Research Project
6691855
DESCRIPTION (provided by applicant): The objective of this proposal is the development of novel methods for the study of the Class II Nuclear Receptors Thyroid hormone receptor (TR), Retinoic Acid Receptor (RAR), and Vitamin D3 Receptor (VDR). Like other members of the nuclear receptor superfamily, these transcription factors directly activate or repress target genes by binding to DNA response elements in the regulatory regions of target genes. A ligand-mediated conformational change in the nuclear receptor serves as the basic mechanism for switching gene repression to activation. Liganded receptors have a high affinity for proteins that activate transcription (co-activators), while un-liganded receptors have a high affinity for transcriptional repressors. Unlike many of the members of the nuclear receptor superfamily, un-liganded TR, RAR, and VDR are not sequestered in the cytoplasm via an interaction with HSP90. In the presence or absence of ligand, these proteins are bound to their response elements on nuclear DNA as heterodimers with Retinoid X Receptor (RXR). In addition, there are no reliable post-translation modifications (such as acetylation or phosphorylation) associated with activation of these receptors. Thus, there are no currently available methods to rapidly quantify ligand-mediated conversion of TR, RAR, and VDR from transcription repressors to transcriptional activators. Recent studies have identified the peptide sequence (LXXLL) of the transcriptional activator SCR-1, which is responsible for this protein's ligand-dependent interaction with nuclear receptors. In this study, we will use this information to develop a peptide-based assay for the quantification of active-conformation receptors contained in nuclear extracts. Further, the peptide sequence (LXX(I/H)IXXX(I/L)) of the transcriptional repressor SMRT (silencing mediator for retinoid and thyroid receptors) responsible for its interaction with non-liganded nuclear receptor has been identified. We will use this peptide to develop an assay to quantify receptors that is in the non-activating conformation.
