Ventura A L, Sibley D R
Molecular Neuropharmacology Section, Experimental Therapeutics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892-1406, USA.
J Pharmacol Exp Ther. 2000 May;293(2):426-34.
To investigate the role of the cAMP-dependent protein kinase (PKA) in the desensitization and down-regulation of the D(1) dopamine receptor, we stably expressed the rat cDNA for this receptor in mutant Chinese hamster ovary (CHO) cell lines deficient in PKA activity. The 10260 mutant CHO cell line has been characterized as expressing less than 10% of type I and type II PKA activities relative to the parental 10001 CHO cell line. The 10248 mutant CHO line lacks type II PKA activity and expresses a defective type I PKA. The transfected parental and mutant cell lines were found to express approximately 1 pmol/mg D(1) receptor binding activity (B(max)) as determined using [(3)H]SCH-23390 binding assays. All three cell lines demonstrated similar levels of dopamine-stimulated adenylyl cyclase activity. Pretreatment of all three CHO cells with dopamine resulted in desensitization of the adenylyl cyclase response, although the maximum desensitization was attenuated by 20 and 40% in the 10260 and 10248 cell lines, respectively. Dopamine also promoted, in a time- and dose-dependent fashion, a >90% down-regulation of D(1) receptors in the parental cell line but only a 50 and 30% decrease in the 10260 and 10248 cells, respectively. Similarly, treatment of the cells with the membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP induced functional desensitization and down-regulation of the D(1) receptor, although it was not as great as that observed with agonist pretreatment. As with the agonist pretreatments, the 8-(4-chlorophenylthio)-induced responses were attenuated in the mutant cells with the 10248 line exhibiting the least desensitization/down-regulation. Our results suggest that PKA significantly contributes to the desensitization and down-regulation of D(1) receptors in CHO cells and that type II PKA may be the more relevant isoform with respect to regulating D(1) receptor function.
为了研究环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）在D1多巴胺受体脱敏和下调中的作用，我们在缺乏PKA活性的突变型中国仓鼠卵巢（CHO）细胞系中稳定表达了该受体的大鼠cDNA。10260突变型CHO细胞系的特征是，相对于亲本10001 CHO细胞系，其I型和II型PKA活性表达低于10%。10248突变型CHO细胞系缺乏II型PKA活性，并表达有缺陷的I型PKA。使用[³H]SCH-23390结合试验测定，发现转染的亲本细胞系和突变细胞系表达的D1受体结合活性（Bmax）约为1 pmol/mg。所有三种细胞系的多巴胺刺激的腺苷酸环化酶活性水平相似。用多巴胺预处理所有三种CHO细胞均导致腺苷酸环化酶反应脱敏，尽管在10260和10248细胞系中最大脱敏分别减弱了20%和40%。多巴胺还以时间和剂量依赖性方式促进亲本细胞系中D1受体>90%的下调，但在10260和10248细胞中仅分别降低50%和30%。同样，用膜通透性cAMP类似物8-（4-氯苯硫基）-cAMP处理细胞会诱导D1受体的功能脱敏和下调，尽管其程度不如激动剂预处理时观察到的那样大。与激动剂预处理一样，8-（4-氯苯硫基）诱导的反应在突变细胞中减弱，10248细胞系表现出最小的脱敏/下调。我们的结果表明，PKA对CHO细胞中D1受体的脱敏和下调有显著贡献，并且就调节D1受体功能而言，II型PKA可能是更相关的同工型。
