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. 2020 Sep 30;25(19):4493.
doi: 10.3390/molecules25194493.

Heteromerization of Endogenous Mu and Delta Opioid Receptors Induces Ligand-Selective Co-Targeting to Lysosomes

Lyes Derouiche  1 , Florian Pierre  1 , Stéphane Doridot  2 , Stéphane Ory  1 , Dominique Massotte  1
Affiliations

Heteromerization of Endogenous Mu and Delta Opioid Receptors Induces Ligand-Selective Co-Targeting to Lysosomes

Lyes Derouiche et al. Molecules. .

Abstract

Increasing evidence indicates that native mu and delta opioid receptors can associate to form heteromers in discrete brain neuronal circuits. However, little is known about their signaling and trafficking. Using double-fluorescent knock-in mice, we investigated the impact of neuronal co-expression on the internalization profile of mu and delta opioid receptors in primary hippocampal cultures. We established ligand selective mu-delta co-internalization upon activation by 1-[[4-(acetylamino)phenyl]methyl]-4-(2-phenylethyl)-4-piperidinecarboxylic acid, ethyl ester (CYM51010), [d-Ala2, NMe-Phe4, Gly-ol5]enkephalin (DAMGO), and deltorphin II, but not (+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80), morphine, or methadone. Co-internalization was driven by the delta opioid receptor, required an active conformation of both receptors, and led to sorting to the lysosomal compartment. Altogether, our data indicate that mu-delta co-expression, likely through heteromerization, alters the intracellular fate of the mu opioid receptor, which provides a way to fine-tune mu opioid receptor signaling. It also represents an interesting emerging concept for the development of novel therapeutic drugs and strategies.

