T he steroid hormone 1␣,25(OH) 2 -vitamin D 3 (1,25D) (Fig. 1A), other steroid hormones, retinoids, and thyroid hormones form the family of ligands for the nuclear receptor (NR) superfamily (1), the members of which produce genomic responses through selective interaction of the liganded receptor with promoters of appropriate genes and basal transcription machinery. Many of these hormones also activate rapid, nongenomic (NG), cellular signaling cascades (2, 3) (except retinoids) that range from activation of ion channels (4, 5) to promoting kinase and other cytosolic signaling cascades (6-9). Defining the structure-function requirements for 1,25D and 17␤-estradiol (E 2 ) rapid actions has been aided by the synthesis of analogs that are NG agonists like 1␣,25(OH) 2 -lumisterol (JN) (ref. 10 and Fig. 1 A) and 4-estren-3␣,17␤-diol (EST) (11) or antagonists like 1␤,25(OH) 2 -vitamin D 3 (HL) (ref. 12 and Fig. 1 A), but that are only weak genomic transactivators (13). Thus, important structural attributes of the sterol dictate its agonistic properties and subsequent genomic vs. NG signaling profile (14, 15).When 1,25D rapid signaling cascades were first discovered, it was hypothesized that the observed activities were propagated by a novel membrane protein(s) (16), because analogs JN and HL did not compete well with [ 3 H]1,25D for binding to the nuclear vitamin D receptor (VDR) (17). Recently, in studies using a VDR knockout (KO) mouse (18) and a naturally occurring human VDR mutation (19), 1,25D-mediated rapid responses were shown to require a functional VDR. Both the VDR and estrogen receptor (ER) have been found localized to the plasma membrane in caveolae (7, 20); therefore, it has been proposed that the VDR and ER propagate some NG signaling (6,9,18,21,22). However, given the poor affinity of JN and HL for the VDR, it is difficult to understand how these sterols can facilitate their activities through the VDR.Results obtained from the modeling (INSIGHT 2000.1) of JN, 1,25D, and HL in the VDR ligand-binding domain (LBD) showed that the VDR could possibly accept and form favorable nonbonding interactions with vitamin D sterols in a distinct ligand-binding pocket [an alternative ligand-binding pocket (A pocket)] from the genomic pocket (G pocket) that was previously defined by x-ray crystallography (23, 24). Our proposed A-pocket accepts ligands that differ in shape from those in the classical G pocket (3,23,24).The data from these models has led to the proposal that the VDR can function as a rapid response receptor through a conformational ensemble mechanism (3, 25) whereby the flexible 1,25D steroid hormone samples an ensemble of energetically similar protein conformations (26). In addition, the ensemble model and existence of an A pocket may provide an explanation for the observed sex-nonspecific, nongenotropic signaling through the ER␣ receptor by EST (3, 9, 11). The physiological relevance of the ensemble model and functional importance of an A pocket within the VDR is further substantiated by applying the m...

Changes in the birth or death of osteoblasts and/or osteoclasts represent fundamental pathophysiologic changes in most acquired metabolic bone diseases, including the osteoporosis that results from sex steroid deficiency, glucocorticoid excess, or old age (1-6). Furthermore, pharmacotherapeutics used commonly for the treatment of metabolic bone diseases exert their beneficial effects on bone by regulating the rate of birth of new osteoclasts or osteoblasts or their apoptosis (6 -8).We have recently shown that estrogens and androgens, acting via their classical nuclear receptors (ER␣, 1 ER␤, or AR), attenuate the apoptosis of several different cell types, including osteoblasts and osteocytes, by rapidly activating the Src/Shc/ ERK and phosphatidylinositol 3-kinase (PI3K) and down-regulating the JNK signaling pathways. This effect requires only the ligand binding, not the DNA binding, domain of the receptor, and, unlike its classical transcriptional action, it is eliminated by nuclear targeting of the receptor (9). Activation of ERKs leads to the rapid translocation of the kinases into the nucleus where they phosphorylate common transcription factors like Elk-1, CCAAT enhancer-binding protein-␤, and cAMPresponse element-binding protein. These transcription factors in turn up-regulate gene expression, as exemplified by the up-regulation of the early growth response-1 protein gene, an ERK/serum response element target gene. Likewise, suppression of the JNK signaling cascade by sex steroids leads to downregulation of c-Jun expression (10). We have earlier used a ligand that potently and selectively activates nongenotropic actions of the classical ER or AR and thereby activates kinases and their downstream transcription factors and target genes with only minimal effects on classical estrogen response element-mediated genotropic transcription. Furthermore, we have demonstrated that, although such classical genotropic actions of sex steroid receptors are essential for their effects on reproductive tissues, they are dispensable for their bone protective effects (2).

