Structure and specificity of nuclear receptor–coactivator interactions

Darimont, Beatrice; Wagner, Richard; Apriletti, James W.; Stallcup, Michael R.; Kushner, Peter J.; Baxter, John D.; Fletterick, Robert J.; Yamamoto, Keith R.

doi:10.1101/gad.12.21.3343

Cited by 875 publications

(867 citation statements)

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“…For in vitro transcription, GR fragments were subcloned into pSG5, which has an integrated T7 promoter. pSG5-N795 (rGR; Darimont et al, 1998) has been described. pSG5-540C was constructed by amplifying the DNA encoding amino acids 540 -795 of pEGFP-N795 using primers containing SalI and BglII and cloning the resulting fragment into the EcoRI/Klenow-BglII sites of pSG5.…”

Section: Plasmid Constructsmentioning

confidence: 99%

“…Beads were washed, and bound proteins were eluted as described previously (Darimont et al, 1998) and immunoblotted with the BuGR2 antibody. For GST pull-downs with in vitro transcribed and translated proteins, GR and truncation derivatives in the pSG5 vector were transcribed and translated (TNT) using the Promega TNT kit as described (Darimont et al, 1998). Immobilized import receptors were incubated with 12 ng GR (as computed from the amount of incorporated 35 S-labeled methionine) in binding buffer for 1 h at 23°C.…”

Section: Binding Assaysmentioning

confidence: 99%

“…Beads were washed three times in pull-down buffer and then incubated with 4 g of GR fragment EX556 (Freedman et al, 1989) containing NL1 for 3 h at 4°C. Bound proteins were resolved and immunoblotted with the BuGR2 antibody.For hormone-dependent binding assays, immobilized import receptors or GRIP were mixed with 12 ng IVT full-length GR that had been translated in the presence or absence of hormone, as described (Darimont et al, 1998). …”

mentioning

confidence: 99%

“…Immobilized importin 7, importin 8, importin ␣, or GST beads were incubated with 240 g extract in the presence or absence of 100 nM dexamethasone for 30 min at room temperature. Bound proteins were washed and resolved as described (Darimont et al, 1998). Gels were immunoblotted with GR-specific polyclonal antibody 283 (a kind gift of Michael Garabedian, NYU).…”

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confidence: 99%

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Importin 7 and Importin α/Importin β Are Nuclear Import Receptors for the Glucocorticoid Receptor

Freedman

2004

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The vertebrate glucocorticoid receptor (GR) is cytoplasmic without hormone and localizes to the nucleus after hormone binding. GR has two nuclear localization signals (NLS): NL1 is similar in sequence to the SV40 NLS; NL2 is poorly defined, residing in the ligand-binding domain. We found that GR displayed similar hormone-regulated compartmentalization in Saccharomyces cerevisiae and required the Sxm1 nuclear import receptor for NL2-mediated import. Two metazoan homologues of Sxm1, importin 7 and importin 8, bound both NL1 and NL2, whereas importin ␣ selectively bound NL1. In an in vitro nuclear import assay, both importin 7 and the importin ␣-importin ␤ heterodimer could import a GR NL1 fragment. Under these conditions, full-length GR localized to nuclei in the presence but not absence of an unidentified component in cell extracts. Interestingly, importin 7, importin 8, and importin ␣ bound GR even in the absence of hormone; thus, hormonal control of localization is exerted at a step downstream of import receptor binding. INTRODUCTIONTo survive in a complex environment, cells sense external cues and commonly respond by altering gene expression, in part by regulating the intracellular localization and activity of transcriptional regulatory factors. For example, the intracellular localization of the yeast Pho4 and mammalian SREBP factors is altered in response to changes in external phosphate and circulating sterol concentrations, respectively (Kaffman et al., 1998b;Nagoshi et al., 1999). Similarly, changes in blood glucose levels and a wide range of physiological stress signals modulate the circulating level of glucocorticoids, changing the activity and localization of the glucocorticoid receptor (GR). Within minutes of glucocorticoid binding, GR enters the nucleus and activates or represses target gene transcription (Picard and Yamamoto, 1987). However, hormone binding is reversible; after hormone withdrawal, GR locates back to the cytoplasm (t 1/2 ϭ 10 h; Hache et al., 1999).Proteins and RNAs enter and exit the nucleus through the nuclear pore, a 125-MDa complex in the nuclear envelope (Stoffler et al., 1999). Small molecules readily diffuse through the nuclear pore, whereas molecules greater than ϳ40 kDa require import and export machinery for transport (Davis, 1995;Pante and Aebi, 1995). The first nuclear localization sequence (NLS) was identified in the SV40 large T-antigen (Kalderon et al., 1984). Development of an in vitro nuclear import assay facilitated the identification of two proteins that import substrates containing SV40 NLS-like domains. The adapter molecule importin ␣ binds to the NLS of the substrate and the importin ␤ transport receptor, creating a trimeric complex that imports the substrate into the nucleus through the nuclear pore (Strom and Weis, 2001).Based on similarity to importin ␤, a family of nuclear import and export receptors was identified and shown to transport a wide variety of proteins and RNAs into and out of the nucleus. After reaching the nucleoplasm, import receptors bind the...

