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In the current study, we investigated the role of receptor phosphorylation and β-arrestins in δ-opioid receptor (DOR) signaling and trafficking by using a DOR mutant in which all Ser/Thr residues in the C terminus were mutated to Ala (DTS). We demonstrated that the DOR agonist D-[Pen²,Pen5]enkephalin could induce receptor internalization and adenylyl cyclase (AC) desensitization of DTS, but with comparatively slower kinetics than those observed with wild type DOR. Blockade of the internalization of DTS by the dominant-negative mutant dynamin, dynamin K44E, did not affect AC desensitization. However, depletion of β-arrestins almost totally blocked both internalization and AC desensitization of DTS. A BRET assay suggested that DOR phosphorylation promotes receptor selectivity for β-arrestin 2 over β-arrestin 1. Furthermore, in mouse embryonic fibroblast (MEF) cells lacking either β-arrestin 1 (βarr1-/-) or β-arrestin 2 (βarr2-/-), agonist-induced DTS desensitization and internalization were similar to that observed in wild type MEFs. In contrast, although DOR internalization decreased in both βarr1-/- MEFs and βarr2-/- MEFs, DPDPE-induced DOR desensitization was significantly reduced in βarr2-/- MEFs, but not in βarr1-/- MEFs. Additionally, the BRET assay suggested that depletion of phosphorylation did not influence the stability of the receptor-β-arrestin complex. Consistent with this observation, DTS did not recycle after internalization, which is like wild type DOR. Taken together, these results indicate that receptor phosphorylation confers DOR selectivity for β-arrestin 2 without affecting the stability of the receptor-β-arrestin complex and the fate of the internalized receptor. [ABSTRACT FROM AUTHOR]

Investigates the intracellular trafficking of opioid receptors directed by carboxyl tail and a di-leucine motif in neuro2A cells. Increase in the rate of agonist-induced receptor internalization and the lysosomal targeting of the wild type and the sigma-opioid receptor carboxyl tail chimera of the mu-opioid reception; Conclusion that the interplay between a di-leucine motif and the carboxyl tail in the lysosomal targeting of the receptor.

Analyzes further the mouse δ-opioid receptor (dor) promoter in EL-4 cells, a mouse T cell line. Correlation of the dor gene with both the expression of DOR on T cells and the capacity of DOR agonists to modulate the immunological functions of the T cell; Indication that Ikaros-2 (Ik-2) homodimers bind to the -378/-374 Ik-binding site and exerts a position-dependent trans-activation effect on the dor promoter.

The lipid raft location of μ-opioid receptor (MOR) determines the receptor activities. However, the manner in which MOR is anchored within the lipid rafts is undetermined. Using the targeted proteomic approach and mass spectrometry analyses, we have identified GRIN1 (G protein-regulated inducer of neurite outgrowth 1) can tether MOR with the G protein a-subunit and subsequently regulate the receptor distribution within the lipid rafts. Glutathione S-transferase fusion pulidown and receptor mutational analyses indicate that GRIN1-MOR interaction involves a receptor sequence 267GSKEK271 within the MOR third intracellular loop that is not involved in Gα interaction. The GRIN1 domains involved in MOR interaction are also distinct from those involved in Ga interaction. Pertussis toxin pretreatment reduced the amount of GRIN1 co-immunoprecipitated with MOR but not the amount with Ga. Furthermore, overexpression of GRIN1 significantly enhanced the amount of MOR in lipid raft and the receptor signaling magnitude as measured by Src kinase activation. Such increase in MOR signaling was demonstrated further by determining the GRIN1-dependent pertussis toxin-sensitive neurite outgrowth. In contrast to minimal neurite outgrowth induced by etorphine in control neuroblastoma N2A cells, overexpression of GRIN1 resulted in the increase in etorphine- and non-morphine-induced neurite outgrowth in these cells. Knocking down endogenous GRIN1 by small interfering RNA attenuated the agonist-induced neurite outgrowth. Disrupting lipid raft by methyl-β-cyclodextrin also blocked neurite outgrowth. Hence, by tethering Gα with MOR, GRIN 1 stabilizes the receptor within the lipid rafts and potenti- ates the receptor signaling in the neurite outgrowth processes. [ABSTRACT FROM AUTHOR]

