scholarly journals Phosphorylation and Intramolecular Stabilization of the Ligand Binding Domain in the Nuclear Receptor Steroidogenic Factor 1

2002 ◽  
Vol 22 (20) ◽  
pp. 7193-7203 ◽  
Author(s):  
Marion Desclozeaux ◽  
Irina N. Krylova ◽  
Florence Horn ◽  
Robert J. Fletterick ◽  
Holly A. Ingraham
Keyword(s):  
Ligand Binding ◽  
Nuclear Receptor ◽  
Activation Function ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Mitogen Activated Protein Kinase ◽  
Orphan Nuclear Receptor ◽  
Steroidogenic Factor 1 ◽  
Study Results ◽  
Biochemical Analyses

ABSTRACT Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor with no known ligand. We showed previously that phosphorylation at serine 203 located N′-terminal to the ligand binding domain (LBD) enhanced cofactor recruitment, analogous to the ligand-mediated recruitment in ligand-dependent receptors. In this study, results of biochemical analyses and an LBD helix assembly assay suggest that the SF-1 LBD adopts an active conformation, with helices 1 and 12 packed against the predicted alpha-helical bundle, in the apparent absence of ligand. Fine mapping of the previously defined proximal activation function in SF-1 showed that the activation function mapped fully to helix 1 of the LBD. Limited proteolyses demonstrate that phosphorylation of S203 in the hinge region mimics the stabilizing effects of ligand on the LBD. Moreover, similar effects were observed in an SF-1/thyroid hormone LBD chimera receptor, illustrating that the S203 phosphorylation effects are transferable to a heterologous ligand-dependent receptor. Our collective data suggest that the hinge together with helix 1 is an individualized specific motif, which is tightly associated with its cognate LBD. For SF-1, we find that this intramolecular association and hence receptor activity are further enhanced by mitogen-activated protein kinase phosphorylation, thus mimicking many of the ligand-induced changes observed for ligand-dependent receptors.

scholarly journals Novel Mechanism of Nuclear Receptor Corepressor Interaction Dictated by Activation Function 2 Helix Determinants

2002 ◽  
Vol 22 (19) ◽  
pp. 6831-6841 ◽  
Author(s):  
Anna N. Moraitis ◽  
Vincent Giguère ◽  
Catherine C. Thompson
Keyword(s):  
Retinoic Acid ◽  
Thyroid Hormone ◽  
Ligand Binding ◽  
Nuclear Receptor ◽  
Thyroid Hormone Receptor ◽  
Activation Function ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Cerebellar Development ◽  
Nuclear Receptor Corepressor

ABSTRACT Transcriptional regulation by nuclear receptors is controlled by the concerted action of coactivator and corepressor proteins. The product of the thyroid hormone-regulated mammalian gene hairless (Hr) was recently shown to function as a thyroid hormone receptor corepressor. Here we report that Hr acts as a potent repressor of transcriptional activation by RORα, an orphan nuclear receptor essential for cerebellar development. In contrast to other corepressor-nuclear receptor interactions, Hr binding to RORα is mediated by two LXXLL-containing motifs, a mechanism associated with coactivator interaction. Mutagenesis of conserved amino acids in the ligand binding domain indicates that RORα activity is ligand-dependent, suggesting that corepressor activity is maintained in the presence of ligand. Despite similar recognition helices shared with coactivators, Hr does not compete for the same molecular determinants at the surface of the RORα ligand binding domain, indicating that Hr-mediated repression is not simply through displacement of coactivators. Remarkably, the specificity of Hr corepressor action can be transferred to a retinoic acid receptor by exchanging the activation function 2 (AF-2) helix. Repression of the chimeric receptor is observed in the presence of retinoic acid, demonstrating that in this context, Hr is indeed a ligand-oblivious nuclear receptor corepressor. These results suggest a novel molecular mechanism for corepressor action and demonstrate that the AF-2 helix can play a dynamic role in controlling corepressor as well as coactivator interactions. The interaction of Hr with RORα provides direct evidence for the convergence of thyroid hormone and RORα-mediated pathways in cerebellar development.

