号要求。因此，拟议的工作具有创新性，可能产生重大影响，具有重要的生物医学健康相关性。

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.