Journal of Molecular Biology
Volume 296, Issue 3, 25 February 2000, Pages 743-756
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Regular article
Ligand-triggered stabilization of vitamin D Receptor/Retinoid X receptor heterodimer conformations on DR4-type response elements1

https://doi.org/10.1006/jmbi.2000.3499Get rights and content

Abstract

Nuclear receptors integrate an incoming signal in the form of a nuclear hormone by undergoing a conformational change that results via co-activator proteins in an activation of the basal transcriptional machinery. The vitamin D3 receptor is the nuclear receptor for 1α,25-dihydroxy vitamin D3 (1α,25(OH)2D3) and is known to function as a heterodimer with the retinoid X receptor on DR3-type 1α,25(OH)2D3 response elements. Here, it could be demonstrated that DR4-type response elements are at least as effective as DR3-type 1α,25(OH)2D3 response elements. Gel shift clipping analysis showed that vitamin D3 receptor-retinoid X receptor heterodimers form in response to 1α,25(OH)2D3 and retinoid X receptor ligands, the pan-agonist 9-cis retinoic acid (9cRA) and the retinoid X receptor-selective retinoid CD2425, different conformations on the DR4-type element of the rat Pit-1 gene. Interestingly, on this response element the heterodimeric complexes of retinoid X receptor with the thyroid hormone receptor, the retinoic acid receptor and the benzoate ester receptor also displayed characteristic individual ligand-dependent complex formation. On the level of complex formation, utilizing DNA affinity and functional assays, only vitamin D3 receptor-retinoid X receptor heterodimers showed a synergistic interaction of both ligands. However, the sensitivity of vitamin D3 receptor-retinoid X receptor heterodimers to 1α,25(OH)2D3 was found to be much higher than to retinoid X receptor ligands. Taken together, this study demonstrates a unique interaction potential of vitamin D3 receptor and retinoid X receptor but also establishes DR4-type response elements as multi-functional DNA binding sites with a potential to integrate various hormone signalling pathways.

Introduction

The biologically active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), is a pleiotropic nuclear hormone being involved in the regulation of calcium homeostasis (DeLuca et al., 1990) and immune function, as well as cellular growth, differentiation and apoptosis (Walters, 1992). Genomic effects of 1α,25(OH)2D3 are mediated through the nuclear receptor 1α,25(CH)2D3 receptor (VDR) (Carlberg & Polly, 1998), which belongs to a transcription factor superfamily (Mangelsdorf et al., 1995). The high homology in their DNA binding domain (DBD) and reasonable homology in their carboxy-terminal ligand binding domain (LBD) between the members of this superfamily suggests similar function in DNA binding, transactivation and contact to other nuclear proteins. The VDR and the receptors for the thyroid hormone 3,5,3′-triiodothyronine (T3), T3R, and the vitamin A derivative all-trans retinoic acid (atRA), RAR, are the classical representatives of a large subgroup (class II) within the nuclear receptor superfamily (Carlberg, 1995). This subgroup has in majority orphan members, which are nuclear receptors that have been cloned before their specific ligands were known (Blumberg & Evans, 1998). A representative example is the orphan nuclear receptor (ONR), which was cloned through its sequence homology to the VDR (Smith et al., 1994). ONR was recently identified as nuclear receptor for benzoate esters, such as 4-amino-butyl-benzoate (4-ABB) (Blumberg et al., 1998a), and was therefore named also benzoate X receptor (BXR).

Hormone responsive genes are defined through the presence and specific recognition of nuclear receptor binding sites, so-called response elements (REs), in their promoter regions. VDR, T3R, RAR and ONR preferentially form heterodimeric complexes with the retinoid X receptor (RXR), which is a nuclear receptor for 9-cis retinoic acid (9cRA) Heyman et al 1992, Levin et al 1992. According to the 3–4–5 rule (Umesono et al., 1991), simple REs are formed by two AGGTCA motifs in a directly repeated (DR) arrangement and the number of spacing nucleotides is the major discriminating parameter for a specific DNA recognition: VDR-RXR heterodimers bind to DR3-type 1α,25(OH)2D3 response elements (VDREs), T3R-RXR heterodimers to DR4-type elements and RAR-RXR heterodimers to DR5-type elements. The orphan partner receptors of RXR are also suggested to have their respective preferential DR-type REs, e.g. ONR-RXR heterodimers were reported to bind to DR3-type REs (Smith et al., 1994).

