Identification of a Novel Nurr1-Interacting Protein

Abstract

The orphan nuclear receptor Nurr1 is required for the development of ventral mesencephalic dopaminergic neurons in mice. One of the possible mechanisms that might contribute to the regulation activity of Nurr1 is through interaction with other proteins. To identify potential partners of Nurr1, we screened a yeast two-hybrid library from developing mouse embryonic mesencephalon with the Nurr1 ligand-binding domain (NLBD). We identified a novel interacting protein, termed the Nurr1-interacting protein (NuIP). We demonstrate that it specifically interacts with NLBD using the mammalian two-hybrid assay and coimmunoprecipitation studies in MN9D cells. In addition, we show that NuIP interacts with Nurr1 in lysates from substantia nigra. Coexpression of NuIP with Nurr1 results in potentiation of the transcriptional activity of Nurr1 on an nerve growth factor inducible-B response element reporter, as well as reporters driven by the endogenous tyrosine hydroxylase promoter. The mechanism underlying the regulatory action of NuIP on Nurr1 is demonstrated to be through assembly of distinct helical domains of the NLBD. Using a NuIP specific antibody, we show that expression of NuIP protein is mainly colocalized with Nurr1 in adult midbrain dopaminergic neurons. Finally, we demonstrate that suppression of NuIP expression in MN9D cells by NuIP-specific small interfering RNA leads to decreased cell division and decreased expression of a Nurr1 target gene, the dopamine transporter. These results suggest NuIP interacts with and positively regulates the activity of Nurr1 protein and modulates the phenotype of dopaminergic cells.