Vitamin D is essential for normal skeletal development and for maintaining the integrity of bone tissue. This role is most evident in the elderly, where abnormalities in vitamin D physiology exacerbate the bone loss in patients with osteoporosis. The fundamental mechanism involves the vitamin D receptor (VDR) and its effects on the transcription of specific gene networks in the intestine and in bone cells. The continuing long-term goal of this project is to understand the molecular mechanism of transcriptional regulation mediated by the VDR with a particular emphasis on proteins that interact with the VDR to mediate the transcriptional response. Key steps in this process are 1,25-(OH)2D3-induced heterodimerization of VDR with retinoid X receptors and interaction with nuclear receptor coactivator proteins. While the molecular details of """"""""firing"""""""" or initiating the 1,25-(OH)2D3-activated transcriptional process are being studied intensively, little is known os the mechanisms which inactivate or down-regulate the activity of liganded VDR in the nucleus. Recently, our laboratory discovered that liganded VDR was degraded by the proteasome in the nucleus of osteoblast cells and that this degradation process was dependent on VDR interaction with Sug1, a protein component of the proteasome. The AF-2 transactivation domain of liganded VDR is required for interaction with Sug1 and this same domain is required for VDR interaction with nuclear receptor coactivators. This suggests a dynamic interplay between two opposing processes in the nucleus. Thus, our working hypothesis states that liganded VDR is directed toward two independent and opposing pathways in the nucleus; 1) coactivator interaction leading to transactivation and 2) sug1 interaction leading to receptor inactivation by proteasome- mediated proteolysis. To test this hypothesis, we propose two specific aims: 1. Examine the competitive interplay between SUG1 and SRC coactivators. We will determine the domains or surfaces that mediate VDR and Sug1 interaction. Quantitative kinetic and equilibrium binding studies and competition studies will be performed in vitro and in vivo. 2. Determine the components of the VDR degradative pathway in osetoblast nuclei. VDR half-life will be assessed under different SUG1 levels. The VDR proteolytic derivative will be identified and analyzed. The role of ubiquitination and the proteasome in the degradation pathway will be determined. These studies will directly test the hypothesis that proteasome- mediated proteolysis via competition between coactivators and proteasome components for liganded VDR is an important negative regulatory step in the mechanism of VDR-mediated transactivation in the nucleus.
