mu-Opioid receptor cell surface expression is regulated by its direct interaction with Ribophorin I

Abstract

The trafficking of the mu-opioid receptor (MOR), a member of the rhodopsin G protein-coupled receptor (GPCR) family, can be regulated by interaction with multiple cellular proteins. To determine the proteins involved in receptor trafficking, using the targeted proteomic approach and mass spectrometry analysis, we have identified that Ribophorin I (RPNI), a component of the oligosaccharide transferase complex, could directly interact with MOR. RPNI can be shown to participate in MOR export by the intracellular retention of the receptor after small interfering RNA knockdown of endogenous RPNI. Overexpression of RPNI rescued the surface expression of the MOR 344KFCTR348 deletion mutant independent of calnexin. Furthermore, RPNI regulation of MOR trafficking is dependent on the glycosylation state of the receptor, as reflected by the inability of overexpression of RPNI to affect the trafficking of the N-glycosylation-deficient mutants, or GPCRs that have minimal glycosylation sites. Hence, this novel RPNI chaperone activity is a consequence of N-glycosylation-dependent direct interaction with MOR.

Fig. 1.

Identification of RPNI as a MOR-associated protein. A, LC MS/MS analysis of the protein band corresponding to RPNI. The bold and underlined sequences represent the RPNI amino acid sequences identified using tandem mass spectrometry after in-gel digestion of the protein-staining band. The protein sequence refers to gi|31543605. B, coimmunoprecipitation of FLAG-RPNI and HA-MOR. a, FLAG-RPNI was detected after HA-MOR was immunoprecipitated (IP) from the cell lysate; b, HA-MOR was detected after FLAG-RPNI was immunoprecipitated from the cell lysates; c, the expression level of FLAG-RPNI was determined in 1/20 of the total lysate used in IP experiments; d, MOR was detected with rabbit polyclonal antibodies directed against the C-tail of MOR after HA-MOR was immunoprecipitated from the cell lysates. C, co-IP of FLAG-RPNI and HA-MOR in mixed cells that individually express FLAG-RPNI and HA-MOR. Lane 1, cells only express HA-MOR as negative control; lane 2, mixed cells that individually express FLAG-RPNI and HA-MOR; lane 3, cells that express both FLAG-RPNI and HA-MOR as positive control. D, gel overlay of RPNI with MOR. a, in vitro translation products of FLAG-RPNI, as indicated by Western analysis using anti-FLAG antibody; b, gel overlay of FLAG-RPNI on membranes containing SDS-PAGE separated N2A extract from cells expressing or not expressing HA-MOR; c, Western analysis of MOR expression.

Fig. 2.

siRNA-mediated knockdown of RPNI decreased cell surface expression of MOR in N2A cells. N2A cells were transfected with GFP-tagged vector, GFP-tagged scramble RPNI siRNA or GFP-tagged RPNI siRNA for 48 h. A, efficiency of siRNA used on knocking down endogenous RPNI. siRNA3 significantly reduced endogenous RPNI expression compared with others. β-Actin was used as control and transfection efficiency was controlled as described under Results. B, transfection of cells with 0.1 μg of scramble GFP-RPNI siRNA did not affect the expression of MOR on cell surface. C, transfection of 0.1 μg of GFP-RPNI siRNA decreased the expression of MOR on cell surface. In both B and C: a, HA-MOR was stained by the mouse anti-HA monoclonal antibody and detected with the goat anti-mouse antibodies conjugated with Alexa Fluor 594; b, merge MOR with GFP-tagged RPNI scramble siRNA or GFP-tagged siRNA. Scale bar equals 10 μm. D, a, expression level of MOR in 1/20 fraction of each sample, which showed total MOR of each sample are almost the same; b, FACS analyses of cell surface MOR expression in N2A cells transfected with RPNI siRNA as described under Materials and Methods. The bars represent the average ± S.E.M. from three separated experiments carried out in triplicate. ^**, p < 0.01 compared with cells transfected with vector.

Fig. 3.

Glycosylation state of MOR when overexpressing or knocking down RPNI. A, Western blot to detect MOR, MOR after EndoH digestion, and MOR after PNGase F digestion. a, MOR in extract from N2A-MOR transfected with vector, after EndoH digestion, and after PNGase F digestion; b, extract from N2A-MOR overexpressing RPNI, after EndoH digestion, and after PNGase F digestion; c, MOR in extract from N2A-MOR transfected with RPNI siRNA, MOR after EndoH digestion, and MOR after PNGase F digestion. B, gel overlay of RPNI with MOR, EndoH-digested MOR, and PNGase F-digested MOR. a, Western blot to detect N2A extract from cells expressing MOR, EndoH-digested MOR, and PNGase F-digested MOR. b, gel overlay of in vitro translated FLAG-RPNI on membrane containing SDS-PAGE separated N2A extract from cells expressing MOR, EndoH-digested MOR, and PNGase F-digested MOR were carried out as described under Materials and Methods. C, gel overlay of in vitro-translated FLAG-RPNI with MOR C2 mutant or MOR5ND mutant. a, gel overlay of FLAG-RPNI on membranes containing SDS-PAGE separated N2A extract from cells expressing MOR C2 mutant or MOR5ND mutant; b, Western analysis was used to detect different glycosylated forms of MOR C2 and MOR5ND mutants using anti-MOR C tail polyclonal antibody.

Fig. 4.