The carboxyl terminus of p53 is a target of a variety of signals for regulation of p53 DNA binding. Growth suppressor c-Abl interacts with p53 in response to DNA damage and overexpression of c-Abl leads to G 1 growth arrest in a p53-dependent manner. Here, we show that c-Abl binds directly to the carboxyl-terminal regulatory domain of p53 and that this interaction requires tetramerization of p53. Importantly, we demonstrate that c-Abl stimulates the DNA-binding activity of wild-type p53 but not of a carboxyl-terminally truncated p53 (p53⌬363C). A deletion mutant of c-Abl that does not bind to p53 is also incapable of activating p53 DNA binding. These data suggest that the binding to the p53 carboxyl terminus is necessary for c-Abl stimulation. To investigate the mechanism for this activation, we have also shown that c-Abl stabilizes the p53-DNA complex. These results led us to hypothesize that the interaction of c-Abl with the C terminus of p53 may stabilize the p53 tetrameric conformation, resulting in a more stable p53-DNA complex. Interestingly, the stimulation of p53 DNA-binding by c-Abl does not require its tyrosine kinase activity, indicating a kinaseindependent function for c-Abl. Together, these results suggest a detailed mechanism by which c-Abl activates p53 DNA-binding via the carboxyl-terminal regulatory domain and tetramerization.p53 exerts its tumor suppression function by inducing growth arrest and apoptosis (11, 13). The biochemical activity of p53 that is required for this relies on its ability to bind to specific DNA sequences and to function as a transcription factor (22). The importance of the activation of transcription by p53 is underscored by the fact that the majority of p53 mutations found in tumors are located within the domain required for sequence-specific DNA binding (11,13). Therefore, it is clear that this activity is critical to the role of p53 in tumor suppression.A contiguous stretch of 30 amino acid residues at the carboxyl terminus of p53 (C terminus; amino acids 363 to 393) constitutes a domain required for regulation of p53 DNA binding. Interference with this domain by modification, including phosphorylation (23) and acetylation (4, 17), or by deletion (5) has been shown to enhance p53 DNA-binding activity. Moreover, several proteins,, have been shown to bind to this region of p53 and enhance the DNA-binding activity of p53. A model for this activation has been proposed in which the C terminus of p53 interacts with the core of the molecule and in which this interaction locks the core into a conformation that is inactive for DNA binding (6). When this interaction is disrupted by modification, deletion, or protein-protein interaction, the core is able to adopt an active conformation. Despite compelling evidences for such a model, the motif on core domain that interacts with the C terminus remains to be identified. Nevertheless, these studies defined the C-terminal domain as a negative regulatory domain that normally results in a latent, low-affinity DNA-binding form of p53. Th...

Recently, we have developed a vitamin D sterol (VDS)-VDR conformational ensemble model. This model can be broken down into three individual, yet interlinked parts: a) the conformationally flexible VDS, b) the apo/holo-VDR helix-12 (H12) conformational ensemble, and c) the presence of two VDR ligand binding pockets (LBPs); one thermodynamically favored (the genomic pocket, Gpocket) and the other kinetically favored by VDSs (the alternative pocket, A-pocket). One focus of this study is to use directed VDR mutagenesis to 1) demonstrate H12 is stabilized in the transcriptionally active closed conformation (hVDR-c1) by three salt-bridges that span the length of H12 (cationic residues R154, K264 and R402), 2) to elucidate the VDR trypsin sites [R173 (hVDRc1), K413 (hVDR-c2) and R402 (hVDR-c3)] and 3) demonstrate the apo-VDR H12 equilibrium can be shifted. The other focus of this study is to apply the model to generate a mechanistic understanding to discrepancies observed in structure-function data obtained with a variety of 1α,25(OH) 2 -vitamin D 3 (1,25D) A-ring and side-chain analogs, and side-chain metabolites. We will demonstrate that these structure-function conundrums can be rationalized, for the most part by focusing on alterations in the VDS conformational flexibility and the elementary interaction between the VDS and the VDR A-and G-pockets, relative to the control, 1,25D.

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The steroid hormone 1␣,25(OH) 2 -vitamin D 3 (1,25D) 2 (see Fig. 1) and the nuclear vitamin D receptor (VDR) forms a complex that modulates the transcription of genes containing a VDR element. This process is termed a genomic response and involves 1,25D binding to the VDR, formation of a heterodimer with retinoid X receptor, and recruitment of nuclear co-activators (NCoAs) and the basal transcription machinery (1).The VDR is a member of the nuclear hormone superfamily of transcription factors, all of which share similar domain partitioning, ligand binding domain (LBD) tertiary fold and localization of their ligand binding pocket (LBP). The current inducedfit model describing nuclear receptor (NR) activation (i.e. the mouse-trap model) is founded on the comparison of apo-and holo-NR x-ray crystal structures. The mouse trap model posits that closure of the NR activation helix (i.e. helix-12) is induced by ligand binding to an opened-like helix-12 apo-NR conformer. The closed helix-12 conformation completes the activation function II domain (AF2), which serves as the high affinity binding site for recruitment of various NCoAs (1-4) (Fig. 2). Thus, the steric blockage of NR helix-12 closure is the basis for the design of traditional NR genomic antagonists (5-9).

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As part of our studies on the membrane-initiated actions of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] and its localization in caveolae membrane fractions, we used a vitamin D receptor (VDR)-knockout (KO) mouse model to study the binding of [(3)H]-1alpha,25(OH)(2)D(3) in the presumed absence of the VDR. In this mouse model, known as the Tokyo strain, the second exon of the VDR gene, which encodes the first of the two zinc fingers responsible for DNA binding, was removed, and the resulting animals have been considered to be VDR-null mice. To our surprise, several tissues in these KO mice showed significant (5-50% of that seen in wild-type animals) specific binding of [(3)H]-1alpha,25(OH)(2)D(3) in nuclear and caveolae membrane fractions. The dissociation constants of this binding in samples from VDR-KO and wild-type mice were indistinguishable. RT-PCR analysis of intestinal mRNA from the VDR-KO animals revealed an mRNA that lacks exon 2 but contains exons 3-9 plus two 5'-untranslated exons. Western analysis of intestinal extracts from VDR-KO mice showed a protein of a size consistent with the use of Met52 as the translational start site. Transfection of a plasmid construct containing the sequence encoding the human analog of this truncated form of the receptor, VDR(52-C), into Cos-1 cells showed that this truncated form of the receptor retains full [(3)H]-1alpha,25(OH)(2)D(3) binding ability. This same construct was inactive in transactivation assays using the osteocalcin promoter in CV1 cells. Thus, we have determined that this widely used strain of the VDR-KO mouse can express a form of the VDR that can bind ligand but not activate gene transcription.