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Section: Plasmid Constructsmentioning

confidence: 99%

Section: Binding Assaysmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Importin 7 and Importin α/Importin β Are Nuclear Import Receptors for the Glucocorticoid Receptor

Freedman

2004

MBoC

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151

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“…The most notable of these are the p160 class of steroid receptor coactivators (SRCs). 2,3 X-ray crystallography has revealed details of these NR-SRC interactions, [4][5][6][7][8][9] which are largely mediated by a short, conserved, pentapeptide LXXLL (L=leucine, X=amino acid) motif of the SRC, termed the NR box. The interaction between the estrogen receptor α (ERα) LBD and a peptide corresponding to NR box 2 of SRC1 (RHKILHRLLQE) is shown in Fig.…”

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confidence: 99%

Bicyclo[2.2.2]octanes: Close structural mimics of the nuclear receptor-binding motif of steroid receptor coactivators

Zhou¹,

Collins²,

Gunther³

et al. 2007

Bioorganic & Medicinal Chemistry Letters

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Nuclear hormone receptor (NR) function relies on association of agonist-bound receptors with steroid receptor coactivator (SRC) proteins through a small pentapeptide motif (LXXLL) of the SRC that binds to a hydrophobic groove on the NR. We have synthesized a series of bicyclo[2.2.2]octanes that are close structural mimics of the two key lysine residues of this SRC sequence as bound in the hydrophobic groove of the estrogen receptor. These bicyclic systems block the NR-SRC interaction with modest potency.Nuclear receptors (NRs) are transcription factors that control many physiological and pathological processes by directly regulating the expression of select target genes. 1 Members of the NR gene superfamily have multi-domain structures, with a conserved DNAbinding domain and a ligand-binding domain (LBD). The transcriptional activity of NRs is initiated by hormone binding that stabilizes the conformation of the LBD. When an agonist ligand binds, conformational stabilization rigidifies surface features that function as docking sites for coactivator protein complexes that are recruited to modify chromatin structure and activate RNA polymerase II. The most notable of these are the p160 class of steroid receptor coactivators (SRCs). 2,3 X-ray crystallography has revealed details of these NR-SRC interactions, 4-9 which are largely mediated by a short, conserved, pentapeptide LXXLL (L=leucine, X=amino acid) motif of the SRC, termed the NR box. The interaction between the estrogen receptor α (ERα) LBD and a peptide corresponding to NR box 2 of SRC1 (RHKILHRLLQE) is shown in Fig. 1. 5 Selected residues of the peptide interact with the LBD through a two-turn amphipathic α-helical motif that places the first and third leucine residues (L690 and L694) in a deep, solvent-exposed hydrophobic groove made up of residues from various helices of the LBD; histidine and arginine residues of the peptide (H691 and R692, removed for clarity) are solvent exposed and appear unnecessary for the interaction. 5 The intrinsic dipole moment of the coactivator α-helix is aligned with charged residues on the surface of the ERα-LBD (E542 interacts with the peptide N-terminus and K362 with its C-terminus), together forming a "charge clamp". 5The traditional strategy to block agonist signaling of NRs involves hormone antagonists (antihormones) that bind to the LBD, displacing the agonist and stabilizing alternative © 2007 Elsevier Ltd. All rights reserved. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. An alternative approach to interrupting NR signaling is the use of compounds capable of blockin...

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Overview of Protein Structural and Functional Folds

2004

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This overview provides an illustrated, comprehensive survey of some commonly observed protein-fold families and structural motifs, chosen for their functional significance. It opens with descriptions and definitions of the various elements of protein structure and associated terminology. Following is an introduction into web-based structural bioinformatics that includes surveys of interactive web servers for protein fold or domain annotation, protein-structure databases, protein-structure-classification databases, structural alignments of proteins, and molecular graphics programs available for personal computers. The rest of the overview describes selected families of protein folds in terms of their secondary, tertiary, and quaternary structural arrangements, including ribbon-diagram examples, tables of representative structures with references, and brief explanations pointing out their respective biological and functional significance.

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Structure and specificity of nuclear receptor–coactivator interactions

Cited by 875 publications

References 56 publications

Importin 7 and Importin α/Importin β Are Nuclear Import Receptors for the Glucocorticoid Receptor

Importin 7 and Importin α/Importin β Are Nuclear Import Receptors for the Glucocorticoid Receptor

Bicyclo[2.2.2]octanes: Close structural mimics of the nuclear receptor-binding motif of steroid receptor coactivators

Overview of Protein Structural and Functional Folds

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