Opioid receptors are expressed in a cell type-specific manner. Here we show that the mouse δ-opioid receptor (mDOR) gene is regulated by promoter region CpG methylation. The tuDOR promoter containing a putative CpG island is highly methylated in Neuro2A cells, correlating with the repression of this gene in these cells. This is in contrast with the unmethylated state of the tuDOR promoter in NS20Y cells, which express a high level of mDOR. Repression of mDOR transcription in Neuro2A cells could be partially relieved by chemically induced demethylation with 5-aza-2'-deoxycytidine. In addition, in vitro methylation of the luciferase reporter gene driven by the mDOR promoter resulted in an inhibition of transcription in NS20Y cells. Methyl-CpG-binding protein complex 1 (MeCP1) has been implicated in methylation-mediated transcriptional repression of several genes. Electrophoretic mobility shift assays showed that fully methylated, but not unmethylated, mDOR promoter fragment formed a MeCP1-like protein complex that contained methyl-CpG-binding domain protein 2 (MBD2) and Sp3. Furthermore, the expression level of Sp3 was decreased when Neuro2A cells were demethylated with 5-aza-2'-deoxycytidine, and increasing Sp3 levels in Schneider's Drosophila line 2 cells led to the repression of mDOR promoter activity when the promotet was methylated. These results demonstrate that Sp3 and MBD2 are involved in the transcriptional repression of mDOR in Neuro2A cells through binding to the methylated CpG sites in the promoter region and may play a role in the cell type-specific expression of mDOR. [ABSTRACT FROM AUTHOR]

Examines the role of receptor phosphorylation in mu-opioid receptor (MOR) internalization and desensitization by truncating the MOR after Ser[sup 363] to remove the putative phosphorylation sites in the carboxyl tail. Expression of the truncated mutant in murine neuroblastoma Neuro2A cells; Examination of the ability of sigma-opioid receptor agonist, etorphine, to induce receptor desensitization and internalization.

A new role of G protein-coupled receptor (GPCR) phosphor- ylation was demonstrated in the current studies by using the μ-opioid receptor (OPRM1) as a model. Morphine induces a low level of receptor phosphorylation and uses the PKC&3x20AC; pathway to induce ERK phosphorylation and receptor desensitization, whereas etorphine, fentanyl, and [o-A1a2,N- Me-Phe4,G1y5-ol]-enkephalin (DAMGO) induce extensive receptor phosphorylation and use the β-arrestin2 pathway. Blocking OPRM1 phosphorylation (by mutating Ser363, Thr370 and Ser375 to Ala) enabled etorphine, fentanyl, and DAMGO to use the PKCE pathway. This was not due to the decreased recruitment of β-arrestin2 to the receptor signal- ing complex, because these agonists were unable to use the PKC&3x20AC; pathway when β-arrestin2 was absent. In addition, overexpressing G protein-coupled receptor kinase 2 (GRK2) decreased the ability of morphine to activate PKC&3x20AC;, whereas overexpressing dominant-negative GRK2 enabled etorphine, fentanyl, and DAMGO to activate PKC&3x20AC;. Furthermore, by overexpressing wild-type OPRM1 and a phosphorylation-deficient mutant in primary cultures of hippocampal neurons, we demonstrated that receptor phosphorylation contributes to the differential effects of agonists on dendritic spine stability. Phosphorylation blockage made etorphine, fentanyl, and DAMGO function as morphine in the primary cultures. Therefore, agonist-dependent phosphorylation of GPCR regulates the activation of the PKC pathway and the subsequent responses. [ABSTRACT FROM AUTHOR]

μ-Opioid receptor regulates microRNA-190 (miR-190) in an agonist-dependent manner; fentanyl, but not morphine, decreases the miR-190 level in rat primary hippocampal neuron cultures and in mouse hippocampi. In this study, the correlation between the cellular content of miR-190 and the mRNA level of its host gene, talin2, suggested that fentanyl decreases the miR-190 level by inhibiting the transcription of talin2. Fentanyl-induced β-arrestin2-mediated ERK phosphorylation led to the phosphorylation of Yin Yang 1 (YY1). In addition, YY1 phosphorylation impaired the association of YY1 with the -208 to -200 region on the Talin2 promoter, and this association was essential for YY1 to stimulate the transcription of talin2. Thus, fentanyl decreased the transcription of talin2 and subsequently the cellular level of miR-190 by inducing YY1 phosphorylation. In contrast, because morphine induces ERK phosphorylation via the protein kinase C pathway, morphine did not induce YY1 phosphorylation and had no effect on the transcription of talin2 and the cellular content of miR-190. This study therefore delineates a signaling pathway that mediates the effects of fentanyl on miR-190 expression. [ABSTRACT FROM AUTHOR]

