Functional analysis of R651 mutations in the putative helix 6 of rat glucocorticoid receptors☆
Introduction
The ligand binding domain (LBD) of steroid receptors effectively translates the information of the cognate ligand into the observed biological effect. Thus, steroid binding to receptors is a pivotal step in steroid receptor regulation of gene transcription. Considerable advances have recently been made in elucidating the steps following steroid binding to receptors, with a major advance coming from the discovery of the involvement of comodulators (CBP), coactivators (TIF2/GRIP-1, SRC-1, and AIB1/ACTR/RAC3/pCIP), and corepressors (SMRT and NCoR) (reviewed in Horwitz et al., 1996). These cofactors can interact with receptor LBDs in ways that are altered by agonist versus antagonist ligands. Furthermore, individual receptors can display preferential binding of specific coactivators (Eng et al., 1998, Feng et al., 1998, Kalkhoven et al., 1998, McInerney et al., 1998, Westin et al., 1998) and corepressors (Wong and Privalsky, 1998), probably with different affinities (Szapary et al., 1999). However, the receptor LBD is usually not sufficient to specify the transcriptional activity of a given steroid-receptor complex. For example, the weak activity of N-terminal truncated glucocorticoid receptors (GRs) containing amino acids 407–795 (Danielsen et al., 1987, Hollenberg et al., 1987, Szapary et al., 1996) argues that ligand-induced effects in LBD must somehow be transmitted to include interactions with the N-terminal A/B domain. Therefore, studies of ligand-induced changes in the LBD will be required for understanding this transmission phenomenon at a molecular level.
The amount of information that can be abstracted from the receptor-bound steroid would be expected to be directly proportional to the surface area of the steroid that is contacted by the receptor. In fact, early calculations on the free energy of binding suggested that the ligand was completely enveloped by the receptor protein (Wolff et al., 1978). This prediction has recently been confirmed by the X-ray crystallographic structures of the LBDs of several steroid receptors, which depict an α-helical sandwich structure with the steroid buried in the middle of the LBD (Bourguet et al., 1995, Renaud et al., 1995, Wagner et al., 1995, Wurtz et al., 1996, Brzozowski et al., 1997, Klaholz et al., 1998, Nolte et al., 1998, Shiau et al., 1998, Tanenbaum et al., 1998, Williams and Sigler, 1998). Recently, it has been proposed that ligand volume, as opposed to molecular weight, is a highly conserved property of steroid/nuclear receptors that may be useful in the design of future, high affinity analogs (Bogan et al., 1998). Nevertheless, it is still not possible to predict the final receptor protein structure that is formed after binding different ligands (Brzozowski et al., 1997, Klaholz et al., 1998, Shiau et al., 1998). Therefore, determinations of the properties of assorted receptor point mutants remain a valuable method for investigating how steroid structure influences receptor structure and biology.
It is well established that ligand binding effects a conformational change in the receptor LBD. A particularly easy method of documenting these changes is to follow the trypsin digestion patterns of receptors±ligand (Simons et al., 1989, Allan et al., 1992, Modarress et al., 1997). Most ligand-free receptors are completely degraded by trypsin but yield several resistant,≈30 kDa fragments if first bound by ligand (Simons et al., 1989, Allan et al., 1992, Beekman et al., 1993, Leng et al., 1993, Modarress et al., 1997, Simons, 1998). The differences in these digestion products, at least for GR, result from cleavage at either ends of the LBD and do not correlate with the agonist or antagonist activity of the steroid (Modarress et al., 1997). Consequently, the cause and nature of these conformational changes is not yet clear. The steroid-free GR appears to be unique in affording a 16 kDa fragment after trypsin digestion. This 16 kDa fragment, which can be covalently labeled by Dex-Mes and retains significant binding affinity and specificity (Simons et al., 1989, Chakraborti et al., 1992), has recently been identified as amino acids 652–795 of the rat GR. In solution, however, this 16 kDa species exists as a non-covalent complex with the other half of the LBD (amino acids 518–651) (Xu et al., 1999).
