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.
Highlights
G protein-coupled receptors (GPCRs) are notoriously difficult to detect in native tissues
HA-MOP knockin mice exhibit reduced antinociceptive responses to morphine, which are similar to heterozygous MOP knockout (MOP+/−) mice (Supplementary Fig. 2f)
HA-MOP was detected in western blots using both an anti-HA antibody directed to the N-terminal tag (Fig. 1b, left panel) as well the well-characterized UMB-3 antibody directed to the carboxyl-terminal tail of the canonical MOP receptor (Fig. 1b, right panel), while no band was detected in WT mice
Summary
G protein-coupled receptors (GPCRs) are notoriously difficult to detect in native tissues. Using polyclonal antisera expression of two C-terminal MOP splice variants (MOP1B4 and MOP1C) could be visualized in various areas of the mouse brain[3,8,9]. Several attempts were made to match specific isoforms with particular opioid compounds or link them to selected physiological opioid effects These studies mostly used isoform-specific RNA knock-down approaches, suffering from inherent limitations of unknown efficacy while working against a background of abundantly expressed canonical receptors. At least 30 C-terminal MOP splice variants have been postulated (mouse, rat, human) based on mRNA sequencing data, out of which 15 are of murine origin. The lack of validated high-affinity antibodies and the low abundance of MOP protein expression in native tissues have made it difficult to conclusively verify the existence of postulated variant MOP isoforms. Our results suggest that C-terminal variants of MOP either do not exist or occur at such low abundance that their physiological significance is questionable
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