scholarly journals The Crystal Structure of the Orphan Nuclear Receptor NR2E3/PNR Ligand Binding Domain Reveals a Dimeric Auto-Repressed Conformation

PLoS ONE ◽  
2013 ◽  
Vol 8 (9) ◽  
pp. e74359 ◽  
Author(s):  
M. H. Eileen Tan ◽  
X. Edward Zhou ◽  
Fen-Fen Soon ◽  
Xiaodan Li ◽  
Jun Li ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Ligand Binding ◽  
Nuclear Receptor ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Orphan Nuclear Receptor

scholarly journals The Orphan Nuclear Receptor SHP Utilizes Conserved LXXLL-Related Motifs for Interactions with Ligand-Activated Estrogen Receptors

2000 ◽  
Vol 20 (4) ◽  
pp. 1124-1133 ◽  
Author(s):  
Lotta Johansson ◽  
Ann Båvner ◽  
Jane S. Thomsen ◽  
MatHias Färnegårdh ◽  
Jan-Åke Gustafsson ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Estrogen Receptors ◽  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Mechanistic Explanation ◽  
Receptor Activation ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Orphan Nuclear Receptor ◽  
Mechanistic Basis

ABSTRACT SHP (short heterodimer partner) is an unusual orphan nuclear receptor consisting only of a ligand-binding domain, and it exhibits unique features of interaction with conventional nuclear receptors. While the mechanistic basis of these interactions has remained enigmatic, SHP has been suggested to inhibit nuclear receptor activation by at least three alternatives; inhibition of DNA binding via dimerization, direct antagonism of coactivator function via competition, and possibly transrepression via recruitment of putative corepressors. We now show that SHP binds directly to estrogen receptors via LXXLL-related motifs. Similar motifs, referred to as NR (nuclear receptor) boxes, are usually critical for the binding of coactivators to the ligand-regulated activation domain AF-2 within nuclear receptors. In concordance with the NR box dependency, SHP requires the intact AF-2 domain of agonist-bound estrogen receptors for interaction. Mutations within the ligand-binding domain helix 12, or binding of antagonistic ligands, which are known to result in an incomplete AF-2 surface, abolish interactions with SHP. Supporting the idea that SHP directly antagonizes receptor activation via AF-2 binding, we demonstrate that SHP variants, carrying either interaction-defective NR box mutations or a deletion of the repressor domain, have lost the capacity to inhibit agonist-dependent transcriptional estrogen receptor activation. Furthermore, our studies indicate that SHP may function as a cofactor via the formation of ternary complexes with dimeric receptors on DNA. These novel insights provide a mechanistic explanation for the inhibitory role of SHP in nuclear receptor signaling, and they may explain how SHP functions as a negative coregulator or corepressor for ligand-activated receptors, a novel and unique function for an orphan nuclear receptor.

scholarly journals Characterization of the Nurr1 ligand-binding domain co-activator interaction surface

2006 ◽  
Vol 37 (2) ◽  
pp. 317-326 ◽  
Author(s):  
Nikolaos Volakakis ◽  
Michal Malewicz ◽  
Banafsheh Kadkhodai ◽  
Thomas Perlmann ◽  
Gerard Benoit
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Close Association ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Site Directed Mutagenesis ◽  
Orphan Nuclear Receptor ◽  
Hydrophobic Region ◽  
Binding Surface

The recently solved crystal structure of the orphan nuclear receptor (NR) Nurr1 ligand-binding domain (LBD) showed that Nurr1 lacks a cavity for ligand binding and a canonical NR co-activator-binding site. Computer modeling of the Nurr1 LBD structure identified a hydrophobic region on the surface of the Nurr1 LBD that was positioned on the opposite side from the classical co-activator-binding site. Site-directed mutagenesis demonstrated that this region is critical for the activity of the Nurr1 LBD. Most mutations introduced in this region reduced or abolished transcriptional activity of the Nurr1 LBD, but mutation at lysine (K577) resulted in a drastically increased activity. Moreover, the activity of the Nurr1 LBD was shown to correlate with a propensity for proteasome-dependent degradation revealing a close association between activity and Nurr1 protein turnover. These data provide novel insights into the mechanisms of transcription via the Nurr1 LBD and identify an alternative co-activator-binding surface that is unique to the NR4A family of NRs.

