Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding

Steven Timmermans; Nicolette J.D. Verhoog; Kelly Van Looveren; Sylviane Dewaele; Tino Hochepied; Melanie Eggermont; Barbara Gilbert; Anne Boerema de Munck; Tineke Vanderhaeghen; Joke Vanden Berghe; Natalia Garcia Gonzalez; Jolien Vandewalle; Yehudi Bloch; Mathias Provost; Savvas N. Savvides; Karolien De Bosscher; Wim Declercq; Robbert J. Rottier; Ann Louw; Claude Libert

doi:10.1016/j.jbc.2022.101574

Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding

Steven Timmermans, Nicolette J.D. Verhoog, Kelly Van Looveren, Sylviane Dewaele, Tino Hochepied, Melanie Eggermont, Barbara Gilbert, Anne Boerema de Munck, Tineke Vanderhaeghen, Joke Vanden Berghe, Natalia Garcia Gonzalez, Jolien Vandewalle, Yehudi Bloch, Mathias Provost, Savvas N. Savvides, Karolien De Bosscher, Wim Declercq, Robbert J. Rottier, Ann Louw, Claude Libert^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

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Abstract

The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GR^dim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GR^D/D) have previously helped to define the functions of GR monomers and dimers. Since GR^D/D retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GR^D+L/D+L mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GR^L/L mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and K_d values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome.

Original language	English
Article number	101574
Journal	Journal of Biological Chemistry
Volume	298
Issue number	2
DOIs	https://doi.org/10.1016/j.jbc.2022.101574
Publication status	Published - 1 Feb 2022

Bibliographical note

Funding Information:
We wish to thank Prof. Jan Tuckermann for providing GRD/D and GR-/- mice. We are thankful to animal house caretakers for animal care. We acknowledge the VIB Nucleomics Core for the RNA sequencing. We would like to thank the VIB Bio-Imaging Core for training, support, and access to the instrument park. Research in the author's laboratories was funded by the Agency for Innovation of Science and Technology in Flanders (IWT), the Research Council of Ghent University (GOA Program), the Research Foundation Flanders (FWO-Vlaanderen), and Flanders Institute for Biotechnology (VIB).

Funding Information:
Acknowledgments—We wish to thank Prof. Jan Tuckermann for providing GRD/D and GR−/− mice. We are thankful to animal house caretakers for animal care. We acknowledge the VIB Nucleomics Core for the RNA sequencing. We would like to thank the VIB Bio-Imaging Core for training, support, and access to the instrument park. Research in the author’s laboratories was funded by the Agency for Innovation of Science and Technology in Flanders (IWT), the Research Council of Ghent University (GOA Program), the Research Foundation Flanders (FWO-Vlaanderen), and Flanders Institute for Biotechnology (VIB).

Publisher Copyright:
© 2022 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand bindingFinal published version, 3.13 MBLicence: CC BY

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Timmermans, S., Verhoog, N. J. D., Van Looveren, K., Dewaele, S., Hochepied, T., Eggermont, M., Gilbert, B., de Munck, A. B., Vanderhaeghen, T., Vanden Berghe, J., Gonzalez, N. G., Vandewalle, J., Bloch, Y., Provost, M., Savvides, S. N., De Bosscher, K., Declercq, W., Rottier, R. J., Louw, A., & Libert, C. (2022). Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding. Journal of Biological Chemistry, 298(2), Article 101574. https://doi.org/10.1016/j.jbc.2022.101574

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title = "Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding",

abstract = "The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GRdim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GRD/D) have previously helped to define the functions of GR monomers and dimers. Since GRD/D retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GRD+L/D+L mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GRL/L mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and Kd values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome.",

author = "Steven Timmermans and Verhoog, {Nicolette J.D.} and {Van Looveren}, Kelly and Sylviane Dewaele and Tino Hochepied and Melanie Eggermont and Barbara Gilbert and {de Munck}, {Anne Boerema} and Tineke Vanderhaeghen and {Vanden Berghe}, Joke and Gonzalez, {Natalia Garcia} and Jolien Vandewalle and Yehudi Bloch and Mathias Provost and Savvides, {Savvas N.} and {De Bosscher}, Karolien and Wim Declercq and Rottier, {Robbert J.} and Ann Louw and Claude Libert",

