Functional studies on the orphan receptor Nurr1 and related retinoid receptors

Abstract: Nuclear receptors (NRs) constitute a large family of ligand-inducible transcription factors which includes receptors for steroid hormones, retinoids (vitamin A derivatives), vitamin D an d thyroid hormone. The family also includes a large number of structurally related proteins that lack identified ligands and are therefore refered to as orphan receptors. Retinoid signaling is mediated by the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), with RAR/RXR being the main functional units. It has been shown that RAR can be activated in RAR/RXR heterodimers, whereas RXR is believed to be a silent partner. Our studies on the retinoid receptors demonstrated that the inhibition of RXR is not obligatory and that RXR can be activated in the RAR/RXR heterodimer, in the presence of RAR ligands. These results provide a base for the synergism observed between RAR and RXR activation in processes such as cell differentiation. Nurr1 is an orphan nuclear receptor expressed in the central nervous system, which plays a critical role in the development of midbrain dopaminergic neurons, the cells that degenerate in Parkinson's disease. The mechanisms whereby Nurr1 mediates this process are not understood. The ligand-binding domain (LBD) of NRs encompasses a ligand dependent activation function (called A172) which, upon ligand binding, is essential for recruitment of transcription mediator proteins called coactivators. Our functional studies on the orphan receptor Nurr1 revealed that Nurr1 LBD has an intrinsic capacity to activate transcription in a cell type dependent fashion, in the absence of exogenously added ligands. The unusual structural features of Nurr1 AF2 suggest an alternative mechanism for coactivator recruitment by this orphan receptor. This was supported by our observation that Nurr1 did not appear to be stimulated by several previously identified NR coactivators. Importantly, our results demonstrated an important role for the Nurr1 AF2 region in transactivation, consistent with the possibility of finding ligands that could regulate Nurr1 activity. We investigated the role of Nuff I in dopaminergic cell differentiation using the MN9D cell line as a model. Nurr1 expression in MN9D cells induced morphological differentiation associated with neurite outgrowth and growth arrest at GI phase of cell cycle. By using different Nurr1 derivatives, we were able to characterize the functional requirements for Nuff I mediating this process, thus generating important insights into the Nurr1 function in vivo. Overall, our results suggest a dual regulatory role for Nurr1 in the control of cell proliferation and dopaminergic differentiation in the CNS. The potential of using Nurr1 for inducing a dopaminergic phenotype in neural stem cells was explored by stably transfecting Nurr I in the neural stem cell line C 17.2. Cells from C 17.2-Nurr I clones could be induced to become dopaminergic when differentiated in the presence of type l astrocytes from the ventral midbrain. Importantly, our results suggest an instructive role for astrocytes in the process of neuronal differentiation in vivo. In addition, they demonstrate the potential for manipulating gene expression in neural stem cells, aimed at generating an unlimited number of neurons of a desired phenotype for cell replacement strategies in the context of neurodegenerative diseases.