Neuropsychopharmacology (2014) 39, 1290–1301 & 2014 American College of Neuropsychopharmacology. All rights reserved 0893-133X/14 www.neuropsychopharmacology.org Protein Kinase D1-Dependent Phosphorylation of Dopamine D1 Receptor Regulates Cocaine-Induced Behavioral Responses 1,3 1,3 1,3 1,3 1 1 1 ,1,2 Ning Wang , Ping Su , Ying Zhang , Jie Lu , Baoming Xing , Kai Kang , Wenqi Li and Yun Wang* 1 Neuroscience Research Institute and Department of Neurobiology, The Key Laboratory for Neuroscience of the Ministry of Education/National 2 Health and Family Planning Commission, Peking University Health Science Center, Beijing, China; PKU-IDG/McGovern Institute for Brain Research, Peking University, Beijing, China The dopamine (DA) D1 receptor (D1R) is critically involved in reward and drug addiction. Phosphorylation-mediated desensitization or internalization of D1R has been extensively investigated. However, the potential for upregulation of D1R function through phosphorylation remains to be determined. Here we report that acute cocaine exposure induces protein kinase D1 (PKD1) activation in the rat striatum, and knockdown of PKD1 in the rat dorsal striatum attenuates cocaine-induced locomotor hyperactivity. Moreover, PKD1-mediated phosphorylation of serine 421 (S421) of D1R promotes surface localization of D1R and enhances downstream extracellular signal-regulated kinase signaling in D1R-transfected HEK 293 cells. Importantly, injection of the peptide Tat-S421, an engineered Tat fusion-peptide targeting S421 (Tat-S421), into the rat dorsal striatum inhibits cocaine-induced locomotor hyperactivity and injection of Tat-S421 into the rat hippocampus or the shell of the nucleus accumbens (NAc) also inhibits cocaine-induced conditioned place preference (CPP). However, injection of Tat-S421 into the rat NAc shell does not establish CPP by itself and injection of Tat-S421 into the hippocampus does not influence spatial learning and memory. Thus, targeting S421 of D1R represents a promising strategy for the development of pharmacotherapeutic treatments for drug addiction and other disorders that result from DA imbalances. Neuropsychopharmacology (2014) 39, 1290–1301; doi:10.1038/npp.2013.341; published online 15 January 2014 Keywords: cocaine addiction; protein kinase D1; dopamine D1 receptor; phosphorylation; conditioned place preference; locomotor activity INTRODUCTION inhibiting DA transporters (Chen et al, 2006; Giros et al, 1996; Schmitt and Reith, 2010). DA exerts its functions via Cocaine is one of the most abused psychostimulants used by binding to DA receptors, which can be grouped into two humans. Chronic, long-term use of cocaine can result in subclasses: D1-like receptors (D1R and D5R) and D2-like addiction, which is a state of compulsive drug use. At receptors (D2R, D3R, and D4R; Beaulieu and Gainetdinov, present, cocaine addiction has become a serious issue 2011). Both D1R and D2R, the most abundant subtypes of worldwide. However, there are currently no effective DA receptors in vivo, are critically involved in drug medications available for the treatment of cocaine addiction addiction and the reward pathway. However, activation of (Shorter and Kosten, 2011). Understanding the neurobiolo- D1R is an absolute requirement for the induction of many gical mechanisms responsible for cocaine addiction and of the cellular and behavioral effects of cocaine (Bateup identifying candidate targets for pharmacotherapeutic et al, 2010; Bateup et al, 2008; Bertran-Gonzalez et al, 2008; intervention is of great importance. Xu et al, 1994). It is well known that the mesolimbic dopamine (DA) The protein kinase D (PKD) family is composed of three system has a critical role in drug addiction and the reward members, PKD1/PKCm, PKD2, and PKD3/PKCn. To date, pathway. Cocaine induces a strong and rapid increase in PKD1 is the best-characterized isoform of this family extracellular DA in the mesolimbic reward circuit by (Rozengurt et al, 2005; Steinberg, 2012). PKD1 has a vital role in various important biological processes, including *Correspondence: Professor Y Wang, Neuroscience Research Institute cell proliferation and differentiation, vesicle trafficking and and Department of Neurobiology, The Key Laboratory for Neu- secretion, immune regulation and cardiac hypertrophy, and roscience of the Ministry of Education/National Health and Family Planning Commission, Peking University Health Science Center, Beijing contraction (Rozengurt, 2011). In the nervous system, we 100191, China, Tel/Fax: +86 10 82801119, E-mail: [email protected] first found that PKD1 has an important role in inflamma- 3These authors contributed equally to this work. tory heat hyperalgesia through an interaction with transient Received 28 August 2013; revised 12 December 2013; accepted 13 receptor potential vanilloid 1 (Wang et al, 2004; Zhu et al, December 2013; accepted article preview online 23 December 2013 2008). Subsequently, studies by us and others have shown PKD1, dopamine D1 receptor, and cocaine addiction N Wang et al 1291 that PKD1 also participates in other nervous system bilaterally into the specific brain regions, and the peptide processes, including neuronal development (Bisbal et al, was administered in 4 min (0.25 ml/min) followed by an 2008; Yin et al, 2008), neuroprotection (Stetler et al, 2012), additional 1 min to allow diffusion before the injection needle and learning (Fu et al, 2009). Although the function of was taken out. PKD1 has been extensively studied in a variety of cellular models, its physiological role, especially in the nervous shRNA Lentivirus system, is largely unknown. Herein, we report the following: (1) PKD1 modulates GFP-PKD1-shRNA lentivirus was constructed and amplified cocaine-induced behaviors via phosphorylating D1R at by Genechem (Shanghai, China). The sequence 50-GGTTCT serine 421 (S421); (2) PKD1 potentiates the surface GGACAGTTCGGAA-30 was connected to the U6-vshRNA- localization of D1R and downstream extracellular signal- UBI-GFP. Lentivirus expressing shRNA targeting a non- regulated kinase (ERK) signaling in D1R-transfected cell specific sequence were used as controls. For in vivo line; (3) the cell-permeable Tat fusion-peptide targeting knockdown of PKD1, 1 Â 108 lentiviral particles were S421 (Tat-S421), targeting phosphorylation of S421 of D1R, injected bilaterally into the dorsal striatum. Five days later, exhibits an inhibitory effect on cocaine-induced behaviors. further experiments were performed. Given the pressing need for effective disease-modifying treatments for addiction, the identification of the molecular Cell Culture and Transfection basis of cocaine addiction and the anti-addiction effects of Tat-S421 are highly valuable findings. HEK 293A cells were maintained in DMEM plus 10% fetal bovine serum (Hyclone, Logan, UT) at 37 1Cin5%CO2. Transfections of cells were performed with Lipofectamine MATERIALS AND METHODS 2000 (Invitrogen). Cells were used 24–36 h post transfection. Antibodies Anti-phospho-D1R (p-S421) antibody was custom-made by Cell Surface Biotinylation Assay and Western Blot GL Biochem (Shanghai, China). Anti-PKD1 antibody (sc-639), Analysis anti-D1R antibody (sc-14001), and anti-ERK1/2 antibody We performed the cell surface biotinylation assay and (sc-93) were purchased from Santa Cruz Biotechno- western blotting as previously described (Xing et al, 2012). logy (Santa Cruz, CA). Anti-phospho-PKD1 (p-S744/748) Immunoreactive bands were scanned and analyzed quanti- antibody (2054S) and anti-phospho-ERK1/2 antibody tatively by densitometry with Quantity One Software (4370S) were purchased from Cell Signaling Technology (Bio-Rad, Hercules, CA). (Danvers, MA). Anti-b-actin antibody (A5316) was pur- chased from Sigma (St Louis, MO). Anti-transferrin receptor antibody (13–6800) was purchased from Invitrogen In Vitro Kinase Assay (Carlsbad, CA). Anti-GFP antibody was purchased from The in vitro kinase assay was conducted as previously Roche Diagnostics (Indianapolis, IN). described (Yang et al, 2007). Briefly, 20 ng of active PKD1 (Upstate Biotechnology, Lake Placid, NY) was resuspended Plasmid Construction and Mutation in assay buffer to a volume of 25 ml and a final concentration of 0.8 ng/ml. To initiate the reaction, 10 ml of the phosphor- The EYFP-N1-D1R construct was a gift from Professor ylation mixture (assay buffer containing 5 mCi of [g-32P]ATP Xuechu Zhen at Soochow University. The construct to fuse and one of the following GST fusion proteins: GST, GST- glutathione S-transferase (GST) with the carboxyl terminus D1R-CT, or GST-D1R-CT S421A) was added. The mixture of the D1R (GST-D1R-CT) was generated using the EcoRI/ was incubated at 30 1C for 30 min, and the reaction was XhoI sites and subcloned into pGEX-5x-1 (GE Healthcare, terminated by the addition of SDS-PAGE sample buffer. Little Chalfont, UK). The construct myc-his-PKD1 was After boiling for 5 min, the samples were subjected to SDS- generated using the XhoI/EcoRI sites and subcloned into PAGE. The gels were stained with Coomassie blue, dried, pcDNA 3.1/myc-His C (Invitrogen). Mutants of GST-D1R- and exposed to X-ray film for autoradiography. CT and myc-his-DNPKD1 (D727A) were created with the QuickChange site-directed mutagenesis kit (Stratagene, Animals La Jolla, CA). All constructs were confirmed by DNA sequencing (BGI, Beijing, China). Male Sprague–Dawley rats (weighing 250–270 g on arrival) were housed in groups of five in a temperature- (23±2 1C) ± Drug and Peptides and humidity- (50 5%) controlled environment. The animals