Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model

G protein-coupled receptors (GPCRs) are important therapeutic targets and therefore extensively studied. Like most transmembrane proteins, there has been considerable difficulty in developing reliable specific antibodies for them. To overcome this, epitope tags are often used to facilitate antibody...

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Main Authors:	Hunter, Morag Rose, Grimsey, Natasha Lillia, Glass, Michelle
Format:	Online
Language:	English
Published:	Nature Publishing Group 2016
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4895180/

id	pubmed-4895180
recordtype	oai_dc
spelling	pubmed-48951802016-06-10 Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model Hunter, Morag Rose Grimsey, Natasha Lillia Glass, Michelle Article G protein-coupled receptors (GPCRs) are important therapeutic targets and therefore extensively studied. Like most transmembrane proteins, there has been considerable difficulty in developing reliable specific antibodies for them. To overcome this, epitope tags are often used to facilitate antibody recognition in studies on fundamental receptor signalling and trafficking. In our study of cannabinoid CB1/dopamine D2 interactions we sought to generate HEK293 cells expressing FLAG-tagged D2 for use in antibody-based assays of GPCR localisation and trafficking activity, however observed that stable FLAG-hD2 expression was particularly challenging to maintain. In contrast, when expressed in cell lines expressing hCB1 robust and stable FLAG-hD2 expression was observed. We hypothesised that co-expression of CB1 might stabilise surface FLAG-hD2 expression, and therefore investigated this further. Here, we describe the observation that co-expression of either cannabinoid CB1 or CB2 receptors in HEK293 decreases the sulfation of a FLAG epitope appended at the N-terminus of the dopamine D2 receptor. Sulfation alters epitope recognition by some anti-FLAG antibodies, leading to the detection of fewer receptors, even though expression is maintained. This demonstrates that cannabinoid receptor expression modifies posttranslational processing of the FLAG-hD2 receptor, and importantly, has wider implications for the utilisation and interpretation of receptor studies involving epitope tags. Nature Publishing Group 2016-06-07 /pmc/articles/PMC4895180/ /pubmed/27273047 http://dx.doi.org/10.1038/srep27316 Text en Copyright © 2016, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Hunter, Morag Rose Grimsey, Natasha Lillia Glass, Michelle
spellingShingle	Hunter, Morag Rose Grimsey, Natasha Lillia Glass, Michelle Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model
author_facet	Hunter, Morag Rose Grimsey, Natasha Lillia Glass, Michelle
author_sort	Hunter, Morag Rose
title	Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model
title_short	Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model
title_full	Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model
title_fullStr	Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model
title_full_unstemmed	Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model
title_sort	sulfation of the flag epitope is affected by co-expression of g protein-coupled receptors in a mammalian cell model
description	G protein-coupled receptors (GPCRs) are important therapeutic targets and therefore extensively studied. Like most transmembrane proteins, there has been considerable difficulty in developing reliable specific antibodies for them. To overcome this, epitope tags are often used to facilitate antibody recognition in studies on fundamental receptor signalling and trafficking. In our study of cannabinoid CB1/dopamine D2 interactions we sought to generate HEK293 cells expressing FLAG-tagged D2 for use in antibody-based assays of GPCR localisation and trafficking activity, however observed that stable FLAG-hD2 expression was particularly challenging to maintain. In contrast, when expressed in cell lines expressing hCB1 robust and stable FLAG-hD2 expression was observed. We hypothesised that co-expression of CB1 might stabilise surface FLAG-hD2 expression, and therefore investigated this further. Here, we describe the observation that co-expression of either cannabinoid CB1 or CB2 receptors in HEK293 decreases the sulfation of a FLAG epitope appended at the N-terminus of the dopamine D2 receptor. Sulfation alters epitope recognition by some anti-FLAG antibodies, leading to the detection of fewer receptors, even though expression is maintained. This demonstrates that cannabinoid receptor expression modifies posttranslational processing of the FLAG-hD2 receptor, and importantly, has wider implications for the utilisation and interpretation of receptor studies involving epitope tags.
publisher	Nature Publishing Group
publishDate	2016
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4895180/
_version_	1613589685340209152

Sulfation of the FLAG epitope is affected by co-expression of G protein-coupled receptors in a mammalian cell model

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