Heteromerization of the μ- and δ-opioid receptors produces ligand-biased antagonism and alters μ-receptor trafficking

Abstract

Heteromerization of opioid receptors has been shown to alter opioid receptor pharmacology. However, how receptor heteromerization affects the processes of endocytosis and postendocytic sorting has not been closely examined. This question is of particular relevance for heteromers of the μ-opioid receptor (MOR) and δ-opioid receptor (DOR), because the MOR is recycled primarily after endocytosis and the DOR is degraded in the lysosome. Here, we examined the endocytic and postendocytic fate of MORs, DORs, and DOR/MOR heteromers in human embryonic kidney 293 cells stably expressing each receptor alone or coexpressing both receptors. We found that the clinically relevant MOR agonist methadone promotes endocytosis of MOR but also the DOR/MOR heteromer. Furthermore, we show that DOR/MOR heteromers that are endocytosed in response to methadone are targeted for degradation, whereas MORs in the same cell are significantly more stable. It is noteworthy that we found that the DOR-selective antagonist naltriben mesylate could block both methadone- and [D-Ala2,NMe-Phe4,Gly-ol5]-enkephalin-induced endocytosis of the DOR/MOR heteromers but did not block signaling from this heteromer. Together, our results suggest that the MOR adopts novel trafficking properties in the context of the DOR/MOR heteromer. In addition, they suggest that the heteromer shows "biased antagonism," whereby DOR antagonist can inhibit trafficking but not signaling of the DOR/MOR heteromer.

Fig. 1.

Agonist-occupied MORs promote coendocytosis of DORs. A to C, HEK293 cells stably expressing FLAG-MOR (A), HA-DOR (B), or FLAG-MOR and HA-DOR (C) were incubated with antibody recognizing the N-terminal epitope tags for 30 min to label surface receptors. Cells were then treated with 1 μM MOR agonist methadone (MD) or MOR agonist DAMGO (DG) for 30 min at 37°C. Cells were fixed and stained as in Materials and Methods. In cells coexpressing DOR and MOR, images were captured consecutively from dual color channels (green and red fluorescence). Images are representative examples of multiple independent experiments. D and E, endocytosis of DOR/MOR heteromers, MOR homomers, and DOR homomers was analyzed by a biotin protection endocytosis assay in cells coexpressing FLAG-MOR and HA-DOR. Cells were biotinylated then treated with 1 μM methadone (MD) or DAMGO (DG) for 30 min. Endocytosed-protected homomeric and heteromeric receptors were separated by serial immunoprecipitation and resolved by SDS-PAGE as three populations: DOR homomers, MOR homomers, and DOR/MOR heteromers (see Materials and Methods). Total refers to the biotinylated receptor signal present in cells after initial labeling and without further manipulation; strip refers to biotinylated cells that were reacted with glutathione without other manipulations, demonstrating the efficiency with which biotin was cleaved from receptors and represents the background. Both total and strip serve as internal controls within each experiment. A representative immunoblot is shown for DOR/MOR heteromers (top), MOR homomers (middle), and DOR homomers (bottom).

Fig. 2.

DOR antagonist NTB inhibits endocytosis in the DOR/MOR cell line. HEK293 cells stably expressing FLAG-MOR (A), HA-DOR (B), or FLAG-MOR and HA-DOR (C) were incubated with antibody recognizing the N-terminal epitope tags for 30 min at 37°C to label surface receptors. The DOR antagonist NTB (1 μM) was then added, followed 20 min later by the MOR agonist methadone (MD; 1 μM) or DAMGO (DG; 1 μM). After 30 min at 37°C, cells were fixed and stained as in Materials and Methods. In cells coexpressing DOR and MOR, images were captured consecutively from dual color channels (green and red fluorescence). Images are representative examples of multiple independent experiments.

Fig. 3.

