Differential desensitization of dopamine D2 receptor isoforms by protein kinase C: the importance of receptor phosphorylation and pseudosubstrate sites.
Altered regulation of dopamine D(2) receptors is implicated in addiction, schizophrenia and movement disorders, as well as lactotroph growth and regulation. Dopamine D(2S) and dopamine D(2L) 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 D(2S) receptor that confer PKC-mediated desensitization. In dopamine D(2L) 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 4beta-phorbol 13alpha-acetate (TPA) completely inhibited calcium mobilization induced by the dopamine D(2S) receptor, but not the dopamine D(2L) variant. Point mutation of i3 PKC sites Ser228/229Gly rendered the dopamine D(2S) receptor resistant to PKC action, with lesser effects of other Ser and Thr mutations. Inactivation of the PKC pseudosubstrate motif in the dopamine D(2L) 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 D(2S), but not D(2L) receptors, in Ltk(-) cells. Conversely, the pseudosubstrate dopamine D(2L) 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 D(2) receptors and mutants. Thus the relative location of phosphorylation and pseudosubstrate sites provides an important determinant substrate sensitivity to PKC.
Morris
Stephen J
SJ
Ottawa Health Research Institute (Neuroscience), University of Ottawa, 451 Smyth Road, Ottawa, Canada K1H-8M5.
Van-Ham
Irit Itzhaki
II
Daigle
Mireille
M
Robillard
Liliane
L
Sajedi
Naghmeh
N
Albert
Paul R
PR
D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D001665:Binding Sites; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D065127:Dopamine D2 Receptor Antagonists; D008024:Ligands; D051379:Mice; D016296:Mutagenesis / Q000502:physiology; D009154:Mutation / Q000502:physiology; D010766:Phosphorylation; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism; D011493:Protein Kinase C / Q000494:pharmacology*; D017448:Receptors, Dopamine D2 / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D019138:Antibodies, Blocking; D065127:Dopamine D2 Receptor Antagonists; D008024:Ligands; D017448:Receptors, Dopamine D2; C113776:dopamine D2L receptor; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium