Live cell imaging unveils multiple domain requirements for in vivo dimerization of the glucocorticoid receptor

Diego M Presman, M Florencia Ogara, Martín Stortz, Lautaro D Alvarez, John R Pooley, R Louis Schiltz, Lars Grøntved, Thomas A Johnson, Paul R Mittelstadt, Jonathan D Ashwell, Sundar Ganesan, Gerardo Burton, Valeria Levi, Gordon L Hager, Adali Pecci

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Glucocorticoids are essential for life, but are also implicated in disease pathogenesis and may produce unwanted effects when given in high doses. Glucocorticoid receptor (GR) transcriptional activity and clinical outcome have been linked to its oligomerization state. Although a point mutation within the GR DNA-binding domain (GRdim mutant) has been reported as crucial for receptor dimerization and DNA binding, this assumption has recently been challenged. Here we have analyzed the GR oligomerization state in vivo using the number and brightness assay. Our results suggest a complete, reversible, and DNA-independent ligand-induced model for GR dimerization. We demonstrate that the GRdim forms dimers in vivo whereas adding another mutation in the ligand-binding domain (I634A) severely compromises homodimer formation. Contrary to dogma, no correlation between the GR monomeric/dimeric state and transcriptional activity was observed. Finally, the state of dimerization affected DNA binding only to a subset of GR binding sites. These results have major implications on future searches for therapeutic glucocorticoids with reduced side effects.

Original languageEnglish
JournalP L o S Biology
Volume12
Issue number3
Pages (from-to)e1001813
ISSN1544-9173
DOIs
Publication statusPublished - Mar 2014

Fingerprint

dimerization
Dimerization
Glucocorticoid Receptors
image analysis
Imaging techniques
Oligomerization
DNA
cells
glucocorticoids
Glucocorticoids
Ligands
DNA-binding domains
point mutation
Point Mutation
Dimers
glucocorticoid receptors
binding sites
Luminance
Assays
pathogenesis

Keywords

  • Animals
  • Cells, Cultured
  • DNA
  • Mice
  • Protein Multimerization
  • Protein Structure, Tertiary
  • Receptors, Glucocorticoid

Cite this

Presman, D. M., Ogara, M. F., Stortz, M., Alvarez, L. D., Pooley, J. R., Schiltz, R. L., ... Pecci, A. (2014). Live cell imaging unveils multiple domain requirements for in vivo dimerization of the glucocorticoid receptor. P L o S Biology, 12(3), e1001813. https://doi.org/10.1371/journal.pbio.1001813
Presman, Diego M ; Ogara, M Florencia ; Stortz, Martín ; Alvarez, Lautaro D ; Pooley, John R ; Schiltz, R Louis ; Grøntved, Lars ; Johnson, Thomas A ; Mittelstadt, Paul R ; Ashwell, Jonathan D ; Ganesan, Sundar ; Burton, Gerardo ; Levi, Valeria ; Hager, Gordon L ; Pecci, Adali. / Live cell imaging unveils multiple domain requirements for in vivo dimerization of the glucocorticoid receptor. In: P L o S Biology. 2014 ; Vol. 12, No. 3. pp. e1001813.
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abstract = "Glucocorticoids are essential for life, but are also implicated in disease pathogenesis and may produce unwanted effects when given in high doses. Glucocorticoid receptor (GR) transcriptional activity and clinical outcome have been linked to its oligomerization state. Although a point mutation within the GR DNA-binding domain (GRdim mutant) has been reported as crucial for receptor dimerization and DNA binding, this assumption has recently been challenged. Here we have analyzed the GR oligomerization state in vivo using the number and brightness assay. Our results suggest a complete, reversible, and DNA-independent ligand-induced model for GR dimerization. We demonstrate that the GRdim forms dimers in vivo whereas adding another mutation in the ligand-binding domain (I634A) severely compromises homodimer formation. Contrary to dogma, no correlation between the GR monomeric/dimeric state and transcriptional activity was observed. Finally, the state of dimerization affected DNA binding only to a subset of GR binding sites. These results have major implications on future searches for therapeutic glucocorticoids with reduced side effects.",
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Presman, DM, Ogara, MF, Stortz, M, Alvarez, LD, Pooley, JR, Schiltz, RL, Grøntved, L, Johnson, TA, Mittelstadt, PR, Ashwell, JD, Ganesan, S, Burton, G, Levi, V, Hager, GL & Pecci, A 2014, 'Live cell imaging unveils multiple domain requirements for in vivo dimerization of the glucocorticoid receptor', P L o S Biology, vol. 12, no. 3, pp. e1001813. https://doi.org/10.1371/journal.pbio.1001813

