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Research ArticleCELLULAR AND MOLECULAR

Altered Regulation of the D1 Dopamine Receptor in Mutant Chinese Hamster Ovary Cells Deficient in Cyclic AMP-Dependent Protein Kinase Activity

Ana Lucia Marques Ventura and David R. Sibley
Journal of Pharmacology and Experimental Therapeutics May 2000, 293 (2) 426-434;
Ana Lucia Marques Ventura
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David R. Sibley
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Abstract

To investigate the role of the cAMP-dependent protein kinase (PKA) in the desensitization and down-regulation of the D1 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 ∼1 pmol/mg D1receptor binding activity (Bmax) as determined using [3H]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 D1receptors 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 D1 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 D1 receptors in CHO cells and that type II PKA may be the more relevant isoform with respect to regulating D1receptor function.

Footnotes

  • Send reprint requests to: Dr. David R. Sibley, Experimental Therapeutics Branch, NINDS/National Institutes of Health, Bldg. 10, Rm. 5C108, 10 Center Dr., MSC 1406, Bethesda, MD 20892-1406. E-mail: sibley{at}helix.nih.gov

  • ↵1 A.L.M.V. is the recipient of a fellowship from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico—CNPq.

  • ↵2 Present address: Departamento de Neurobiologia, Universidade Federal Fluminense, Niteroi, Cx. Postal 100180-RJ, 24001-970 Brasil.

  • Abbreviations:
    GPCR
    G protein-coupled receptor
    PKA
    protein kinase A
    GRK
    G protein-coupled receptor kinase
    EBSS
    Earle's balanced salt solution
    CPT
    8-(4-chlorophenylthio)
    CHO
    Chinese hamster ovary
    • Received October 5, 1999.
    • Accepted January 18, 2000.
  • U.S. Government
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Journal of Pharmacology and Experimental Therapeutics: 293 (2)
Journal of Pharmacology and Experimental Therapeutics
Vol. 293, Issue 2
1 May 2000
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Research ArticleCELLULAR AND MOLECULAR

Altered Regulation of the D1 Dopamine Receptor in Mutant Chinese Hamster Ovary Cells Deficient in Cyclic AMP-Dependent Protein Kinase Activity

Ana Lucia Marques Ventura and David R. Sibley
Journal of Pharmacology and Experimental Therapeutics May 1, 2000, 293 (2) 426-434;

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Research ArticleCELLULAR AND MOLECULAR

Altered Regulation of the D1 Dopamine Receptor in Mutant Chinese Hamster Ovary Cells Deficient in Cyclic AMP-Dependent Protein Kinase Activity

Ana Lucia Marques Ventura and David R. Sibley
Journal of Pharmacology and Experimental Therapeutics May 1, 2000, 293 (2) 426-434;
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