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Molecular Characterization of ADP-ribosyl Cyclase Coupled with Receptors

与受体偶联的 ADP-核糖基环化酶的分子表征

基本信息

批准号：
12480228
负责人：
HIGASHIDA Haruhiro
金额：
$ 8.45万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12480228/
关键词：
Ca signaling cyclic ADP-ribose Ca pool second messenger Signal transduction NAD ADPR cyclase cADPR サイクリックADPリボナース

项目摘要

We previously reported that stimulation of β-adrenergic and angiotensin II receptors modulates activity of ADP-ribosyl cyclase, a synthetic enzyme of cADP-ribose, and showed that cADP-ribose is a critical molecule in sympathetic potentiation of heart contraction, post-natal heart growth, and neuron-glia interaction. In the present study, we used the same strategy to explore the idea mat cADP-ribose is a second messenger downstream of the mGluRs. We first measured ADP-ribosyl cyclase activity in crude membranes from various rat and mouse nervous tissues in the presence or absence of glutamate and/or GTP. Second, the coupling preference of mGluRs to ADP-ribosyl cyclase was examined in NG108-15 neuroblastoma x glioma hybrid cells over-expressing each subtype.Glutamate stimulates ADP-ribosyl cyclase activity in rat or mouse crude membranes of retina via group III mGIuRs or in superior cervical ganglion via group I mGluRs. The retina of mGluR6 deficient mice showed no increase in the ADP-ribosyl cyclase level in response to glutamate. GTP enhanced the initial rate of basal and glutamate-stimutated cyclase activity. GTP-γ-S also stimulated basal activity. To determine whether the coupling mode of mGluRs to ADP-ribosyl cyciase is a feature common to individual cloned mGIuRs, we expressed each mGluR subtype in NG108-15 neuroblastoma x glioma hybrid cells. The glutamate-induced stimulation of the cyclase occurs preferentially in NG108-15 cells over-expressing mGluRs1, 3, 5, and 6. Cells expressing mGluR2 or mGluRs4 and 7 exhibit inhibition or no coupling, respectively. Glutamate-induced activation or inhibition of the cyclase activity was eliminated after pretreatment with cholera or pertussis toxin, respectively. Thus, the subtype-specific coupling of mGIuRs to ADP-ribosyl cyclase via G proteins suggests that some glutamate-evoked neuronal function is mediated by cADP-ribose.

我们先前报道了刺激β-肾上腺素能和血管紧张素II受体调节ADP-核糖基环化酶（cADP-核糖的合成酶）的活性，并表明cADP-核糖是心脏收缩交感神经增强、出生后心脏生长和神经元-胶质细胞相互作用的关键分子。在本研究中，我们使用相同的策略来探索cADP-核糖是mGluRs下游的第二信使的想法。我们首先测量ADP-核糖环化酶活性的粗膜从各种大鼠和小鼠的神经组织中存在或不存在的谷氨酸和/或GTP。第二，在NG 108 -15神经母细胞瘤×胶质瘤杂交细胞中检测了mGluRs对ADP-核糖基环化酶的偶联偏好，谷氨酸通过III组mGluRs刺激大鼠或小鼠视网膜粗膜中的ADP-核糖基环化酶活性，或通过I组mGluRs刺激上级颈神经节中的ADP-核糖基环化酶活性。mGluR 6缺陷小鼠的视网膜显示响应谷氨酸的ADP-核糖基环化酶水平没有增加。GTP增强了基础和谷氨酸刺激的环化酶活性的初始速率。GTP-γ-S对基础活性也有促进作用。为了确定mGluR与ADP-核糖基环化酶的偶联模式是否是单个克隆的mGluR的共同特征，我们在NG 108 -15神经母细胞瘤x神经胶质瘤杂交细胞中表达了每种mGluR亚型。谷氨酸诱导的环化酶的刺激优先发生在NG 108 -15细胞过表达mGluRs 1，3，5和6。表达mGluR 2或mGluR 4和7的细胞分别表现出抑制或无偶联。谷氨酸诱导的环化酶活性的激活或抑制被消除后，分别与霍乱或百日咳毒素预处理。因此，亚型特异性偶联mGIuRs ADP-核糖基环化酶通过G蛋白表明，一些谷氨酸诱发的神经元功能介导的cADP-核糖。