Snaprud P, Gerwins P, Caron M G, Libert F, Persson H, Fredholm B B, Fuxe K
Department of Neuroscience, Karolinska Institute, Stockholm, Sweden.
Biochem Pharmacol. 1994 Nov 29;48(11):2043-7. doi: 10.1016/0006-2952(94)90503-7.
The rat D2 receptor and the dog A2a receptor subcloned into the pXM vector were transiently transfected into COS-7 cells using the DEAE-dextran method. The transfected cells expressed approx. 200 fmol D2 receptors/mg protein and approx. 5 pmol/mg protein of the A2a receptor as judged by binding experiments with [3H]raclopride [or[3H]-N-propyl-apomorphine (NPA)] and [3H]-CGS 21680, respectively. The high affinity KD values were 0.43 and 19 nM for D2 and A2a receptors, respectively, in agreement with results obtained from other cells and tissues. The non-selective adenosine receptor agonist NECA stimulated cAMP accumulation both in non-transfected and transfected COS-7 cells with only a slight difference in potency, suggesting that most of the stimulation is due to activation of A2b receptors known to be present on virtually every cell. The two A2a selective agonists CGS 21680 and CV-1808 were essentially inactive in transfected COS-7 cells, but were very active in PC-12 cells known to possess functional A2a receptors. Dopamine did not decrease cAMP accumulation in the transfected COS-7 cells. CGS 21680 (30 nM) did not affect the binding characteristics of D2 receptors in the co-transfected COS-7 cells in contrast to the increased KH, KL and RH values found previously in rat striatal membranes after CGS 21680 treatment. The present findings indicate that transiently transfected A2a and D2 receptors in COS-7 cells have normal binding properties, but couple poorly to adenylyl cyclase, despite the presence of Gs protein and adenylyl cyclase in these cells. Our results also demonstrate that the previously reported interactions between A2a receptors and D2 receptors do not occur when only the receptor proteins are expressed in COS-7 cells, suggesting that the two receptor molecules do not interact directly to influence binding characteristics.
将亚克隆到pXM载体中的大鼠D2受体和犬A2a受体，采用DEAE-葡聚糖法瞬时转染到COS-7细胞中。通过用[3H]雷氯必利[或[3H]-N-丙基阿扑吗啡（NPA）]和[3H]-CGS 21680进行结合实验判断，转染细胞分别表达约200 fmol D2受体/毫克蛋白和约5 pmol/毫克蛋白的A2a受体。D2和A2a受体的高亲和力KD值分别为0.43和19 nM，与从其他细胞和组织获得的结果一致。非选择性腺苷受体激动剂NECA在未转染和转染的COS-7细胞中均刺激cAMP积累，效力仅略有差异，这表明大部分刺激是由于几乎存在于每个细胞上的A2b受体的激活。两种A2a选择性激动剂CGS 21680和CV-1808在转染的COS-7细胞中基本无活性，但在已知具有功能性A2a受体的PC-12细胞中非常活跃。多巴胺不会降低转染的COS-7细胞中的cAMP积累。与先前CGS 21680处理后大鼠纹状体膜中发现的KH、KL和RH值增加相反，CGS 21680（30 nM）不影响共转染的COS-7细胞中D2受体的结合特性。目前的研究结果表明，COS-7细胞中瞬时转染的A2a和D2受体具有正常的结合特性，但与腺苷酸环化酶的偶联较差，尽管这些细胞中存在Gs蛋白和腺苷酸环化酶。我们的结果还表明，当仅在COS-7细胞中表达受体蛋白时，先前报道的A2a受体与D2受体之间的相互作用不会发生，这表明这两种受体分子不会直接相互作用以影响结合特性。