scholarly journals Adrenocorticotropic Hormone Regulates the Activities of the Orphan Nuclear Receptor Nur77 through Modulation of Phosphorylation*

Endocrinology ◽  
1997 ◽  
Vol 138 (10) ◽  
pp. 4138-4146 ◽  
Author(s):  
Yanzhuang Li ◽  
Lester F. Lau
Keyword(s):  
Dna Binding ◽  
Nuclear Receptor ◽  
Binding Domain ◽  
Mitogen Activated Protein Kinase ◽  
Dna Binding Domain ◽  
Orphan Nuclear Receptor ◽  
Expression Of Genes ◽  
Acth Treatment ◽  
Mitogen Activated Protein

Abstract ACTH treatment of Y1 adrenocortical cells induces the synthesis of Nur77, an orphan nuclear receptor that can act as a potent trans-activator for such genes as 21-hydroxylase (CYP21). Nur77 has thus been proposed to be a mediator of ACTH action in activating the expression of genes that encode steroidogenic enzymes. Here we show that ACTH regulates the activity of Nur77 at the level of phosphorylation. ACTH induces the synthesis of transcriptionally active, DNA-binding Nur77 that is unphosphorylated at Ser354, which resides within the DNA-binding domain. By contrast, the Nur77 population that is constitutively present in Y1 cells is phosphorylated at Ser354 and does not bind DNA. Substitutions of Ser354 with negatively charged amino acids, such as Asp or Glu, dramatically decreased Nur77 DNA-binding and trans-activation activities, whereas mutation to the neutral Ala had no effect. Aside from phosphorylation within the DNA-binding domain, ACTH treatment does not induce modifications in the N- and C-terminal domains of Nur77 that significantly affect activity. Although the specific kinases that phosphorylate Nur77 in vivo are not known, the mitogen-activated protein kinase/pp90RSK pathway is not critical to Nur77 regulation. We propose that ACTH treatment of Y1 cells results in modulation of the activities of both kinases and phosphatases, which, in turn, regulate the activities of such transcription factors as Nur77.

scholarly journals The AF1 and AF2 Domains of the Androgen Receptor Interact with Distinct Regions of SRC1

1999 ◽  
Vol 19 (12) ◽  
pp. 8383-8392 ◽  
Author(s):  
Charlotte L. Bevan ◽  
Sue Hoare ◽  
Frank Claessens ◽  
David M. Heery ◽  
Malcolm G. Parker
Keyword(s):  
Androgen Receptor ◽  
Ligand Binding ◽  
Activation Function ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Rich Region ◽  
Independent Manner ◽  
N Terminus ◽  
Androgen Receptor Activity ◽  
Glutamine Rich Region

ABSTRACT The androgen receptor is unusual among nuclear receptors in that most, if not all, of its activity is mediated via the constitutive activation function in the N terminus. Here we demonstrate that p160 coactivators such as SRC1 (steroid receptor coactivator 1) interact directly with the N terminus in a ligand-independent manner via a conserved glutamine-rich region between residues 1053 and 1123. Although SRC1 is capable of interacting with the ligand-binding domain by means of LXXLL motifs, this interaction is not essential since an SRC1 mutant with no functional LXXLL motifs retains its ability to potentiate androgen receptor activity. In contrast, mutants lacking the glutamine-rich region are inactive, indicating that this region is both necessary and sufficient for recruitment of SRC1 to the androgen receptor. This recruitment is in direct contrast to the recruitment of SRC1 to the estrogen receptor, which requires interaction with the ligand-binding domain.

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