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J. Biol. Chem., Vol. 279, Issue 47, 49533-49541, November 19, 2004

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Protein Kinase C Mediates Phosphorylation, Desensitization, and Trafficking of the D₂ Dopamine Receptor^*

Yoon Namkung and David R. Sibley

From the Molecular Neuropharmacology Section, NINDS, National Institutes of Health, Bethesda, Maryland 20892-1406

Previously, D₂ dopamine receptors (D₂ DARs) have been shown to undergo G-protein-coupled receptor kinase phosphorylation in an agonist-specific fashion. We have now investigated the ability of the second messenger-activated protein kinases, protein kinase A (PKA) and protein kinase C (PKC), to mediate phosphorylation and desensitization of the D₂ DAR. HEK293T cells were transiently transfected with the D₂ DAR and then treated with intracellular activators and inhibitors of PKA or PKC. Treatment with agents that increase cAMP, and activate PKA, had no effect on the phosphorylation state of the D₂ DAR, suggesting that PKA does not phosphorylate the D₂ DAR in HEK293T cells. In contrast, cellular treatment with phorbol 12-myristate 13-acetate (PMA), a PKC activator, resulted in an 3-fold increase in D₂ DAR phosphorylation. The phosphorylation was specific for PKC as the PMA effect was mimicked by phorbol 12,13-dibutyrate, but not by 4-phorbol 12,13-didecanoate, active and inactive, phorbol diesters, respectively. The PMA-mediated D₂ DAR phosphorylation was completely blocked by co-treatment with the PKC inhibitor, bisindolylmaleimide II, and augmented by co-transfection with PKCI. In contrast, PKC inhibition had no effect on agonist-promoted phosphorylation, suggesting that PKC is not involved in this response. PKC phosphorylation of the D₂ DAR was found to promote receptor desensitization as reflected by a decrease in agonist potency for inhibiting cAMP accumulation. Most interestingly, PKC phosphorylation also promoted internalization of the D₂ DAR through a -arrestin- and dynamin-dependent pathway, a response not usually associated with PKC phosphorylation of G-protein-coupled receptors. Site-directed mutagenesis experiments resulted in the identification of two domains of PKC phosphorylation sites within the third intracellular loop of the receptor. Both of these domains are involved in regulating sequestration of the D₂ DAR, whereas only one domain is involved in receptor desensitization. These results indicate that PKC can mediate phosphorylation of the D₂ DAR, resulting in both functional desensitization and receptor internalization.

Received for publication, July 22, 2004 , and in revised form, September 2, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by an NINDS competitive fellowship from the National Institutes of Health.

To whom correspondence should be addressed: Molecular Neuropharmacology Section, NINDS/National Institutes of Health, Bldg. 10, Rm. 5C108, 10 Center Dr., MSC 1406, Bethesda, MD 20892-1406. Tel.: 301-496-9316; Fax: 301-496-6609; E-mail: sibley{at}helix.nih.gov.

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