A Nurr1 point mutant, implicated in Parkinson’s disease, uncouples ERK1/2-dependent regulation of tyrosine hydroxylase transcription Kirsten X. Jacobsen, a,b,e Heather MacDonald, a Sylvie Lemonde, a Mireille Daigle, a David A. Grimes, a,c,e Dennis E. Bulman, a,c,d,e and Paul R. Albert a,c,e, ⁎ a Ottawa Health Research Institute, Ottawa, Ontario, Canada b Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H-8M5 c Department of Medicine, Division of Neurology, University of Ottawa, Ottawa, Canada d Center for Neuromuscular Disease, University of Ottawa, Ottawa, Ontario, Canada e Parkinson’s Research Consortium, University of Ottawa and Ottawa Health Research Institute, Canada Received 7 June 2007; revised 19 July 2007; accepted 12 August 2007 Available online 21 August 2007 The orphan nuclear receptor NURR1 is critical for the development of mesencephalic dopamine neurons and directly regulates tyrosine hydroxylase (TH) via specific NGFI-B response elements (NBRE). We identified a Parkinson’s disease patient with a NURR1 mutation, resulting in a p.Ser125Cys change, immediately adjacent to the putative ERK1/2 phosphorylation site. Here we show, in dopaminergic SK-N- AS human neuroblastoma cells, that this substitution markedly attenuated NURR1-induced transcriptional activation through a human TH promoter NBRE. Furthermore, in SK-N-AS cells co-transfected with the dopamine-D2S receptor and NURR1, the dopamine-D2 agonist quinpirole stimulated ERK1/2 phosphorylation and enhanced tran- scriptional activation by wild-type NURR1 but not the p.Ser125Cys NURR1 mutant, and these actions were blocked by the specific MEK1/2 inhibitor PD98059. These results indicate that Ser125 is critical for basal and ERK1/2-induced NURR1 activity and suggest a role for this and other NURR1 mutations in the regulation of dopamine synthesis and predisposition to Parkinson’s disease. © 2007 Elsevier Inc. All rights reserved. Keywords: Dopamine; Development; Transcription; Orphan nuclear receptor; Point Mutation; Phosphorylation Introduction NURR1 (Nur-related factor 1, a.k.a. NR4A2/NOT1/RNR-1/ HZF-3/TINUR) (Mages et al., 1994) is an orphan nuclear receptor transcriptional activator that is required for the development of mesencephalic dopamine neurons (Jorgensen et al., 2006; Law et al., 1992; Wang et al., 2003). As with other NR4A orphan nuclear receptors, NURR1 lacks a cavity for ligand binding and adopts a transcriptionally active conformation owing to the presence of several large hydrophobic residues that occupy this space (Wang et al., 2003). NURR1 contains N- and C-terminal activation function domains (AF-1 and AF-2, respectively) thought to regulate its transcriptional activity (Castro et al., 1999; Martinez-Gonzalez and Badimon, 2005; Nordzell et al., 2004; Wang et al., 2003). NURR1 activity is positively regulated by mitogen-activated protein kinase (ERK1/2) signaling via the N-terminal AF-1 region, and putative ERK1/2 phosphorylation sites have been identified proximal to the AF-1 core of NURR1 (Kim et al., 2006; Kovalovsky et al., 2002; Nordzell et al., 2004). NURR1 (NR4A2) is highly expressed in midbrain dopamine neurons of the substantia nigra (Jorgensen et al., 2006; Maheux et al., 2005; Martinez-Gonzalez and Badimon, 2005; Zetterstrom et al., 1996), and the progressive degeneration of these neurons is implicated in Parkinson’ s disease (Jankovic et al., 2005; Missale et al., 1998; Vallone et al., 2000). NURR1 is implicated in the differentiation, survival, connectivity and migration of dopamine neurons, and in mice lacking NURR1, mesencephalic precursors fail to undergo terminal differentiation and adopt a mature dopaminergic phenotype, dying as development progresses (Eells et al., 2002; Saucedo- Cardenas et al., 1998; Witta et al., 2000). Importantly, NURR1 directly induces transcription of tyrosine hydroxylase (TH), the rate limiting enzyme in the synthesis of dopamine (Kessler et al., 2003; Kim et al., 2003; Sakurada et al., 1999), as well as other important dopaminergic markers, including the dopamine transporter (DAT) and vesicular monoamine transporter 2 (Smits et al., 2003). Taken together, these studies provide compelling evidence for an essential regulatory role of NURR1 in dopamine neuron development. The important role NURR1 plays in dopaminergic development has been underscored by the identification of several mutations in this gene that associate with Parkinson’ s disease (PD) (Grimes et www.elsevier.com/locate/ynbdi Neurobiology of Disease 29 (2008) 117 – 122 ⁎ Corresponding author. Ottawa Health Research Institute, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H-8M5. E-mail address: palbert@uottawa.ca (P.R. Albert). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2007.08.003