note = "Funding Information: We wish to thank Prof. Jan Tuckermann for providing GRD/D and GR-/- mice. We are thankful to animal house caretakers for animal care. We acknowledge the VIB Nucleomics Core for the RNA sequencing. We would like to thank the VIB Bio-Imaging Core for training, support, and access to the instrument park. Research in the author's laboratories was funded by the Agency for Innovation of Science and Technology in Flanders (IWT), the Research Council of Ghent University (GOA Program), the Research Foundation Flanders (FWO-Vlaanderen), and Flanders Institute for Biotechnology (VIB). Funding Information: Acknowledgments—We wish to thank Prof. Jan Tuckermann for providing GRD/D and GR−/− mice. We are thankful to animal house caretakers for animal care. We acknowledge the VIB Nucleomics Core for the RNA sequencing. We would like to thank the VIB Bio-Imaging Core for training, support, and access to the instrument park. Research in the author{\textquoteright}s laboratories was funded by the Agency for Innovation of Science and Technology in Flanders (IWT), the Research Council of Ghent University (GOA Program), the Research Foundation Flanders (FWO-Vlaanderen), and Flanders Institute for Biotechnology (VIB). Publisher Copyright: {\textcopyright} 2022 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).",

year = "2022",

month = feb,

day = "1",

doi = "10.1016/j.jbc.2022.101574",

language = "English",

volume = "298",

journal = "Journal of Biological Chemistry",

issn = "0021-9258",

publisher = "American Society for Biochemistry and Molecular Biology Inc.",

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Timmermans, S, Verhoog, NJD, Van Looveren, K, Dewaele, S, Hochepied, T, Eggermont, M, Gilbert, B, de Munck, AB, Vanderhaeghen, T, Vanden Berghe, J, Gonzalez, NG, Vandewalle, J, Bloch, Y, Provost, M, Savvides, SN, De Bosscher, K, Declercq, W, Rottier, RJ, Louw, A & Libert, C 2022, 'Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding', Journal of Biological Chemistry, vol. 298, no. 2, 101574. https://doi.org/10.1016/j.jbc.2022.101574

TY - JOUR

T1 - Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding

AU - Timmermans, Steven

AU - Verhoog, Nicolette J.D.

AU - Van Looveren, Kelly

AU - Dewaele, Sylviane

AU - Hochepied, Tino

AU - Eggermont, Melanie

AU - Gilbert, Barbara

AU - de Munck, Anne Boerema

AU - Vanderhaeghen, Tineke

AU - Vanden Berghe, Joke

AU - Gonzalez, Natalia Garcia

AU - Vandewalle, Jolien

AU - Bloch, Yehudi

AU - Provost, Mathias

AU - Savvides, Savvas N.

AU - De Bosscher, Karolien

AU - Declercq, Wim

AU - Rottier, Robbert J.

AU - Louw, Ann

AU - Libert, Claude

N1 - Funding Information: We wish to thank Prof. Jan Tuckermann for providing GRD/D and GR-/- mice. We are thankful to animal house caretakers for animal care. We acknowledge the VIB Nucleomics Core for the RNA sequencing. We would like to thank the VIB Bio-Imaging Core for training, support, and access to the instrument park. Research in the author's laboratories was funded by the Agency for Innovation of Science and Technology in Flanders (IWT), the Research Council of Ghent University (GOA Program), the Research Foundation Flanders (FWO-Vlaanderen), and Flanders Institute for Biotechnology (VIB). Funding Information: Acknowledgments—We wish to thank Prof. Jan Tuckermann for providing GRD/D and GR−/− mice. We are thankful to animal house caretakers for animal care. We acknowledge the VIB Nucleomics Core for the RNA sequencing. We would like to thank the VIB Bio-Imaging Core for training, support, and access to the instrument park. Research in the author’s laboratories was funded by the Agency for Innovation of Science and Technology in Flanders (IWT), the Research Council of Ghent University (GOA Program), the Research Foundation Flanders (FWO-Vlaanderen), and Flanders Institute for Biotechnology (VIB). Publisher Copyright: © 2022 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PY - 2022/2/1

Y1 - 2022/2/1

N2 - The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GRdim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GRD/D) have previously helped to define the functions of GR monomers and dimers. Since GRD/D retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GRD+L/D+L mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GRL/L mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and Kd values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome.

AB - The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GRdim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GRD/D) have previously helped to define the functions of GR monomers and dimers. Since GRD/D retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GRD+L/D+L mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GRL/L mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and Kd values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome.

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U2 - 10.1016/j.jbc.2022.101574

DO - 10.1016/j.jbc.2022.101574

M3 - Article

C2 - 35007536

AN - SCOPUS:85123681069

SN - 0021-9258

VL - 298

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 2

M1 - 101574

ER -

Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding

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