A common single nucleotide polymorphism A118G of the μ opioid receptor alters its N-glycosylation and protein stability.

The amino acid sequences of the N-terminal domains of human and mouse MOPRsPotential N-linked glycosylation sites are in red. The common amino acids are highlighted in yellow. The conservative substitutions are shaded in grey.

Immunoblotting of the MOPRs in the thalami and striata of A/A mice, G/G mice and MOPR(-/-) miceBrain membranes were prepared from thalami or striata of adult female mice. Membrane proteins were resolved with 8% SDS–PAGE and immunoblotted with anti-mu C (1:5000), an affinity-purified polyclonal anti-MOR antibody we generated, as described in Materials and Methods. Each figure is a representative of the three independent experiments performed with tissues from different cohorts of female mice. Male mice yielded similar results (not shown).

Deglycosylation with PNGase F of MOPRs from thalami of A/A and G/G miceThalamic membranes of A/A or G/G mice (mixed gender: 50% of each sex) were pooled and solubilized with 2% Triton X-100. The solubilized preparations were incubated with the wheat germ lectin (WGL) Sepharose 6MB beads and the bound glycoproteins were eluted with 2 × Laemmli sample buffer. The amount of eluate was adjusted to have similar MOPR levels. The eluate was left untreated or treated with PNGase F, resolved with 8% SDS–PAGE, and immunoblotted with anti-mu C (1:5000) as described in Fig.1. This figure is from one of the two independent experiments performed with similar results.

Immunoblotting of the wild-type and A118G mutant of hMOPR (A118/N40-hMOPR and G118/D40-hMOPR, respectively) stably expressed in CHO and HEK293 cellsCHO cells were stably transfected with 3HA tagged A118/N40- or G118/D40-hMOPR cDNA in the mammalian expression vector pcDNA3. Cells from stable mixed clones were collected and the protein contents resolved with 8% SDS–PAGE and immunoblotted with monoclonal anti-HA antibody HA.11. After stripping, the same blot was then processed for immunoblotting with mouse anti-GAPDH-HRP-conjugated. Each figure is from one of two independent experiments performed with similar results.

Deglycosylation with PNGase F of the wild-type and A118G mutant of hMOPR (A118/N40-hMOPR and G118/N40-hMOPR, respectively) stably expressed in CHO cellsCells were solubilized with 2% Triton X-100. The A118/N40- or G118/D40-hMOPR was partially purified by WGL affinity chromatography, left untreated or treated with PNGase F and resolved with 8% SDS–PAGE as described in the Fig. 3 legend, and immunoblotted with HA.11 described in the Fig.4 legend. The amount of eluate was adjusted to have similar MOPR levels. Each figure shown is from one of three independent experiments with similar results.

Pulse-chase experiments of wildtype and A118G mutant of hMOPR (A118/N40-hMOPR and G118/D40-hMOPR, respectively) stably expressed in CHO cellsCells were metabolically labeled with [35S]Met/Cys at 37°C for 60 min (pulse). Medium was aspirated, and cells were incubated with complete medium (chase) for specified time periods. Cells were solubilized and immunoprecipitated with HA.11 antibody twice in tandem. Immunoprecipitated materials were resolved with SDS-PAGE and gels were dried. The gels were then exposed to a storage phosphor screen for 5 days, and the autoradiograms were acquired. (A) shows a representative autoradiogram. The intensities of [35S]-labeled protein bands were analyzed and quantified for the mature forms (B) and the precursors (C) as described in Methods. The signals for mature forms and precursors were normalized against their maximal signals observed at time points 2h and 0h, respectively. The experiment was performed three times with similar results. Each value in (B) and (C) is mean ± s.e.m. (n=3).