Desensitization, phosphorylation and palmitoylation of the human dopamine D1 receptor.
The regulation and post-translational modifications of the human dopamine D1 receptor were studied in the baculovirus-eukaryotic cell expression system. Baculovirus constructs containing either the DNA encoding the dopamine D1 receptor or a DNA encoding a c-myc epitope tagged dopamine D1 receptor (c-myc-dopamine D1 receptor) were used to infect Spodoptera frugiperda (Sf9) insect cells. Expressed dopamine D1 and c-myc-dopamine D1 receptors bound agonists and antagonists with affinities and a rank order of potency characteristic of a classical dopamine D1 receptor pharmacological profile. In membrane preparations from cells expressing c-myc-dopamine D1 receptor, the photoaffinity label [125I](3-methyl-2-[4'-azidophenyl]-2,3,5-tetrahydro-2H-3-benzazepine) ([125I]MAB) bound specifically upon photolysis. A major broad band of approximately 48 kDa was detected. This species was identified in immunoblots by the monoclonal antibody raised against the c-myc epitope of c-myc-dopamine D1 receptor was isolated by immunoprecipitation from whole cells and was shown to be post-translationally modified by phosphorylation and palmitoylation. Exposure of cells expressing c-myc-dopamine D1 receptor to dopamine for 15 min resulted in a reduction in the maximal dopamine stimulated adenylyl cyclase activity, which was accompanied by an increased phosphorylation of the receptor and a rapid redistribution of surface c-myc-dopamine D1 receptor as detected by in situ immunofluorescence. Dopamine exposure also resulted in an increased level of incorporation of [3H]palmitic acid into the receptor. Thus, we provide the first evidence that the human dopamine D1 receptor undergoes agonist-dependent desensitization, phosphorylation and palmitoylation.
Ng
G Y
GY
Department of Pharmacology, University of Toronto, Ontario, Canada.
Mouillac
B
B
George
S R
SR
Caron
M
M
Dennis
M
M
Bouvier
M
M
O'Dowd
B F
BF
D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine / Q000378:metabolism / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000032:analysis; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001552:Benzazepines / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D003888:Desensitization, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009036:Moths; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000494:pharmacology; D010766:Phosphorylation; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D017447:Receptors, Dopamine D1 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic
D001552:Benzazepines; D000939:Epitopes; D010169:Palmitic Acids; D016271:Proto-Oncogene Proteins c-myc; D017447:Receptors, Dopamine D1; D019308:Palmitic Acid; D015647:2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; D000262:Adenylyl Cyclases