Endocytosis of the DOR/MOR heteromer can be inhibited by NTB in a dose-dependent manner. A, endocytosis of DOR/MOR heteromers and MOR homomers was analyzed by biotin protection endocytosis assay in cells coexpressing FLAG-MOR and HA-DOR. Cells were biotinylated then pretreated with different concentrations of NTB (or left untreated) for 20 min as indicated. Next, cells were treated with 1 μM methadone (MD) or DAMGO (DG) for an additional 30 min. Endocytosed protected receptors were separately resolved by SDS-PAGE as two populations, MOR homomers and DOR/MOR heteromers, after serial immunoprecipitation (see Materials and Methods). Total refers to the biotinylated receptor signal present in cells after initial labeling and without further manipulation; strip refers to biotinylated cells that were reacted with glutathione without other manipulations, demonstrating the efficiency with which biotin was cleaved from receptors and represents the background. Both total and strip serve as internal controls within each experiment. A representative immunoblot is shown for DOR/MOR heteromers (top) and MOR homomers (bottom). B, quantification of multiple experiments performed as in A. Histogram shows the endocytosis produced in the presence of antagonist as a percentage of that produced with methadone or DAMGO alone. Shown are the mean ± S.E.M. of n = three to five independent experiments (two-way ANOVA, Bonferroni post-test: **, p < 0.01; ***, p < 0.001).

Fig. 4.

Endocytosis of the DOR/MOR heteromer can be inhibited by several DOR antagonists. A, endocytosis was assessed as in Fig. 3A but in the presence of the DOR-selective antagonists, NTI and BNTX (both at 1 μM). B, quantification of A was performed as in Fig. 3B. Shown are the mean ± S.E.M. of n = three to seven independent experiments (one-way ANOVA, Dunnet post-test compared with agonist treatment without any pretreatment: *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Fig. 5.

NTB shows significantly reduced antagonism of signaling on DOR/MOR heteromers. Cells coexpressing DOR/MOR (▴), DOR only (■), or MOR only (●) were pretreated with increasing concentrations of the DOR antagonist NTB for 20 min. Calcium release caused by chimeric G protein Δ6-G_qi4-myr activation (see Materials and Methods) was measured in a Flex apparatus upon stimulation with methadone (A) or DAMGO (B) (1 μM). Maximal effects for methadone (RFU) were: MOR (1038 ± 88), DOR (565 ± 32), and DOR/MOR (843 ± 76). Maximal effects for DAMGO (RFU) were: MOR (735 ± 51), DOR (582 ± 55), and DOR/MOR (956 ± 56). Data represent means ± S.E.M. of n = three to five experiments carried out in triplicate (two-way ANOVA and Bonferroni post-test: **, p < 0.01; ***, p < 0.001 DOR/MOR compared with DOR-only cells).

Fig. 6.

Reduced antagonism of DOR/MOR heteromer signaling is NTB specific. A and B, cells coexpressing DOR/MOR were pretreated with increasing concentrations of NTB (■), BNTX (□), or NTI (▵) for 20 min and signaling in response to methadone (A) or DAMGO (B) (both 1 μM) was measured as in Fig. 5. C and D, cells expressing only DOR are shown as a control for each antagonist upon methadone (C) or DAMGO (D) (both 1 μM) stimulation. Data represent means ± S.E.M. of n = four to five experiments carried out in triplicate (two-way ANOVA and Bonferroni post-test: *, p < 0.05, **, p < 0.01, ***, p < 0.001 NTB compared with BNTX; ###, p < 0.001 NTB compared with NTI). See Table 2.

Fig. 7.

Endocytosis of the DOR/MOR heteromer leads to the degradation of the receptor complex. Postendocytic stability of DOR/MOR heteromers and MOR homomers from the same cell line were analyzed by biotin protection-degradation assay. Cells coexpressing FLAG-MOR and HA-DOR were biotinylated, then left untreated or treated with 1 μM methadone for 30, 60, 90, and 120 min before stripping. Total refers to the biotinylated receptor signal present in cells after initial labeling and without further manipulation; strip refers to biotinylated cells that were reacted with glutathione without other manipulations, demonstrating the efficiency with which biotin can be cleaved from surface receptors and represents the background. The stability of the protected endocytosed DOR/MOR heteromers (top) and MOR homomers (bottom) was assessed by serial immunoprecipitation followed by SDS-PAGE and streptavidin overlay (see Materials and Methods) at the time points stated. B, quantification of experiments in A is shown for DOR/MOR heteromers versus MOR homomers. Histogram shows the mean stability of the biotinylated endocytosed receptors relative to the endocytosed pool seen after 30 min of stimulation. Shown are the mean ± S.E.M. n = 4 to 10 independent experiments (two-way ANOVA, Bonferroni post-test: *, p < 0.05; **, p < 0.01; ***, p < 0.001).