Live cell imaging unveils multiple domain requirements for in vivo dimerization of the glucocorticoid receptor. / Presman, Diego M; Ogara, M Florencia; Stortz, Martín; Alvarez, Lautaro D; Pooley, John R; Schiltz, R Louis; Grøntved, Lars; Johnson, Thomas A; Mittelstadt, Paul R; Ashwell, Jonathan D; Ganesan, Sundar; Burton, Gerardo; Levi, Valeria; Hager, Gordon L; Pecci, Adali.

In: P L o S Biology, Vol. 12, No. 3, 03.2014, p. e1001813.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Live cell imaging unveils multiple domain requirements for in vivo dimerization of the glucocorticoid receptor

AU - Presman, Diego M

AU - Ogara, M Florencia

AU - Stortz, Martín

AU - Alvarez, Lautaro D

AU - Pooley, John R

AU - Schiltz, R Louis

AU - Grøntved, Lars

AU - Johnson, Thomas A

AU - Mittelstadt, Paul R

AU - Ashwell, Jonathan D

AU - Ganesan, Sundar

AU - Burton, Gerardo

AU - Levi, Valeria

AU - Hager, Gordon L

AU - Pecci, Adali

PY - 2014/3

Y1 - 2014/3

N2 - Glucocorticoids are essential for life, but are also implicated in disease pathogenesis and may produce unwanted effects when given in high doses. Glucocorticoid receptor (GR) transcriptional activity and clinical outcome have been linked to its oligomerization state. Although a point mutation within the GR DNA-binding domain (GRdim mutant) has been reported as crucial for receptor dimerization and DNA binding, this assumption has recently been challenged. Here we have analyzed the GR oligomerization state in vivo using the number and brightness assay. Our results suggest a complete, reversible, and DNA-independent ligand-induced model for GR dimerization. We demonstrate that the GRdim forms dimers in vivo whereas adding another mutation in the ligand-binding domain (I634A) severely compromises homodimer formation. Contrary to dogma, no correlation between the GR monomeric/dimeric state and transcriptional activity was observed. Finally, the state of dimerization affected DNA binding only to a subset of GR binding sites. These results have major implications on future searches for therapeutic glucocorticoids with reduced side effects.

AB - Glucocorticoids are essential for life, but are also implicated in disease pathogenesis and may produce unwanted effects when given in high doses. Glucocorticoid receptor (GR) transcriptional activity and clinical outcome have been linked to its oligomerization state. Although a point mutation within the GR DNA-binding domain (GRdim mutant) has been reported as crucial for receptor dimerization and DNA binding, this assumption has recently been challenged. Here we have analyzed the GR oligomerization state in vivo using the number and brightness assay. Our results suggest a complete, reversible, and DNA-independent ligand-induced model for GR dimerization. We demonstrate that the GRdim forms dimers in vivo whereas adding another mutation in the ligand-binding domain (I634A) severely compromises homodimer formation. Contrary to dogma, no correlation between the GR monomeric/dimeric state and transcriptional activity was observed. Finally, the state of dimerization affected DNA binding only to a subset of GR binding sites. These results have major implications on future searches for therapeutic glucocorticoids with reduced side effects.

KW - Animals

KW - Cells, Cultured

KW - DNA

KW - Mice

KW - Protein Multimerization

KW - Protein Structure, Tertiary

KW - Receptors, Glucocorticoid

U2 - 10.1371/journal.pbio.1001813

DO - 10.1371/journal.pbio.1001813

M3 - Journal article

C2 - 24642507

VL - 12

SP - e1001813

JO - P L o S Biology

JF - P L o S Biology

SN - 1544-9173

IS - 3

ER -