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Structure and function of a new purinergic receptor

新型嘌呤受体的结构和功能

基本信息

批准号：
10670104
负责人：
NAKATA Hiroyasu
金额：
$ 2.11万
依托单位：
Tokyo Metropolitan Organization for Medical Research (1999-2000)Tokyo Metropolitan Institute for Neuroscience (1998)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670104/
关键词：
adenosine purinergic cellular signal transduction mechanism heterodimer cross-talk ATP receptor P3 receptor NECA 情報伝達情報伝達機構

项目摘要

In 1996, we found a unique adenosine-binding protein, termed as P3LP, which is sensitive not only to adenosine but also to ATP in rat brain membranes. Because properties of P3LP seems to be similar to presumed P3 purinergic receptors, the structure and properties of P3LP have been extensively investigated in this project. First, we developed a specific ligand for P3LP in order to determine the content of P3LP in various tissues and cells and also for the functional assays. As NECA, a non-specific adenosine receptor ligand, was found to bind to P3LP, various derivatives of NECA were synthesized for the screening of P3LP ligand. One of the compounds tested, 9-(6,7-dideoxy-β-D-allo-hept-5-ynofuranosyl) adenine(HAK2701) was found to be a potent ligand for P3LP.Essentially no binding activity was detected with other adenosine receptors. A relativelysimple assay method for P3LP was then developed using HAK2701 as the specific ligand. In the functional studies, HAK2701 was found to facilitate the release of noradrenaline in the rabbit ear artery where the presence of P3 receptor has been reported. This result suggests that HAK2701 may be a potent and selective a agonist for P3 receptor. From the detailed ligand pharmacology of P3LP, it was found that the specificity of P3LP seems to be the hybrid of A1 adenosine receptor and P2Y1 receptor. We therefore tried to form heterodimer between A1 adenosine receptor and P2Y1 receptor that shows a hybrid phamacology by transfection of each cDNA into HEK293T cells. The oligomeric association between these two receptors was shown by co-immunoprecipitation experiments. Functionally, the co-transfected cells revealed A1 adenosine receptor activity with P2Y_1R-like agonistic pharmacology. These studies strongly suggest P3 purinergic receptor or P3LP is really a hybrid purinoceptor between purinergic receptors.

1996年，我们发现了一种独特的腺苷结合蛋白，称为P3LP，它不仅对腺苷敏感，而且对大鼠脑膜中的ATP也敏感。由于P3LP的性质似乎与推测的P3嘌呤能受体相似，因此本项目对P3LP的结构和性质进行了广泛的研究。首先，我们开发了一种P3LP的特异性配体，用于测定各种组织和细胞中P3LP的含量，并用于功能分析。由于NECA是一种非特异性的腺苷受体配体，被发现与P3LP结合，因此人们合成了多种NECA的衍生物来筛选P3LP配体。其中9-(6，7-二脱氧-β-D-异亮氨酸-5-呋喃葡萄糖基)腺嘌呤(HAK2701)是P3LP的有效配基，与其他腺苷受体无结合活性。以HAK2701为特异性配基，建立了一种相对简单的P3LP检测方法。在功能研究中，HAK2701被发现促进P3受体存在的兔耳动脉去甲肾上腺素的释放。提示HAK2701可能是一种有效的、选择性的P3受体激动剂。从P3LP的详细配基药理研究发现，P3LP的特异性似乎是A1腺苷受体和P2Y1受体的杂交。因此，我们试图在A1腺苷受体和P2Y1受体之间形成异源二聚体，通过将各自的cDNA分别导入HEK293T细胞，从而显示出一种杂交的药理。免疫共沉淀实验表明这两种受体之间存在寡聚结合。在功能上，共转染细胞显示A1腺苷受体活性与P2Y1R类似的激动剂药理作用。这些研究有力地表明，P3嘌呤能受体或P3LP实际上是嘌呤能受体之间的杂交嘌呤受体。