Altered regulation of the D-1 dopamine receptor in mutant Chinese hamster ovary cells deficient in cyclic AMP-dependent protein kinase activity

To investigate the role of the cAMP-dependent protein kinase (PKA) in the d esensitization and down-regulation of the D-1 dopamine receptor, we stably expressed the rat cDNA for this receptor in mutant Chinese hamster ovary (C HO) cell lines deficient in PKA activity. The 10260 mutant CHO cell line ha s 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 C HO line lacks type II PKA activity and expresses a defective type I PKA. Th e transfected parental and mutant cell lines were found to express similar to 1 pmol/mg D-1 receptor binding activity (B-max) as determined using [H-3 ]SCH-23390 binding assays. All three cell lines demonstrated similar levels of dopamine-stimulated adenylyl cyclase activity. Pretreatment of all thre e CHO cells with dopamine resulted in desensitization of the adenylyl cycla se 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 rece ptors 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 function al desensitization and down-regulation of the D-1 receptor, although it was not as great as that observed with agonist pretreatment. As with the agoni st pretreatments, the 8-(4-chlorophenylthio) induced responses were attenua ted in the mutant cells with the 10248 line exhibiting the least desensitiz ation/down-regulation. Our results suggest that PKA significantly contribut es 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 regu lating D-1 receptor function.