STUDIES ON THE MOLECULAR MECHANISM OF CELLULAR TRANSDUCTION VIA ADENOSINE RECEPTORS

利用细胞培养系统对信号转导机制进行了研究。我们找到了几种能抑制腺苷受体的细胞系。我们发现，DDT1MF-2（叙利亚仓鼠平滑肌来源）、ECV304（人内皮细胞）和N1E115（小鼠神经母细胞瘤）等细胞系表达A1腺苷受体。ECV304和N1E115还具有另一种腺苷受体亚型A2b。在这些细胞系中，通过特异性激动剂CPA激活A1腺苷受体，发现对基因表达或细胞增殖起重要作用的丝裂原活化蛋白激酶（MAP激酶）被刺激数倍。这种激活是短暂的，并在30分钟内恢复到基础水平。添加几种蛋白激酶C或pi -3激酶抑制剂抑制了MAP激酶的刺激，表明蛋白激酶C和pi -3激酶参与了上述细胞系中发现的MAP激酶级联反应。在转染了大鼠A1腺苷受体cDNA的CHO细胞中，观察到MAP激酶有更多的激活。这些结果表明，在培养细胞中，除了众所周知的cAMP系统外，腺苷受体，特别是A1亚型的信号还可以控制基因表达或细胞增殖。为了解释腺苷在各种细胞或组织中的多种作用，我们还研究了腺苷受体的另一种亚型。以[3H] NECA结合为标记，我们在大鼠脑膜中发现了一种新的腺苷结合蛋白，该蛋白具有独特的配体特异性，不属于任何已知的腺苷受体。我们开发了该蛋白的光亲和标记系统，并在SDS-PAGE上测定了其分子量（54 kDa）。这种蛋白位于突触膜上。该蛋白的纯化正在进行中，以获得部分氨基酸序列。因此，确定其整个序列和功能的分子克隆将很快进行。少

Studies on the signal transduction mechanism was performed using a cell culture system.We searched several cell lines which ecpress adenosine receptors.We found that cell lines such as DDT1MF-2 (syrian hamster smooth muscle-derived), ECV304 (human endothelial ) and N1E115 (mouse neuroblastoma) expressed A1 adenosine receptors.ECV304 and N1E115 also possessed another adenosine receptor subtype, A2b.By activation of A1 adenosine receptors by specific agonist, CPA,in these cell lines, it was found that mitogen-activated protein kinase (MAP kinase) which is known to play an essential role for the gene expression or cell proliferation was stimulated several fold.This activation was transient and retuned to the basal level in 30 min.Addition of several inhibitors for protein kinase C or PI-3-kinase inhibited the stimulation of MAP kinase, suggesting that protein kinase C and PI-3-kinase are involved in the MAP kinase cascade found in these cell lines described above.The similar agonist-induc … More ed activation of MAP kinase was observed in CHO cells which had been transfected with cDNA of rat A1 adenosine receptor.These results suggest that signals via adenosine receptors, especially A1 subtype, can control gene expression or cell proliferation in addition to the well-known cAMP system in cultured cells.We have also searched another subtype of adenosine receptors in order to explain many kinds of actions of adenosine in various cells or tissues.By using [3H] NECA binding as the marker, we could find a new adenosine binding protein in rat brain membranes which showed a unique ligand specificity, which is not classified into any known adenosine receptors.We developed a photoaffinity labeling system for this protein and determined the molecular mass (54 kDa) on SDS-PAGE.This protein was located in synaptic membranes.Purification of this protein is in progress to obtain partial amino acid sequence.Molecular cloning to determine its whole sequence and function will thus be preformed soon. Less