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HA-MOP knockin mice express the canonical µ-opioid receptor but lack detectable splice variants

Fritzwanker, S; Moulédous, L; Mollereau, C; Froment, C; Burlet-Schiltz, O; Effah, F; Bailey, A; Spetea, M; Reinscheid, RK; Schulz, S; et al. Fritzwanker, S; Moulédous, L; Mollereau, C; Froment, C; Burlet-Schiltz, O; Effah, F; Bailey, A; Spetea, M; Reinscheid, RK; Schulz, S; Kliewer, A (2021) HA-MOP knockin mice express the canonical µ-opioid receptor but lack detectable splice variants. Communications Biology, 4 (1). p. 1070. ISSN 2399-3642 https://doi.org/10.1038/s42003-021-02580-6
SGUL Authors: Bailey, Alexis

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Abstract

G protein-coupled receptors (GPCRs) are notoriously difficult to detect in native tissues. In an effort to resolve this problem, we have developed a novel mouse model by fusing the hemagglutinin (HA)-epitope tag sequence to the amino-terminus of the µ-opioid receptor (MOP). Although HA-MOP knock-in mice exhibit reduced receptor expression, we found that this approach allowed for highly efficient immunodetection of low abundant GPCR targets. We also show that the HA-tag facilitates both high-resolution imaging and immunoisolation of MOP. Mass spectrometry (MS) confirmed post-translational modifications, most notably agonist-selective phosphorylation of carboxyl-terminal serine and threonine residues. MS also unequivocally identified the carboxyl-terminal 387LENLEAETAPLP398 motif, which is part of the canonical MOP sequence. Unexpectedly, MS analysis of brain lysates failed to detect any of the 15 MOP isoforms that have been proposed to arise from alternative splicing of the MOP carboxyl-terminus. For quantitative analysis, we performed multiple successive rounds of immunodepletion using the well-characterized rabbit monoclonal antibody UMB-3 that selectively detects the 387LENLEAETAPLP398 motif. We found that >98% of HA-tagged MOP contain the UMB-3 epitope indicating that virtually all MOP expressed in the mouse brain exhibit the canonical amino acid sequence.

Item Type: Article
Additional Information: © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
SGUL Research Institute / Research Centre: Academic Structure > Institute of Medical & Biomedical Education (IMBE)
Academic Structure > Institute of Medical & Biomedical Education (IMBE) > Centre for Biomedical Education (INMEBE)
Journal or Publication Title: Communications Biology
ISSN: 2399-3642
Language: en
Dates:
DateEvent
December 2021Published
14 September 2021Published Online
17 August 2021Accepted
Publisher License: Creative Commons: Attribution 4.0
Projects:
Project IDFunderFunder ID
2019_A68Else Kröner-Fresenius-Stiftunghttp://dx.doi.org/10.13039/501100003042
AMSP 03Interdisciplinary Center for Clinical Research JenaUNSPECIFIED
SFB/TR166-TPC5Deutsche Forschungsgemeinschafthttp://dx.doi.org/10.13039/501100001659
SCHU924/15-1Deutsche Forschungsgemeinschafthttp://dx.doi.org/10.13039/501100001659
SCHU924/18-1Deutsche Forschungsgemeinschafthttp://dx.doi.org/10.13039/501100001659
ANR-10-INBS-08French Ministry of ResearchUNSPECIFIED
I2463-B21Austrian Science Fundhttp://dx.doi.org/10.13039/501100002428
URI: https://openaccess.sgul.ac.uk/id/eprint/113654
Publisher's version: https://doi.org/10.1038/s42003-021-02580-6

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