A Quantitative Study of Internal and External Interactions of Homodimeric Glucocorticoid Receptor Using Fluorescence Cross-Correlation Spectroscopy in a Live Cell

Abstract Glucocorticoid receptor (GRα) is a well-known ligand-dependent transcription-regulatory protein. The classic view is that unliganded GRα resides in the cytoplasm, relocates to the nucleus after ligand binding, and then associates with a specific DNA sequence, namely a glucocorticoid respons...

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Autores principales:	Manisha Tiwari, Sho Oasa, Johtaro Yamamoto, Shintaro Mikuni, Masataka Kinjo
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f774e0e9eec54b11b779ba2ad8de42b3
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spelling	oai:doaj.org-article:f774e0e9eec54b11b779ba2ad8de42b32021-12-02T16:06:57ZA Quantitative Study of Internal and External Interactions of Homodimeric Glucocorticoid Receptor Using Fluorescence Cross-Correlation Spectroscopy in a Live Cell10.1038/s41598-017-04499-72045-2322https://doaj.org/article/f774e0e9eec54b11b779ba2ad8de42b32017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-04499-7https://doaj.org/toc/2045-2322Abstract Glucocorticoid receptor (GRα) is a well-known ligand-dependent transcription-regulatory protein. The classic view is that unliganded GRα resides in the cytoplasm, relocates to the nucleus after ligand binding, and then associates with a specific DNA sequence, namely a glucocorticoid response element (GRE), to activate a specific gene as a homodimer. It is still a puzzle, however, whether GRα forms the homodimer in the cytoplasm or in the nucleus before DNA binding or after that. To quantify the homodimerization of GRα, we constructed the spectrally different fluorescent protein tagged hGRα and applied fluorescence cross-correlation spectroscopy. First, the dissociation constant (Kd) of mCherry2-fused hGRα or EGFP-fused hGRα was determined in vitro. Then, Kd of wild-type hGRα was found to be 3.00 μM in the nucleus, which was higher than that in vitro. Kd of a DNA-binding-deficient mutant was 3.51 μM in the nucleus. This similarity indicated that GRα homodimerization was not necessary for DNA binding but could take place on GRE by means of GRE as a scaffold. Moreover, cytoplasmic homodimerization was also observed using GRα mutated in the nuclear localization signal. These findings support the existence of a dynamic monomer pathway and regulation of GRα function both in the cytoplasm and nucleus.Manisha TiwariSho OasaJohtaro YamamotoShintaro MikuniMasataka KinjoNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-16 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Manisha Tiwari Sho Oasa Johtaro Yamamoto Shintaro Mikuni Masataka Kinjo A Quantitative Study of Internal and External Interactions of Homodimeric Glucocorticoid Receptor Using Fluorescence Cross-Correlation Spectroscopy in a Live Cell
description	Abstract Glucocorticoid receptor (GRα) is a well-known ligand-dependent transcription-regulatory protein. The classic view is that unliganded GRα resides in the cytoplasm, relocates to the nucleus after ligand binding, and then associates with a specific DNA sequence, namely a glucocorticoid response element (GRE), to activate a specific gene as a homodimer. It is still a puzzle, however, whether GRα forms the homodimer in the cytoplasm or in the nucleus before DNA binding or after that. To quantify the homodimerization of GRα, we constructed the spectrally different fluorescent protein tagged hGRα and applied fluorescence cross-correlation spectroscopy. First, the dissociation constant (Kd) of mCherry2-fused hGRα or EGFP-fused hGRα was determined in vitro. Then, Kd of wild-type hGRα was found to be 3.00 μM in the nucleus, which was higher than that in vitro. Kd of a DNA-binding-deficient mutant was 3.51 μM in the nucleus. This similarity indicated that GRα homodimerization was not necessary for DNA binding but could take place on GRE by means of GRE as a scaffold. Moreover, cytoplasmic homodimerization was also observed using GRα mutated in the nuclear localization signal. These findings support the existence of a dynamic monomer pathway and regulation of GRα function both in the cytoplasm and nucleus.
format	article
author	Manisha Tiwari Sho Oasa Johtaro Yamamoto Shintaro Mikuni Masataka Kinjo
author_facet	Manisha Tiwari Sho Oasa Johtaro Yamamoto Shintaro Mikuni Masataka Kinjo
author_sort	Manisha Tiwari
title	A Quantitative Study of Internal and External Interactions of Homodimeric Glucocorticoid Receptor Using Fluorescence Cross-Correlation Spectroscopy in a Live Cell
title_short	A Quantitative Study of Internal and External Interactions of Homodimeric Glucocorticoid Receptor Using Fluorescence Cross-Correlation Spectroscopy in a Live Cell
title_full	A Quantitative Study of Internal and External Interactions of Homodimeric Glucocorticoid Receptor Using Fluorescence Cross-Correlation Spectroscopy in a Live Cell
title_fullStr	A Quantitative Study of Internal and External Interactions of Homodimeric Glucocorticoid Receptor Using Fluorescence Cross-Correlation Spectroscopy in a Live Cell
title_full_unstemmed	A Quantitative Study of Internal and External Interactions of Homodimeric Glucocorticoid Receptor Using Fluorescence Cross-Correlation Spectroscopy in a Live Cell
title_sort	quantitative study of internal and external interactions of homodimeric glucocorticoid receptor using fluorescence cross-correlation spectroscopy in a live cell
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/f774e0e9eec54b11b779ba2ad8de42b3
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A Quantitative Study of Internal and External Interactions of Homodimeric Glucocorticoid Receptor Using Fluorescence Cross-Correlation Spectroscopy in a Live Cell

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