Molecular Characterization of Purinergic Receptor and its Signal Transduction in the Vascular System

众所周知，各种嘌呤通过P_1和P_2嘌呤受体在多种组织中发挥不同的生物活性。我们尝试了血管组织中表达的嘌呤受体的分子特征和信号转导。首先，以PCR扩增的狗A_1受体cDNA为探针，通过杂交筛选，从大鼠肝cDNA文库中克隆了大鼠P_1（A_1亚型）受体cDNA全长。以大鼠A_1受体cDNA为探针，筛选大鼠主动脉平滑肌cDNA文库。然而，我们未能检测到代表P_2受体的克隆。我们采用表达克隆的策略，通过测定转染pCDM 8 cDNA文库亚组的Ltk^-细胞中磷酸肌醇的产量，筛选了构建在pCDM 8表达载体上的大鼠主动脉平滑肌cDNA文库。我们无法检测到任何赋予阳性反应的文库亚组。最后，我们尝试了用相应于不同G蛋白偶联受体之间高度保守的第3和第6跨膜区的简并寡核苷酸引物，通过PCR方法克隆P_2受体。我们分离了40多个代表G蛋白偶联受体的克隆，其中包括A_1受体、ET_A内皮素受体、α_1和β_1肾上腺素能受体。其中两个被认为是代表新的G蛋白偶联受体。我们分离了这两种受体的全长克隆。将两个cDNA克隆中的任何一个转染到哺乳动物细胞中均不赋予对P_2配体的反应性。我们现在正试图找到这两种新受体的配体。

It is well known that various purines exert diverse biological activities through P_1 and P_2 purinergic receptors in a variety of tissues. We attempted the molecular characterization of a purinergic receptor expressed in the vascular tissue and the signal transduction. Firstly, we cloned a full length rat P_1(A_1 subtype)receptor cDNA from rat liver lambdagt 10 cDNA library by hybridization screening using PCR-amplified dog A_1 receptor cDNA as a probe. Then, we screened rat aortic smooth muscle cDNA library by using rat A_1 receptor cDNA as a probe. However, we failed to detect a clone which represents P_2 receptors. We, then adapted the strategy of the expression cloning, and screened rat aortic smooth muscle cDNA library constructed in the expression vector pCDM8 by measuring the inositol phosphate production in Ltk^-cells transfected with subgroups of the pCDM8 cDNA library. We could not detect any library subgroup which confered a positive response. Finally, we tried molecular cloning of P_2 receptors by PCR using degenerate oligonucleotide primers corresponding to the 3rd and the 6th transmembrane domains which are well conserved among different G protein-coupled receptors. We isolated more than 40 clones which represented G protein-coupled receptors, among which A_1 receptor, ET_A endothelin receptor and alpha_1 and beta_1 adrenergic receptors were included. Two of them were considered to represent novel G protein-coupled receptors. We isolated full length clones of these two receptors. The transfection of either of the two cDNA clones into mammalian cells did not confer responsiveness to the P_2 ligand. We are now trying to find ligands for these two novel receptors.