STUDIES ON THE MOLECULAR MECHANISM OF CELLULAR TRANSDUCTION VIA ADENOSINE RECEPTORS

利用细胞培养系统对腺苷受体的信号转导机制进行了研究，我们筛选了几种表达腺苷受体的细胞系，发现DDT 1 MF-2等细胞系，（叙利亚仓鼠平滑肌衍生），ECV 304（人内皮）和N1 E115（小鼠神经母细胞瘤）表达A1腺苷受体，ECV 304和N1 E115还具有另一种腺苷受体亚型，A2 b.在这些细胞系中，通过特异性激动剂CPA激活A1腺苷受体，发现丝裂原活化蛋白激酶（MAP激酶）这种激活是短暂的，在30分钟内恢复到基础水平。几种蛋白激酶C或PI-3-激酶的抑制剂抑制MAP激酶的刺激，表明蛋白激酶C和PI-3-激酶参与上述细胞系中发现的MAP激酶级联反应。 关于我们 在转染大鼠腺苷A1受体cDNA的CHO细胞中观察到MAP激酶的艾德激活，提示腺苷受体，尤其是A1亚型，可以控制基因表达或细胞增殖，我们还寻找了腺苷受体的另一种亚型，以解释腺苷在各种细胞中的多种作用，以[~ 3 H] NECA结合为标记，我们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，我们开发了一种用于该蛋白的光亲和标记系统，并在SDS-聚丙烯酰胺凝胶上测定了其分子量（54 kDa）。该蛋白定位于突触膜，目前正在纯化该蛋白，以获得部分氨基酸序列，因此，不久将进行分子克隆，以确定其完整序列和功能。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