Vol. 60, Issue 4, 857-864, October 2001

D₄ Dopamine Receptor Differentially Regulates Akt/Nuclear Factor-B and Extracellular Signal-Regulated Kinase Pathways in D₄MN9D Cells

Xuechu Zhen, Jie Zhang, Gerard P. Johnson, and Eitan Friedman

Laboratory of Molecular Pharmacology, Department of Pharmacology and Physiology, MCP Hahnemann School of Medicine, Philadelphia, Pennsylvania

The present study was designed to investigate the role of D₄ dopamine receptors in regulating the Akt/nuclear factor-B (NF-B) and extracellular signal-regulated kinase (ERK) signaling pathways. The D₄ dopamine receptor agonist PD168077 induced time- and dose-dependent activation of Akt and ERK in D₄MN9D cells that stably express D₄ dopamine receptors. Maximal Akt and ERK stimulation was achieved at 1 µM PD168077. The agonist-mediated stimulations of Akt and ERK were abolished when cells were preincubated with 50 ng/ml PTX or with 1 µM L745,870, a D₄ dopamine receptor antagonist, indicating that activation of the Akt or ERK pathways is mediated by D₄ dopamine receptors and require a pertussis toxin-sensitive G protein. We also detected a time- and dose-dependent activation of NF-B. Activation of NF-B by 1 µM PD168077 was attenuated in D₄MN9D cells that were transfected with a kinase-deficient Akt but not in cells transfected with a dominant negative Ras (N17Ras), suggesting that NF-B activation requires Akt but is independent of Ras. In contrast, the transfection of N17Ras into D₄MN9D cells blunted D₄ dopamine receptor-mediated ERK activation, indicating a Ras-dependent mechanism. Moreover, PP2 (20 nM), an inhibitor of Src, blocked D₄ receptor-mediated SHC phosphorylation and ERK activation. In contrast, transfection of a kinase-dead Akt did not alter D₄ receptor-stimulated ERK. However, PP2 and the mitogen activated protein kinase kinase inhibitor PD98059 did not change D₄ receptor-mediated Akt/NF-B activation. All these indicate that distinct mechanisms mediate ERK and Akt/NF-B activation by D₄ dopamine receptor stimulation. We also demonstrated that D₄ receptor-stimulated cell proliferation is mediated by the Src/SHC/Ras/ERK pathway.