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

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这样的克隆。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）检测是否存在腺苷结合蛋白，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，我们可以检测到不同的腺苷结合活性，既不是A1也不是A2腺苷受体。该结合位点已被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层析。有趣的是，纯化的腺苷结合位点除了对NECA和腺苷外，还对腺苷核苷酸如AMP或ADP具有高亲和力。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。进一步的研究包括完全纯化和cDNA克隆正在进行中。通过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，研究腺苷受体基因表达。地塞米松诱导[j]。[) A1腺苷受体mRNA稳态水平升高50%，同时A1腺苷受体结合活性显著激活。A2腺苷受体激动剂也可调节A2腺苷受体的表达。NECA或CGS-21680在PC12细胞中引起A2腺苷受体mRNA的短暂升高后又降低。Forskolin也引起了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细胞中的A2腺苷受体基因表达受A2腺苷受体激活的调控，其机制涉及第二信使cAMP。对受体基因中cAMP或其他反应因子的进一步分析目前正在研究中。少

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