Differential desensitization of dopamine D2 receptor isoforms by protein kinase C: The importance of receptor phosphorylation and pseudosubstrate sites

Altered regulation of dopamine D2 receptors is implicated in addiction, schizophrenia and movement disorders, as well as lactotroph growth and regulation. Dopamine D2S and dopamine D2L receptors are alternately-spliced variants that differ by 29 amino acids in the third intracellular (i3) domain and display different sensitivity to desensitization by protein kinase C (PKC). In the present studies we determined the specific phosphorylation sites on the dopamine D2S receptor that confer PKC-mediated desensitization. In dopamine D2L receptors, we identified a PKC pseudosubstrate site responsible for the relative insensitivity of the receptor to PKC-induced uncoupling. In transiently transfected Ltk− fibroblast cells, 2-min preactivation of PKC with 12-O-tetradecanoyl 4β-phorbol 13α-acetate (TPA) completely inhibited calcium mobilization induced by the dopamine D2S receptor, but not the dopamine D2L variant. Point mutation of i3 PKC sites Ser228/229Gly rendered the dopamine D2S receptor resistant to PKC action, with lesser effects of other Ser and Thr mutations. Inactivation of the PKC pseudosubstrate motif in the dopamine D2L receptor sensitized the receptor to PKC, and this was reversed by mutation of i3 PKC sites Ser228/229. A phospho-specific antibody generated against phospho-Ser228/229 demonstrated PKC-induced phosphorylation at these sites of dopamine D2S, but not D2L receptors, in Ltk− cells. Conversely, the pseudosubstrate dopamine D2L receptor mutant displayed PKC-induced phosphorylation at Ser228/229, which was abolished when these sites were mutated. Similar phosphorylation results were observed using GH4 cells stably transfected with dopamine D2 receptors and mutants. Thus the relative location of phosphorylation and pseudosubstrate sites provides an important determinant substrate sensitivity to PKC.