Abstract
Nurr1, an orphan nuclear receptor mainly expressed in the central nervous system, is essential for the development of the midbrain dopaminergic neurons. Nurr1 binds DNA as a monomer and exhibits constitutive transcriptional activity. Nurr1 can also regulate transcription as a heterodimer with the retinoid X receptor (RXR) and activate transcription in response to RXR ligands. However, the specific physiological roles of Nurr1 monomers and RXR-Nurr1 heterodimers remain to be elucidated. The aim of this study was to define structural requirements for RXR-Nurr1 heterodimerization. Several amino acid substitutions were introduced in both Nurr1 and RXR in the I-box, a region previously shown to be important for nuclear receptor dimerization. Single amino acid substitutions introduced in either Nurr1 or RXR abolished heterodimerization. Importantly, heterodimerization-deficient Nurr1 mutants exhibited normal activities as monomers. Thus, by introducing specific amino acid substitutions in Nurr1, monomeric and heterodimeric properties of Nurr1 can be distinguished. Interestingly, substitutions in the RXR I-box differentially affected heterodimerization with Nurr1, retinoic acid receptor, thyroid hormone receptor, and constitutive androstane receptor demonstrating that the dimerization interfaces in these different heterodimers are functionally unique. Furthermore, heterodimerization between RXR and Nurr1 had a profound influence on the constitutive activity of Nurr1, which was diminished as a result of RXR interaction. In conclusion, our data show unique structural and functional properties of RXR-Nurr1 heterodimers and also demonstrate that specific mutations in Nurr1 can abolish heterodimerization without affecting other essential functions.
Highlights
Nuclear receptors (NRs)1 constitute a large family of ligandregulated transcription factors including receptors for a variety of small lipophilic ligands such as steroid hormones, retinoids, thyroid hormone, and vitamin D
Nurr1 (NR4A2), which belongs to the category of orphan receptors, is mainly expressed in the central nervous system where it plays a critical role in the development of the midbrain dopamine (DA) cells as shown from analyses of gene-targeted mice [3,4,5,6,7,8]
In these experiments expression vectors encoding wild-type and mutated Nurr1 derivatives were cotransfected with a luciferase reporter gene containing three copies of the retinoic acid receptor response element from the human RAR2 gene (RE), a DNA-binding site previously shown to be efficiently activated by retinoid X receptor (RXR)-Nurr1 in response to RXR ligands [12]
Summary
Nuclear receptors (NRs) constitute a large family of ligandregulated transcription factors including receptors for a variety of small lipophilic ligands such as steroid hormones, retinoids, thyroid hormone, and vitamin D. Two closely related orphan receptors, NGFI-B (NR4A1) and Nor (NR4A3), are expressed in the central nervous system as well as in several peripheral tissues in both overlapping and distinct regions and cell types. These three orphan receptors are likely to exert both unique and redundant functions in vivo. Nurr can form heterodimers with the 9-cis-retinoic acid receptor RXR. These heterodimers recognize a DNA-binding site composed of two consensus NR binding motifs organized as direct repeats separated by five nucleotides (DR5). The purpose of this study has been to identify residues in both Nurr and RXR that are important for heterodimeriza-
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