DNA binding properties of the vitamin D3 receptor zinc finger region.

The DNA binding domains of the nuclear receptor superfamily are highly conserved and consist of residues that fold into two zinc finger-like motifs, suggesting that the structures of this region among the members of the superfamily are likely to be very similar. Furthermore, the response elements that these receptors bind to are similar in sequence and organization. Nevertheless, these receptors selectively recognize target response elements and differentially regulate linked genes. In order to study the details of receptor:DNA binding, we have overexpressed and purified the vitamin D3 receptor DNA binding domain (VDRF) and have begun characterizing its DNA binding properties. We find that the VDRF protein binds strongly and specifically to direct repeats constituting a vitamin D response element from the mouse osteopontin (Spp-1) promoter region but weakly to the human osteocalcin vitamin D response element. Unlike receptors that recognize hormone response elements oriented as inverted repeats, such as the glucocorticoid receptor (GR) and estrogen receptor, VDRF appears to bind half-sites noncooperatively, without the free energy contribution of dimerization seen when the glucocorticoid receptor DNA binding domain associates with a glucocorticoid response element. By comparing and contrasting the DNA binding properties of the vitamin D and glucocorticoid receptors, we suggest a model for how receptors that prefer direct repeats differ in their binding strategy from those that recognize inverted repeats.     

Hormone-free mouse glucocorticoid receptors overexpressed in Chinese hamster ovary cells are localized to the nucleus and are associated with both hsp70 and hsp90

In this work, we examine the cellular localization and protein interactions of mouse glucocorticoid receptors that have been overexpressed in Chinese hamster ovary (CHO) cells (Hirst, M. A., Northrop, J. P., Danielsen, M., and Ringold, G. M. (1990) Mol. Endocrinol. 4, 162-170). We demonstrate that wild-type unliganded mouse glucocorticoid receptor, which is expressed in CHO cells to a level approximately 10 times that of L cells, is localized entirely to the nucleus by indirect immunofluorescence with the BuGR antireceptor monoclonal antibody. Overexpressed receptors that have either no hormone binding activity or no DNA binding activity because of point mutations also localize to the nucleus, providing genetic proof that the nuclear localization cannot reflect a steroid-mediated shift of the receptor from the cytoplasm to the nucleus and that DNA binding activity is not required for nuclear localization. Like unliganded progesterone receptors, which also associate in a loosely bound "docking" complex with the nucleus, the mouse glucocorticoid receptor overexpressed in CHO cells is associated with both hsp90 and hsp70. This is in contrast to the untransformed mouse glucocorticoid receptor in L cell cytosol, which is associated with hsp90 but not hsp70. The difference in hsp70 association between cell types could reflect overexpression of the receptor in CHO cells. However, like receptors in CHO cells selected for very high levels of overexpression, receptors in CHO cells selected for an intermediate level of receptor expression that is comparable to that of L cells are also bound to hsp70. This observation argues against an explanation of hsp70 association based purely on receptor overexpression, and we speculate that association of the unliganded glucocorticoid receptor with hsp70 might be a consequence of its nuclear localization in the CHO cells. Although there are differences between the mouse receptor in CHO cells and L cells, the nuclear localization signal of the untransformed mouse receptor reacts equivalently with the AP64 antibody against NL1 in cytosols prepared from both cell types.     

On the mechanism of DNA binding by nuclear hormone receptors: A structural and functional perspective

The nuclear hormone receptor DNA-binding domain consists of two zinc finger-like modules whose amino acids are highly conserved among the members of the receptor superfamily. In this review, we describe the various genetic, biochemical, and structural experiments that have been carried out primarily for the DNA-binding domains of the glucocorticoid and estrogen receptors. We describe how the structural and functional information have permitted us to predict properties of the DNA-binding domains of other nuclear receptors. We postulate how receptors discriminate closely related response elements through sequence-specific contacts and distinguish symmetry of target sites through protein-protein interactions. This mechanism explains in part how the receptors regulate diverse sets of genes from a limited repertoire of core response elements. Lastly, we describe the stereochemical basis of nuclear receptor dysfunction in certain clinical disorders. © 1993 Wiley-Liss, Inc.     

