Three-dimensional model of the ligand binding domain of the nuclear receptor for 1alpha,25-dihydroxy-vitamin D(3).

A three-dimensional model for residues 142-427 of the ligand binding domain (LBD) of the human nuclear receptor for 1alpha, 25-dihydroxy-vitamin D(3) [VDR] has been generated based on the X-ray crystallographic atomic coordinates of the LBD of the rat alpha1 thyroid receptor (TR). The VDR LBD model is an elongated globular shape comprised of an antiparallel alpha-helical triple sandwich topology, made up of 12 alpha-helical elements linked by short loop structures; collectively these structural features are similar to the characteristic secondary and tertiary structures for six nuclear receptors with known X-ray structures. The model has been used to describe the interaction of the conformationally flexible natural hormone, 1alpha,25-dihydroxy-vitamin D(3) [1alpha, 25(OH)(2)D(3)], and a number of related analogs with the VDR LBD. The optimal orientation of the 1alpha,25(OH)(2)D(3) in the LBD is with its A-ring directed towards the interior and its flexible side chain pointing towards and interacting with helix-12, site of the activation function-2 domain (AF-2) of the VDR. Mapping of four natural and one experimental point mutations of the VDR LBD, which result in ligand-related receptor dysfunction, indicates the close proximity of these amino acids to the bound ligand.     

An investigation into CAG repeat length variation and N/C terminal interactions in the T877A mutant androgen receptor found in prostate cancer

Prostate cancer may progress by circumventing ablation therapy due to mutations in the androgen receptor (AR) gene. The most intensively studied is the T877A mutation in the ligand binding domain (LBD), which causes the AR to become promiscuous, i.e., respond to a number of different ligands. Our investigations have shown that the T877A mutation alters the inverse relationship between CAG repeat length and transactivation in a noticeable albeit minor manner, while increasing N/C terminal interactions. In the presence of beta-catenin, a coactivator over-expressed in prostate cancer, the inverse relationship between CAG repeat length and transactivation is reversed for the wild type (wt) AR as well. We have also used molecular modeling with the AR and FXXLF and LXXLL peptides to investigate N/C terminal and coactivator interactions. In T877A, this approach revealed an increase in the flexibility of amino acid residues in the activation function 2 (AF-2) domain in the LBD, and a larger solvent accessible surface in T877A compared to the wt AR AF-2 domain. Thus, the improved induced fit of the AR N-terminal domain FXXLF-containing peptide into the T877A LBD could be due to the increased flexibility and solvent accessibility of the AF-2 domain. These new observations suggest that the AR CAG effect can be overridden by prostate cancer mutations, and also further our understanding of hormone-refractory prostate cancer by helping to explain the promiscuity of the T877A mutation.     

Degradation of RXRs influences sensitivity of rat osteosarcoma cells to the antiproliferative effects of calcitriol

Several cell lines, including ROS17/2.8 rat osteosarcoma (ROS) cells, contain functional VDRs and RXRs but are resistant to the antiproliferative effects of calcitriol and retinoids. We explored the role of receptor degradation in this hormone resistance. Results of transactivation assays indicated that ROS cells contain insufficient amounts of RXR to activate a DR-1 reporter, and Western blot analyses of cell extracts showed that the degradation of RXR is accelerated and produces an aberrant 45-kDa RXR. We stably expressed functional fluorescent chimeras of VDR and RXR [green fluorescent protein (GFP)-VDR; yellow fluorescent protein (YFP)-RXR] to evaluate degradation mechanisms and the impact of excess receptor expression on antiproliferative effects. Microscopy showed a diminished expression of YFP-RXR in ROS cells compared with the expression in CV-1 cells. Treatment with inhibitors of proteasomal degradation (lactacystin and MG132) selectively enhanced GFP-VDR and YFP-RXR expression and also increased the endogenous levels of VDR and RXR. Expression of GFP-VDR had no effect on the sensitivity of ROS cells to calcitriol. Increases of RXR levels by YFP-RXR expression, drug treatments, or the combination of the two, however, restored the growth-inhibitory effects of calcitriol and 9-cis-RA and restored p21 induction by calcitriol. These studies revealed that an accelerated and aberrant RXR degradation could cause resistance to the antiproliferative effects of calcitriol and retinoids in ROS cells.     

Hydrogen/deuterium exchange reveals distinct agonist/partial agonist receptor dynamics within vitamin D receptor/retinoid X receptor heterodimer

Regulation of nuclear receptor (NR) activity is driven by alterations in the conformational dynamics of the receptor upon ligand binding. Previously, we demonstrated that hydrogen/deuterium exchange (HDX) can be applied to determine novel mechanism of action of PPARγ ligands and in predicting tissue specificity of selective estrogen receptor modulators. Here, we applied HDX to probe the conformational dynamics of the ligand binding domain (LBD) of the vitamin D receptor (VDR) upon binding its natural ligand 1α,25-dihydroxyvitamin D3 (1,25D3), and two analogs, alfacalcidol and ED-71. Comparison of HDX profiles from ligands in complex with the LBD with full-length receptor bound to its cognate receptor retinoid X receptor (RXR) revealed unique receptor dynamics that could not be inferred from static crystal structures. These results demonstrate that ligands modulate the dynamics of the heterodimer interface as well as provide insight into the role of AF-2 dynamics in the action of VDR partial agonists.