The fact that steroid-bound GR no longer permits trypsin cutting at R651 of rat GR, as seen by the absence of the 16 kDa fragment (Simons et al., 1989, Modarress et al., 1997), argues that steroid binding has a major effect on the accessibility of this amino acid. One possibility, given the general lack of cleavage of amino acids present in an α-helix (Fontana et al., 1986), is that the short sequence containing R651, and predicted to be present as an α-helix in steroid-bound GR (Wurtz et al., 1996), does not attain the α-helical structure until after steroid binds to the receptor. In this case, prevention of the formation of helix 6 might have significant repercussions for the biological activity of GR. Other reasons for suspecting that R651 might be important in GR action are that R651 of rGR is close to C656, which is affinity labeled by Dex-Mes (Simons et al., 1987), and that R651 is relatively near the steroid in models of steroid-bound GR (Wurtz et al., 1996, Williams and Sigler, 1998).
The purpose of this study was to investigate the hypothesis that R651 plays a significant role in, and is part of a sequence involved in a steroid-induced conformational change that influences, steroid-regulated gene expression by GR. Further interest derived from the fact that little information exists on structure versus function for this region of GR ( Simons, 1994, Martinez et al., 1998). The fact that R651 is in the middle of a predicted short α-helix (Wurtz et al., 1996, Williams and Sigler, 1998) makes it ideal for mutational analyses. We therefore examined the consequences of several mutations on ligand binding and the biological activity of agonist and antagonist steroids with two different enhancer/promoter constructs. Trypsin digestion was performed to probe the structural consequences of the mutations. The results argue that mutations of R651 influence GR activity in ways that are inconsistent with the presence of helix 6 in either steroid-free or steroid-bound GR. The significance of these results with two different enhancers is discussed. The results with steroids of very different sizes were consistent with a computer analysis suggesting that bulky ligands can be useful, active hormones.
Section snippets
Materials and methods
Unless otherwise indicated, all operations were performed at 0°C.
Properties of mutant receptors
Four different mutations were selected for the basic amino acid, arginine, at position 651 of the rat GR: alanine, glycine, proline, and tryptophan. All mutations were to neutral amino acids in order to assess the importance of the lone basic amino acid in the putative helix 6. Alanine is considered to constitute a neutral replacement (Cunningham, 1989) while tryptophan is the most hydrophobic. Proline, and to a lesser extent glycine, are disrupters of α-helices (Chakrabartty et al., 1991,
Discussion
This study examined the existence and importance of helix 6 in GR functions. The extensive homology between the LBDs of GR and progesterone receptors (PRs) led to the suggestion that both proteins may also have highly similar tertiary structures (Wurtz et al., 1996, Williams and Sigler, 1998). In this case, it would be predicted that R651 would lie in the middle of a small α-helix, helix 6, and near both C656, which is affinity labeled by Dex-Mes (Simons et al., 1987), and the bound ligand (
Acknowledgements
We thank Bernd Groner (Frankfurt am Main, Germany), Gordon Hager (NCI/NIH), Keith Yamamoto (University of California, San Francisco, CA) for their generous gifts of research materials, Keith Yamamoto for sharing unpublished data, Paul Sigler (Yale University) for discussions about the PR X-ray structure, David Wheeler (NLM/NIH) for assistance with RasMac, Susan Chacko (NIH) for invaluable help with retrieving structures from the Cambridge Structural Database and with volume calculations by
References (77)
- et al.
Hormone and antihormone induce distinct conformational changes which are central to steroid receptor activation
J. Biol. Chem.
(1992) - et al.
Creation of ‘super’ glucocorticoid receptors by point mutations in the steroid binding domain
J. Biol. Chem.
(1991) - et al.
Role of cysteines 640, 656, and 661 in steroid binding to rat glucocorticoid receptors
J. Biol. Chem.
(1992) - et al.
Evaluation of the major metabolites of raloxifene as modulators of tissue selectivity
J. Steroid. Biochem. Mol. Biol.
(1997) - et al.
Different classes of coactivators recognize distinct but overlapping binding sites on the estrogen receptor ligand binding domain
J. Biol. Chem.
(1998) - et al.
Unique response pathways are established by allosteric interactions among nuclear hormone receptors
Cell
(1995) - et al.
Site-directed mutagenesis by overlap extension using the polymerase chain reaction
Gene
(1989) - et al.
