Differential regulation of dopamine D1A receptor responsiveness by various G protein-coupled receptor kinases.
The role of G protein-coupled receptor kinases (GRKs) in the regulation of dopamine D1A receptor responsiveness is poorly understood. To explore the potential role played by the GRKs in the regulation of the rat dopamine D1A receptor, we performed whole cell phosphorylation experiments and cAMP assays in 293 cells cotransfected with the receptor alone or with various GRKs (GRK2, GRK3, and GRK5). The agonist-dependent phosphorylation of the rat D1A receptor was substantially increased in cells overexpressing GRK2, GRK3, or GRK5. Moreover, we report that cAMP formation upon receptor activation was differentially regulated in cells overexpressing either GRK2, GRK3, and GRK5 under conditions that elicited similar levels of GRK-mediated receptor phosphorylation. Cells expressing the rat D1A receptor with GRK2 and GRK3 displayed a rightward shift of the dopamine dose-response curve with little effect on the maximal activation when compared with cells expressing the receptor alone. In contrast, cells expressing GRK5 displayed a rightward shift in the EC50 value with an additional 40% reduction in the maximal activation when compared with cells expressing the receptor alone. Thus, we show that the dopamine D1A receptor can serve as a substrate for various GRKs and that GRK-phosphorylated D1A receptors display a differential reduction of functional coupling to adenylyl cyclase. These results suggest that the cellular complement of G protein-coupled receptor kinases may determine the properties and extent of agonist-mediated responsiveness and desensitization.
Tiberi
M
M
Howard Hughes Medical Institute Laboratories, Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Nash
S R
SR
Bertrand
L
L
Lefkowitz
R J
RJ
Caron
M G
MG
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis / Q000378:metabolism*; D004298:Dopamine / Q000494:pharmacology; D004789:Enzyme Activation; D054770:G-Protein-Coupled Receptor Kinase 3; D054774:G-Protein-Coupled Receptor Kinase 5; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000378:metabolism*; D017447:Receptors, Dopamine D1 / Q000096:biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D016324:Sequence Tagged Sites; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases; D001616:beta-Galactosidase / Q000096:biosynthesis
D017447:Receptors, Dopamine D1; D011994:Recombinant Proteins; C098593:dopamine D1A receptor; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D054770:G-Protein-Coupled Receptor Kinase 3; C517848:GRK3 protein, human; C517851:Grk3 protein, rat; D051552:beta-Adrenergic Receptor Kinases; D054774:G-Protein-Coupled Receptor Kinase 5; C517898:GRK5 protein, human; C517897:Grk5 protein, rat; D001616:beta-Galactosidase; D000262:Adenylyl Cyclases; D004298:Dopamine