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- Title
HA-MOP knockin mice express the canonical µ-opioid receptor but lack detectable splice variants.
- Authors
Fritzwanker, Sebastian; Moulédous, Lionel; Mollereau, Catherine; Froment, Carine; Burlet-Schiltz, Odile; Effah, Felix; Bailey, Alexis; Spetea, Mariana; Reinscheid, Rainer K.; Schulz, Stefan; Kliewer, Andrea
- 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. Fritzwanker et al. develop a knock-in transgenic mouse line in which the hemagglutinin epitope tag sequence is fused with the amino-terminus of the µ-opioid receptor. Their model enables more efficient immunodetection of G protein-coupled receptors.
- Subjects
G protein coupled receptors; HEMAGGLUTININ; THREONINE; SERINE; MASS spectrometry
- Publication
Communications Biology, 2021, Vol 4, Issue 1, p1
- ISSN
2399-3642
- Publication type
Article
- DOI
10.1038/s42003-021-02580-6