Colocalization of DNA-binding and transcriptional activation functions in the human glucocorticoid receptor
Cell
(1987) - et al.
Interaction between the amino- and carboxyl-terminal regions of the rat androgen receptor modulates transcriptional activity and is influenced by nuclear receptor coactivators
J. Biol. Chem.
(1997) - et al.
Three amino acid substitutions selectively disrupt the activation but not the repression function of the glucocorticoid receptor N terminus
J. Biol. Chem.
(1997)
Cooperativity of glucocorticoid response elements located far upstream of the tyrosine aminotransferase gene
Cell
Intermolecular NH2-/carboxyl-terminal interactions in androgen receptor dimerization revealed by mutations that cause androgen insensitivity
J. Biol. Chem.
Ligand-dependent conformational changes in thyroid hormone and retinoic acid receptors are potentially enhanced by heterodimerization with retinoic X receptor
J. Steroid Biochem. Molec. Biol.
Steroid-induced conformational changes at ends of the hormone binding domain in the rat glucocorticoid receptor are independent of agonist versus antagonist activity
J. Biol. Chem.
Glucocorticoid-receptor complexes and the earliest steps in the action of glucocorticoids on thymus cells
J. Steroid Biochem.
Breaking the camel’s back: proline-induced turns in a model transmembrane helix
J. Mol. Biol.
Sequence-selective interactions of transcription factor elements with tandem glucocorticoid-responsive elements at physiological steroid concentrations
J. Biol. Chem.
Quantity of partial agonist activity for antiglucocorticoids complexed with mutant glucocorticoid receptors is constant in two different transactivation assays but not predictable from steroid structure
J. Steroid Biochem. Mol. Biol.
The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen
Cell
Induction properties of a transiently transfected glucocorticoid-responsive gene vary with glucocorticoid receptor concentration
J. Biol. Chem.
The ligand-binding site of the estradiol receptor resides in a non-covalent complex of two consecutive peptides of 17 and 7 kDa
Biochem. Biophys. Res. Commun.
Interplay of steroid hormone receptors and transcription factors on the mouse mammary tumor virus promoter
J. Steroid Biochem. Molec. Biol.
Modular structure of glucocorticoid receptor domains is not equivalent to functional independence. Stability and activity of the steroid binding domain are controlled by sequences in separate domains
J. Biol. Chem.
Transcription factor loading on the MMTV promoter: a bimodal mechanism for promoter activation
Science
Transcriptional activation by the estrogen receptor requires a conformational change in the ligand binding domain
Mol. Endo.
Steroid-binding properties and stabilization of cytoplasmic glucocorticoid receptors from rat thymus cells
Biochem. J.
Structure and dynamics of the glucocorticoid receptor DNA-binding domain: comparison of wild type and a mutant with altered specificity
Biochemistry
Effect of triphenylacrylonitrile derivatives on estradiol-receptor binding and on human breast cancer cell growth
J. Med. Chem.
Natural ligands of nuclear receptors have conserved volumes
Nat. Struct. Biol.
Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-alpha
Nature
Glucocorticoid receptor-dependent disruption of a specific nucleosome on the mouse mammary tumor virus promoter is prevented by sodium butyrate
Proc. Natl. Acad. Sci. USA
Molecular basis of agonism and antagonism in the oestrogen receptor
Nature
Large differences in the helix propensities of alanine and glycine
Nature
High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis
Science
Domains of the glucocorticoid receptor involved in specific and nonspecific deoxyribonucleic acid binding, hormone activation, and transcriptional enhancement
Mol. Endo.
Hormone-dependent coactivator binding to a hydrophobic cleft on nuclear receptors
Science
Correlation between sites of limited proteolysis and segmental mobility in thermolysin
Biochemistry
Cited by (5)
Structure and Function of the Glucocorticoid Receptor Ligand Binding Domain
2004, Vitamins and HormonesDrug-regulatable cancer cell death induced by BID under control of the tissue-specific, lung cancer-targeted TTS promoter system
2009, International Journal of Cancer
- ☆
The X-ray coordinates, and the thermal parameters, for the human PR LBD were obtained from the Protein Data Bank (ID No. 1A52).
- 1
Present address: Genetic therapy, Novartis, 938 Clopper Road, Gaithersburg, MD, USA