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Studies on the molecular mechanism of cellular signal transduction via adenosine receptors

腺苷受体细胞信号转导分子机制研究

基本信息

批准号：
04454603
负责人：
NAKATA Hiroyasu
金额：
$ 3.71万
依托单位：
TOKYO METOROPOLITAN INSTITUTE FOR NEUROSCIENCES
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

We first screened cDNA libraries from mouse brains in order to find a new subtype adenosine receptor. However, no such clones were detected in our screening. We then examined if there is an adenosine binding protein which shows a unique adenosine ligand specificity on rat brain membrane fractions using [H^3N]NECA as a tracer, which is a non-selective adenosine receptor agonist. After several trials, we could detect a distinct adenosine binding activity which is neither A1 nor A2 adenosine receptor. This binding site has been highly purified by several chromatographical steps including adenine nucleotide-coupled agarose affinity chromatography. Interestingly, the purified adenosine binding site shows high affinity to adenine nucleotides such as AMP or ADP in addition to NECA and adenosine. The order of the affinity for various adenosine receptor ligands of the purified adenosine binding site was defferent from any known purinergic receptors. Further studies including complete purificati … More on and cDNA cloning are now in progress.Gene expression of adenosine receptor was also investigated by studying the effect of glucocorticoid or adenosine receptor agonist treatment of cultured DDT_1MF-2 or PC12 cells on mRNA expression. Dexamethasone induced (〕SY.apprxeq.〔) 50% increase in the steady-state level of A1 adenosine receptor mRNA accompanied with a significant activation of A1 adenosine receptor binding activity. A2 adenosine receptor expression was also modulated by A2 adenosine receptor agonists. NECA or CGS-21680 caused a transient increase followed by a decrease of A2 adenosine receptor mRNA in PC12 cells. Forskolin also induced similar changes in the level of A2 mRNA.These changes were blocked by an adenosine receptor antagonist. These results suggest that A2 adenosine receptor gene expression in PC12 cells is regulated by an activation of A2 adenosine receptor via a mechanism involving the second messenger, cAMP.Further analysis of the cAMP or other response elements in the receptor gene is now under investigation. Less

我们首先从小鼠大脑中筛选 cDNA 文库，以寻找新的腺苷受体亚型。然而，在我们的筛选中没有检测到此类克隆。然后，我们使用 [H^3N]NECA 作为示踪剂（一种非选择性腺苷受体激动剂）检查是否存在一种腺苷结合蛋白，该蛋白在大鼠脑膜组分上显示出独特的腺苷配体特异性。经过多次试验，我们可以检测到一种独特的腺苷结合活性，它既不是 A1 也不是 A2 腺苷受体。该结合位点已通过几个色谱步骤高度纯化，包括腺嘌呤核苷酸偶联琼脂糖亲和色谱。有趣的是，除了 NECA 和腺苷之外，纯化的腺苷结合位点还对腺嘌呤核苷酸（例如 AMP 或 ADP）显示出高亲和力。纯化的腺苷结合位点对各种腺苷受体配体的亲和力顺序不同于任何已知的嘌呤能受体。包括完全纯化和cDNA克隆在内的进一步研究正在进行中。还通过研究糖皮质激素或腺苷受体激动剂处理培养的DDT_1MF-2或PC12细胞对mRNA表达的影响来研究腺苷受体的基因表达。地塞米松诱导 (]SY.apprxeq.〔) A1 腺苷受体 mRNA 稳态水平增加 50%，同时显着激活 A1 腺苷受体结合活性。 A2 腺苷受体的表达也受到A2 腺苷受体激动剂的调节。 NECA 或 CGS-21680 引起 PC12 细胞中 A2 腺苷受体 mRNA 短暂增加，随后减少。 Forskolin 也引起 A2 mRNA 水平的类似变化。这些变化被腺苷受体拮抗剂阻断。这些结果表明，PC12 细胞中 A2 腺苷受体基因表达是通过涉及第二信使 cAMP 的机制，通过 A2 腺苷受体的激活来调节的。目前正在研究对 cAMP 或受体基因中其他反应元件的进一步分析。较少的