title: Altered regulation of the D(1) dopamine receptor in mutant Chinese hamster ovary cells deficient in cyclic AMP-dependent protein kinase activity.
abstract: 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.
authors.affiliation: Molecular Neuropharmacology Section, Experimental Therapeutics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892-1406, USA.
authors.forename: A L
authors.initials: AL
authors.lastname: Ventura
authors.forename: D R
authors.initials: DR
authors.lastname: Sibley
mesh_terms: D000818:Animals; D001552:Benzazepines / Q000378:metabolism / Q000494:pharmacology; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000494:pharmacology; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000172:deficiency* / Q000235:genetics*; D004298:Dopamine / Q000096:biosynthesis / Q000494:pharmacology; D018491:Dopamine Agonists / Q000378:metabolism / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D007527:Isoenzymes / Q000172:deficiency / Q000235:genetics; D009154:Mutation / Q000235:genetics; D018696:Neuroprotective Agents / Q000494:pharmacology; D010766:Phosphorylation; D011869:Radioligand Assay; D051381:Rats; D017447:Receptors, Dopamine D1 / Q000096:biosynthesis / Q000235:genetics*; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection / Q000235:genetics
chemical_list: D001552:Benzazepines; D018491:Dopamine Agonists; D007527:Isoenzymes; D018696:Neuroprotective Agents; D017447:Receptors, Dopamine D1; D013873:Thionucleotides; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; D004298:Dopamine