The stabilization of a functional conformation of the nuclear receptor LBD is, beside the specific recognition of DNA, the second important factor in nuclear hormone signalling Nayeri et al 1996, Nayeri and Carlberg 1997. According to LBD crystal structures, e.g. of apo-RXRα (Bourguet et al., 1995) and atRA-bound RARγ (Renaud et al., 1995), the binding of ligand mainly results in changing the position of the most carboxy-terminal α-helix that contains the so-called activation function 2 (AF-2) domain. With the VDR, traditional ligand competition assays using radiolabelled ligand do not allow for visualization of such receptor conformation changes (Mørk Hansen et al., 1996). In contrast, the limited protease digestion assay has been demonstrated to be a powerful method for characterizing functional VDR conformations Peleg et al 1995, Nayeri et al 1996, Nayeri and Carlberg 1997, Peleg et al 1998. However, in its classical form the method describes only monomeric receptors in solution, but a recently developed method, called gel shift clipping assay Quack et al 1998b, Quack and Carlberg 1999, combines the advantages of the limited protease digestion assay and of the well-known DNA-dependent gel shift assay.

The model of the multiple 1α,25-dihycoxy vitamin D3 (1α,25(OH)2D3) signalling pathways (Carlberg, 1996) suggests that the different physiological functions of the hormone may be represented by different types of VDR-VDRE complexes. This may include different types of heterodimeric complexes (Green, 1993) and different RE structures (Carlberg, 1995), but also different conformations of the same heterodimer complex type. Non-DR3-type VDREs, such as direct repeats with six spacing nucleotides (DR6) Carlberg et al 1993, Polly et al 1996, Xie and Bikle 1997 and inverted palindromic arrangements of the core binding sites with nine intervening nucleotides (IP9) Schrader et al 1995, Schrader et al 1997, have been known for some time and have been demonstrated to mediate some aspects of the selective functional profile of 1α,25(OH)2D3 analogues Carlberg et al 1994, Nayeri et al 1995, Quack and Carlberg 1999.

A VDRE type that has not been intensively investigated thus far are DR4-type elements, i.e. direct repeats with four intervening nucleotides. This study shows that DR4-type VDREs are at least as potent as DR3-type VDREs, but also demonstrates that DR4-type REs are recognized by a variety of other heterodimeric complexes, including T3R-RXR, RAR-RXR and ONR-RXR. These different heterodimers show individual ligand-induced complex formation. However, concerning an interaction with RXR ligands, VDR-RXR heterodimers show a unique interaction potential.

Section snippets

Results

The DNA binding profile of VDR-RXR, T3R-RXR, RAR-RXR and ONR-RXR heterodimers (and RXR homodimers as a control) was analysed by gel shift experiments on REs that each consist of two copies of the core binding motifs AGTTCA (DRXT, Figure 1(a)-(e)) or AGGTCA (DRXG, Figure 1(f)-(j)) in a directly repeated orientation with two to six intervening nucleotides (DR2T to DR6T or DR2G to DR6G, see Table 1). The protein-DNA complexes were separated from free probe on non-denaturing 10 % polyacrylamide

Discussion

The members of the nuclear receptor superfamily are complex regulators of transcription that are themselves regulated in a number of different ways. In this study, the heterodimeric complexes VDR-RXR, T3R-RXR, RAR-RXR and ONR-RXR have been chosen as representatives for different classes of dimeric nuclear receptor complexes. According to the 3–4–5 rule (Umesono et al., 1991), complexes of VDR, T3R and RAR should bind to DR3, DR4 and DR5-type REs and RAR is also known to bind to DR2-type REs

Compounds

The 1α,25(OH)2D3 (kindly provided by C. Mørk Hansen, Leo Pharmaceutical Products, Ballerup, Denmark) was dissolved and diluted in ethanol, CD2425 (kindly provided by U. Reichert, Galderma R&D, Sophia Antipolis, France), T3, atRA, 9cRA and 4-ABB (all from Sigma, Deisenhofen, Germany) were dissolved and diluted in DMSO.

DNA constructs

The cDNA for human VDR (Carlberg et al., 1993), chicken T3Rα (Sap et al., 1986), human RARα (Levin et al., 1992) and human RXRα (Levin et al., 1992) were subcloned into the

Acknowledgements

We thank P. Polly for discussions and C. Mørk Hansen for 1α,25(OH)2D3, U. Reichert for CD2425 and B. Old for the ONR construct. This work was supported by the Medical Faculty of the Heinrich-Heine-University Düsseldorf, the Fonds der Chemischen Industrie and the LEO Research Foundation.

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