Glucocorticoid receptor dimerization in the cytoplasm might be essential for nuclear localization

Su-Jun Lee; Ryota Shizu; Masahiko Negishi

doi:10.1016/j.bbrc.2021.03.071

Glucocorticoid receptor dimerization in the cytoplasm might be essential for nuclear localization

Biochem Biophys Res Commun. 2021 May 14:553:154-159. doi: 10.1016/j.bbrc.2021.03.071. Epub 2021 Mar 24.

Authors

Su-Jun Lee¹, Ryota Shizu², Masahiko Negishi³

Affiliations

¹ Pharmacogenetic Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, USA; Department of Pharmacology and Pharmacogenomics Research Center, Inje University College of Medicine, Inje University, Busan, South Korea. Electronic address: 2sujun@inje.ac.kr.
² Pharmacogenetic Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, USA; Laboratory of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
³ Pharmacogenetic Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, USA.

PMID: 33773137
DOI: 10.1016/j.bbrc.2021.03.071

Abstract

The glucocorticoid receptor (GR) plays an important role in steroid-dependent regulation of metabolism, development, and the immune response in humans. Although GR is known to be activated by the binding of glucocorticoid, the mechanism of action is poorly understood. We investigated dimerization of GR in the cytoplasm and nuclear trans-localization in response to treatment with the ligand dexamethasone. GFP-tagged GR and FLAG-tagged GR were co-expressed in COS-1 cells, and cell lysates were subjected to co-immunoprecipitation assay with anti-GFP antibody to determine their dimerization. FLAG-GR was co-precipitated with GFP-GR in the cytoplasmic fraction of COS-1 cells. Treatment with the GR agonist dexamethasone significantly decreased the cytoplasmic interaction between FLAG- and GFP-GR, and significantly increased interaction of the GRs in the nuclear fraction. The two amino acids, Pro625 and Ile628 known to be located in GR-GR dimer interface, were mutated to alanine and the influence of the mutation on dimerization, ligand-dependent nuclear localization, and transcriptional activities were determined. Mutant GR showed a dramatic decrease in interaction in the cytoplasmic fraction and no detectable nuclear translocation in the presence or absence of dexamethasone. Furthermore, luciferase assays showed that mutant GR showed no detectable transcriptional activation via the GR-responsive DNA element (GRE) compared to the wild-type. Our results suggest that GR exists as a dimer in the cytoplasm and this dimerization may be essential for GRE-mediated transcriptional activation following ligand binding.

Keywords: Dexamethasone; Glucocorticoid receptor (GR); Homodimer; Ligand binding domain; Nuclear translocation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
COS Cells
Cell Nucleus / drug effects
Cell Nucleus / metabolism*
Chlorocebus aethiops
Cytoplasm / drug effects
Cytoplasm / metabolism*
Dexamethasone / metabolism
Dexamethasone / pharmacology
Humans
Ligands
Models, Molecular
Mutation
Protein Multimerization* / drug effects
Protein Transport / drug effects
Receptors, Glucocorticoid / chemistry*
Receptors, Glucocorticoid / genetics
Receptors, Glucocorticoid / metabolism*

Substances

Ligands
Receptors, Glucocorticoid
Dexamethasone