PXD027825-1

PXD027825 is an original dataset announced via ProteomeXchange.

Title	HA-MOP knockin mice express the canonical µ-opioid receptor but lack detectable splice variants
Description	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.
HostingRepository	PRIDE
AnnounceDate	2022-12-07
AnnouncementXML	Submission_2022-12-07_07:15:12.964.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	CarineFroment
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	phosphorylated residue
Instrument	LTQ Orbitrap Velos

Revision	Datetime	Status	ChangeLog Entry
0	2021-08-09 08:46:19	ID requested
⏵ 1	2022-12-07 07:15:13	announced
2	2023-11-14 08:53:28	announced	2023-11-14: Updated project metadata.

submitter keyword: brain,Mouse, pharmacology, MOP splice variants, opioid receptor family, NanoLC-MS/MS CID/ETD, Phosphorylation

StefanSchulz
contact affiliation	Department of Pharmacology and Toxicology, Jena UniversityHospital - Friedrich Schiller University Jena, Drackendorfer Straße 1, Jena, Germany
contact email	stefan.schulz@med.uni-jena.de
lab head
CarineFroment
contact affiliation	IPBS-CNRS
contact email	carine.froment@ipbs.fr
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD027825
     1. Label: PRIDE project
     2. Name: HA-MOP knockin mice express the canonical µ-opioid receptor but lack detectable splice variants