Desensitization, phosphorylation and palmitoylation of the human dopamine D 1 receptor

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The regulation and post-translational modifications of the human dopamine D 1 receptor were studied in the baculovirus-eukaryotic cell expression system. Baculovirus constructs containing either the DNA encoding the dopamine D 1 receptor or a DNA encoding a c-myc epitope tagged dopamine D 1 receptor (c-myc-dopamine D 1 receptor) were used to infect Spodoptera frugiperda (Sf9) insect cells. Expressed dopamine D 1 and c-myc-dopamine D 1 receptors bound agonists and antagonists with affinities and a rank order of potency characteristic of a classical dopamine D 1 receptor pharmacological profile. In membrane preparations from cells expressing c-myc-dopamine D 1 receptor, the photoaffinity label [ 125 I](3- methyl-2-[4′- azidophenyl]-2,3,5- tetrahydro-2H-3- benzazepine) ([ 125I]MAB) bound specifically upon photolysis. A major broad band of ∼48 kDa was detected. This species was identified in immunoblots by the monoclonal antibody raised against the c-myc epitope of c-myc-dopamine D 1 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 D 1 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 D 1 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 D 1 receptor undergoes agonist-dependent desensitization, phosphorylation and palmitoylation.