The interaction site for tamoxifen aziridine with the bovine estrogen receptor

Calf uterine estrogen receptor was covalently labeled with [3H]tamoxifen aziridine during affinity chromatography purification. After carboxymethylation, affinity labeled receptor was digested with trypsin under limit conditions and the labeled peptides were fractionated by reversed-phase high performance liquid chromatography into one major and two minor components. Sequence analysis of the dominant labeled fragment indicated the facile cleavage of label during Edman degradation but identified two peptides, both derived from the extreme carboxyl terminus of the steroid-binding domain. The 17 residues of one peptide were fully conserved in all estrogen receptors. This fragment contained five nucleophilic amino acids and was considered as the more favored interaction site for tamoxifen aziridine. A corresponding region of the glucocorticoid receptor has recently been identified as one of three major contact sites for glucocorticoids (Carlstedt-Duke, J., Str?mstedt, P.-E., Persson, B., Cederlund, E., Gustafsson, J.-A., and J?rnvall, H. (1988) J. Biol. Chem. 263, 6842-6846). A comparison of amino acid physical characteristics in the hormone-binding domains of human estrogen and glucocorticoid receptors demonstrated an excellent structural correlation between the two regions and delineated elements in the estrogen receptor which may be directly involved in estradiol binding.     

Retinoic acid receptor belongs to a subclass of nuclear receptors that do not form "docking" complexes with hsp90.

We have recently reported that, in contrast to the glucocorticoid receptor, the thyroid hormone receptor does not bind to hsp90 when the receptor is translated in rabbit reticulocyte lysate [Dalman, F. C., Koenig, R. J., Perdew, G. H., Massa, E., & Pratt, W. B. (1990) J. Biol. Chem. 265, 3615-3618]. All of the steroid receptors that are known to bind hsp90 are recovered in the cytosolic fraction when hormone-free cells are ruptured in hypotonic buffer. In contrast, unliganded thyroid hormone receptors and retinoic acid receptors are tightly associated with nuclear components. In this paper, we translated the human estrogen receptor and the human retinoic acid receptor in reticulocyte lysate and then immunoadsorbed the [35S]methionine-labeled translation products with the 8D3 monoclonal antibody against hsp90. The estrogen receptor is bound to hsp90, as indicated by coimmunoadsorption, but the retinoic acid receptor is not. Translation and immunoadsorption of chimeric proteins containing the DNA binding domain of one receptor and the N-terminal and COOH-terminal segments of the other show that the DNA binding finger region of the estrogen receptor is neither necessary nor sufficient for hsp90 binding. These observations suggest that there are two classes within the steroid receptor family. In one class (e.g., glucocorticoid, mineralocorticoid, sex hormone, and dioxin receptors), the receptors bind to hsp90 and remain in some kind of inactive "docking" mode until hormone-triggered release of hsp90 occurs. In the retinoic acid/thyroid hormone class, the unligated receptors do not bind to hsp90, and the receptors appear to proceed directly to their high-affinity nuclear acceptor sites without entering the "docking" state.     

Nuclear and nuclear envelope binding proteins of the glucocorticoid receptor nuclear localization peptide identified by crosslinking

The molecular mechanisms underlying the nuclear entry of steroid receptors and possible regulation of steroid hormone action during receptor passage across the nuclear envelope have not been elucidated. A nuclear localization signal has been identified in the hinge region of the glucocorticoid receptor. A synthetic peptide corresponding to this sequence was radio-iodinated and incubated with high salt- and detergent-extracted rat liver nuclei or nuclear envelope in the presence of crosslinker. After SDS-PAGE, two nuclear polypeptides of 60 and 76 kDa which had been specifically crosslinked were identified by autoradiography. A 60 kDa polypeptide was also crosslinked in the nuclear envelope fraction. ATP and elevated temperatures enhanced the crosslinking of both nuclear peptides. Finally, we showed that the pattern of crosslinking of the simian virus 40 large tumour antigen nuclear localization signal was identical to that of the glucocorticoid receptor signal to the nuclear polypeptides. The crosslinked peptides are good candidates for nuclear importers of the glucocorticoid receptor. In addition, the data suggest that these binding sites may be part of a general mechanism for nuclear entry of proteins.