Keywords: delta opioid receptor; heteromer; internalization; lysosomes; mu opioid receptor; primary hippocampal culture.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Mu and delta opioid receptors co-internalize upon CYM51010 activation in primary hippocampal cultures. (A) Representative confocal images showing mu-mCherry and delta-eGFP fluorescence localized at the plasma membrane (arrowheads) under basal condition or internalized in vesicle-like structures 15 or 60 min after CYM51010 (400 nM) application (arrows). Scale bar = 10 μm. (B) Receptor internalization induced by CYM51010 application (400 nM) expressed as a ratio of membrane-associated versus intracellular fluorescence densities for each receptor. Two-way ANOVA Ftreatment (3, 94) = 17.98; p < 0.0001. Freceptor (1, 94) = 1.06; Finteraction (3, 94) = 0.54. Tukey’s post hoc test for mu-mCherry, *** p < 0.001, ** p = 0.01. Tukey’s post hoc test for delta-eGFP, * p = 0.02, ** p = 0.002, *** p < 0.001; n = 10 to 20 neurons per group from at least three independent cultures. (C) Subcellular redistribution of mu–delta heteromers expressed as a ratio of membrane-associated versus intracellular fluorescence densities for co-localized mu-mCherry and delta-eGFP receptors. One-way ANOVA (F (3, 48) = 13.64; p < 0.0001) followed by multiple-comparison Dunn’s post hoc test. * p = 0.03, *** p < 0.001; n = 10–20 neurons per group from at least three independent cultures. (D) Fraction of cytoplasmic mu-delta heteromers expressed as the percentage of mu-mCherry and delta-eGFP overlapping objects detected in vesicle-like structures at the different times. Kruskal Wallis test (p < 0.0001) followed with multiple comparisons Dunn’s test. ** p < 0.01, 30 min vs basal, *** p < 0.001 15 min and 60 min vs basal. N = 10 to 20 neurons per group from at least 3 independent cultures.
Figure 2
Figure 2
Mu and delta opioid receptors co-localize in the lysosomal compartment upon CYM51010 activation in primary hippocampal cultures. (A) Representative confocal images showing mu-mCherry–delta-eGFP colocalization with LAMP1 immunoreactive compartment under basal conditions or 60 min after CYM51010 application (400 nM). Scale bar = 10 μm (inset scale bar = 2.5 μm). (B) Drug treatment induces statistically significant increase in the amount of colocalization of mu-mCherry/delta-eGFP colocalization with LAMP1 labeling. Two-way ANOVA Fdrug treatment (1, 49) = 62.70; p < 0.0001. Freceptor (1, 49) = 2.12, p = 0.15; Finteraction (1, 49) = 3.65, p = 0.2. Tukey’s post hoc test: *** p < 0.001 for both mu-mCherry and delta-eGFP; n = 10–20 neurons per group from at least three independent cultures.
Figure 3
Figure 3
CYM51010 internalization of mu or delta opioid receptors in primary hippocampal cultures from mice deficient for one of the receptors. (A) Representative confocal images showing that mu-mCherry is associated with the plasma membrane (arrows) in basal conditions and 30 min after CYM51010 (400 nM) addition in delta-knockout (KO) mice. Scale bar = 10 μm. (B) Mu-mCherry internalization induced by CYM51010 application expressed as a ratio of membrane-associated versus intracellular fluorescence densities. Mann–Whitney test, p = 0.20; n = 13 to 20 neurons per group from at least three independent cultures. (C) Representative confocal images showing that delta-eGFP is predominantly associated with the plasma membrane in basal conditions (arrows) in mu-KO mice, whereas the association is mostly intracellular at 30 and 60 min after CYM51010 (400 nM) addition (arrowheads). Scale bar = 10 μm. (D) Delta-eGFP internalization induced by CYM51010 application expressed as a ratio of membrane-associated versus intracellular fluorescence densities. Kruskal–Wallis test (p < 0.0001) followed by Dunn’s multiple comparison test. Significant differences after multiple-comparison tests are expressed as p < 0.001 (***) compared to basal group; n = 9–20 neurons per group from at least three independent cultures.
Figure 4
Figure 4
Antagonist pretreatment abolishes mu–delta opioid receptor co-internalization by CYM51010 in primary hippocampal cultures. (A) Representative confocal images showing mu-mCherry predominant localization at the plasma membrane and delta-eGFP extensive internalization after pretreatment with the mu antagonist β-FNA (200 nM) for 15 min, followed by incubation for 60 min with CYM51010 (400 nM). Scale bar = 10 μm. (B) Representative confocal images showing mu-mCherry and delta-eGFP predominant localization at the plasma membrane after pretreatment with delta antagonist naltrindole (200 nM) (NTI) for 15 min, followed by incubation for 60 min with CYM51010 (400 nM). Scale bar = 10 μm. (C) Pretreatment with the mu antagonists β-FNA or CTAP (200 nM) blocks mu-mCherry but not delta-eGFP internalization, whereas pretreatment with the delta antagonists naltrindole (NTI) and tic-deltorphin (tic) (200 nM) prevent internalization of both mu-mCherry and delta-eGFP. Receptor internalization is expressed as a ratio of membrane-associated versus intracellular fluorescence densities for each receptor. Two-way ANOVA F treatment (5, 104) = 4.73, p = 0.0001. Freceptor (1, 104) = 0.1, p = 0.84; Finteraction (5, 100) = 1.96; p = 0.0006. Multiple comparisons with Tukey’s post hoc test, * p = 0.04 basal vs. CYM51010 for mu-mCherry, * p = 0.04 basal vs. CYM51010, * p = 0.04 basal vs. β-FNA, * p = 0.04 basal vs. CTAP; n = 9–20 neurons per group from at least three independent cultures. (D) Mu-mCherry/delta-eGFP co-internalization is prevented by treatment with either mu or delta antagonists. Percentage of colocalized receptors in the cytoplasm after drug treatment. The fraction of cytoplasmic mu–delta heteromers is expressed as the percentage of mu-mCherry and delta-eGFP overlapping objects detected in vesicle-like structures 60 min after CYM51010 application. One-way ANOVA (p < 0.0001) followed by multiple-comparison Dunnett’s test. Significant differences after multiple comparisons tests are expressed as *** p < 0.001 when compared to basal group and ### p < 0.001 when compared to CYM51010 without antagonists; n = 9–20 neurons per group from at least three independent cultures.
Figure 5
Figure 5
Mu–delta opioid receptor co-internalization is ligand-selective in primary hippocampal cultures. Representative confocal images showing mu-mCherry and delta-eGFP fluorescence at the plasma membrane (arrowheads) under basal conditions or co-internalized in vesicle-like structures (arrows) 30 min after DAMGO (1 μM) or deltorphin II (100 nM), but not SNC80 (100 nM), morphine (10 μM), or methadone (1 μM). Scale bar = 10 μm.
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References

    1. Gaveriaux-Ruff C., Kieffer B.L. Opioid receptor genes inactivated in mice: The highlights. Neuropeptides. 2002;36:62–71. doi: 10.1054/npep.2002.0900. - DOI - PubMed
    1. Feng Y., He X., Yang Y., Chao D., H Lazarus L., Xia Y. Current research on opioid receptor function. Curr. Drug Targets. 2012;13:230–246. doi: 10.2174/138945012799201612. - DOI - PMC - PubMed
    1. Charbogne P., Kieffer B.L., Befort K. 15 years of genetic approaches in vivo for addiction research: Opioid receptor and peptide gene knockout in mouse models of drug abuse. Pt BNeuropharmacology. 2014;76:204–217. doi: 10.1016/j.neuropharm.2013.08.028. - DOI - PMC - PubMed
    1. Zhang Z., Pan Z.Z. Synaptic mechanism for functional synergism between delta- and mu-opioid receptors. J. Neurosci. 2010;30:4735–4745. doi: 10.1523/JNEUROSCI.5968-09.2010. - DOI - PMC - PubMed
    1. Gendron L., Mittal N., Beaudry H., Walwyn W. Recent advances on the delta opioid receptor: From trafficking to function. Br. J. Pharmacol. 2015;172:403–419. doi: 10.1111/bph.12706. - DOI - PMC - PubMed