RPNI rescues C2 cell surface expression but not five N-glycosylation-site mutants of MOR. A, RPNI up-regulate the cell surface expression of export deficient MOR C2 mutant. a, wild-type (WT) N2A cells were cotransfected with HA-MOR C2 mutant and FLAG-RPNI. C2 mutant expression was detected by rabbit anti-HA antibody conjugated with Alexa Fluor 488; b, merge of MOR C2 mutant and FLAG-RPNI that was determined by staining with mouse anti-FLAG monoclonal antibody and detected with goat anti-mouse antibodies conjugated with Alexa Fluor 594. Scale bar equals 10 μm. B, FACS analyses of C2 cell surface expression in the presence of naloxone and overexpression of RPNI. N2A cells were transiently transfected with vector or 0.5 μg of RPNI or 1 μg of RPNI and MOR C2 mutant. The bars represent averages ± S.E.M. of immunofluorescence in cells treated and not treated with 1 μM naloxone after transfection in n = 3 experiments. C, RPNI failed to regulate the export of five N-glycosylation-site mutants of MOR (MOR5ND). a, WT N2A cells were cotransfected with (His)6-MOR5ND mutant and FLAG-RPNI. MOR5ND mutant expression was detected by rabbit anti-(His)6 antibody conjugated with Alexa Fluor 488; b, merge of MOR5ND mutant and FLAG-RPNI that was determined by staining with mouse anti-FLAG monoclonal antibody and detected with goat anti-mouse antibodies conjugated with Alexa Fluor 594. Scale bar, 10 μm. D, inability of RPNI or naloxone to rescue cell surface expression of MOR5ND as determined by FACS analyses. The bars represent the averages ± S.E.M. of three separate experiments carried out in triplicate.

Fig. 5.

Inability of RPNI to affect the export of five single N-glycosylationsite mutants. A, N2A cells were transiently transfected either with MOR mutant or MOR for 48 h before cell surface receptor levels were determined by FACS analyses, as described under Materials and Methods. The bars represent the averages ± S.E.M. of three separate experiments carried out in triplicate. B, N2A cells were transiently transfected either with MOR mutant and vector or with MOR mutant and 1 μg of RPNI for 48 h before cell surface receptor levels were determined by FACS analyses, as described under Materials and Methods. The □, , and bars represent the averages ± S.E.M. in three separated experiments carried out in triplicate in the absence and presence of RPNI overexpression or treatment with 1 μM naloxone, respectively; ^**, p < 0.01 compared with cells transfected with vector.

Fig. 6.

RPNI did not increase C2 and calnexin interaction. N2A cells were transfected either with HA-MOR and vector (A, lane 1), HA-C2 mutant and FLAG-RPNI (A, lane 2), HA-C2 mutant and vector treated with naloxone (A, lane 3), HA-C2 mutant and vector treated with 10 μM MG132 16 h before harvesting (A, lane 4), or HA-C2 mutant and vector (A, lane 5). Immunoprecipitation and Western analyses were carried out as described under Materials and Methods. B, relative intensities of C2 mutant levels using HA-MOR as a reference, and the amount of β-actin immunoactivity in each lane was used for loading control. The bars represent the average ± S.E.M. of three separate experiments. C, FACS analyses of cell surface MOR C2 mutant after overexpression of RPNI, or the addition of MG132. D, relative intensities of calnexin co-IP with HA-MOR using HA-MOR immunoreactivity as a reference, and β-actin immunoactivity in each lane was used for loading control. The bars represent the average ± S.E.M. of three separate experiments.

Fig. 7.

Interaction between RPNI and DOR and KOR affects receptor export. A, co-IP of RPNI with DOR. N2A cells were transiently transfected either with vector and HA-DOR, vector and FLAG-RPNI, or FLAG-RPNI and HA-DOR, as described under Materials and Methods. a, RPNI expression was detected with mouse anti-FLAG antibody in 1/20 total cell lysates; b, RPNI was detected with rabbit anti-FLAG after HA-DOR was immunoprecipitated from the cell lysates using mouse anti-HA antibodies; c, the amount of HA-DOR immunoprecipitated and loaded on each lane was demonstrated after stripping the blots used to detect RPNI. B, increase of DOR export by RPNI. N2A cells were transiently transfected either with HA-DOR and vector, HA-DOR and RPNI, or HA-DOR and RPNI siRNA for 48 h before cell-surface receptor levels were determined by FACS analyses, as described under Materials and Methods. The bars represent the averages ± S.E.M. of n = 3 experiments carried out in triplicate. ^*, p < 0.05; ^**, p < 0.01 compared with cells transfected with vector. C, co-IP of RPNI with KOR. N2A cells were transfected either with vector and HA-KOR, vector and FLAG-RPNI, or FLAG-RPNI and HA-KOR, as described under Materials and Methods. a, RPNI expression was detected with mouse anti-FLAG antibody in 1/20 total cell lysates; b, RPNI was detected with rabbit anti-FLAG after HA-KOR was immunopreciptated from the cell lysates using mouse anti-HA antibodies; c, the amount of HA-KOR immunoprecipitated and loaded on each lane was demonstrated after stripping the blots used to detect RPNI. D, decrease in KOR export after RPNI knockdown. N2A cells were transiently transfected either with HA-KOR and vector, HA-KOR and FLAG-RPNI, or HA-KOR and RPNI siRNA for 48 h before cell surface receptor levels were determined by FACS analyses, as described under Materials and Methods. The bars represent the averages ± S.E.M. of n = 3 experiments carried out in triplicate. ^*, p < 0.05 compared with cells transfected with vector.