Recent studies have revealed that in G protein-coupled receptor signalings switching between G protein- and β-arrestin (βArr)-dependent pathways occurs. In the case of opioid receptors, the signal is switched from the initial inhibition of adenylyl cyclase (AC) to an increase in AC activity (AC activation) during prolonged agonist treatment. The mechanism of such AC activation has been suggested to involve the switching of G proteins activated by the receptor, phosphorylation of signaling molecules, or receptor-dependent recruitment of cellular proteins. Using protein kinase inhibitors, dominant negative mutant studies and mouse embryonic fibroblast cells isolated from Src kinase knock-out mice, we demonstrated that μ-opioid receptor (OPRM1)-mediated AC activation requires direct association and activation of Src kinase by lipid raft-located OPRM1. Such Src activation was independent of βArr as indicated by the ability of OPRM1 to activate Src and AC after prolonged agonist treatment in mouse embryonic fibroblast cells lacking both βArr-1 and -2. Instead the switching of OPRM1 signals was dependent on the heterotrimeric G protein, specifically G[subi2] α-subunit. Among the Src kinase substrates, OPRM1 was phosphorylated at Tyr[sup336] within NPXXY motif by Src during AC activation. Mutation of this Tyr residue, together with mutation of Tyr[sup166] within the DRY motif to Phe, resulted in the complete blunting of AC activation. Thus, the recruitment and activation of Src kinase by OPRM1 during chronic agonist treatment, which eventually results in the receptor tyrosine phosphorylation, is the key for switching the opioid receptor signals from its initial AC inhibition to subsequent AC activation. [ABSTRACT FROM AUTHOR]

Homo- and heterodimerization of the opioid receptors with functional consequences were reported previously. However, the exact nature of these putative dimers has not been identified. In current studies, the nature of the heterodimers was investigated by producing the phenotypes of the 1:1 heterodimers formed between the constitutively expressed μ-opioid receptor (MOR) and the ponasterone A-induced expression of δ-opioid receptor (DOR) in EcR293 cells. By examining the trafficking of the cell surface-located MOR and DOR, we determined that these two receptors endocytosed independently. Using cell surface expression-deficient mutants of MOR and DOR, we observed that the corresponding wild types of these receptors could not rescue the cell surface expression of the mutants, whereas the antagonist naloxone could. Furthermore, studies with constitutive or agonist-induced receptor internalization also indicated that MOR and DOR endocytosed independently and could not "drag in" the corresponding wild types or endocytosis-deficient mutants. Additionally, the heterodimer phenotypes could be eliminated by the pretreatment of the EcR293 cells with pertussis toxin and could be modulated by the deletion of the RRITR sequence in the third intracellular loop that is involved in the receptor-G protein interaction and activation. These data suggest that MOR and DOR heterodimerize only at the cell surface and that the oligomers of opioid receptors and heterotrimeric G protein are the bases for the observed MOR-DOR heterodimer phenotypes. [ABSTRACT FROM AUTHOR]

The μ opioid receptor (MOR) is expressed in the central nervous system and specific cell lines with varying expression levels perhaps playing important roles. One of the neuronal-specific transcription regulators, neuron-restrictive silencer factor (NRSF), has been shown to repress the expression of neuron-specific genes in non-neuronal cells. However, we showed here that the neuron-restrictive silencer element (NRSE) of MOR functions as a critical regulator to repress the MOR gene expression in specific neuronal cells depending on NRSF expression level. Using cotransfection studies, we showed that the NRSE of the MOR promoter is functional in NRSF-positive cells (NS20Y and HeLa) but not in NRSF-negative cells (PC12). NRSF binds to the NRSE of the MOR gene in a sequence-specific manner confirmed by supershift and chromatin immunoprecipitation assays, respectively. The suppression of NRSF activity with either trichostatin A or a dominant-negative NRSF induced MOR promoter activity and transcription of the MOR gene. When the NRSF was disrupted in NS20Y and HeLa cells using small interfering RNA, the transcription of the endogenous target MOR gene increased significantly. This provides direct evidence the role of NRSF in the cells and also indicates that NRSF expression is regulated by post-translational modification in neuronal NMB cells. Our data suggested that NRSF can function as a repressor of MOR transcription in specific cells, via a mechanism dependent on the MOR NRSE. [ABSTRACT FROM AUTHOR]

We previously reported that the 34-bp cis-acting element of the mouse μ opioid receptor (MOR) gene represses transcription of the MOR gene from the distal promoter. Using a yeast one-hybrid screen to identify potential transcription factors of the MOR promoter, we have identified PU.1 as one of the candidate genes. PU.1 is a member of the ets family of transcription factors, expressed predominantly in hematopoietic cells and microglia of brain. PU.1 plays an essential role in the development of both lymphoid and myeloid lineages. Opioids exert neuromodulatory as well as immunomodulatory effects, which are transduced by MOR. Moreover, MOR-deficient mice exhibit increased proliferation of hematopoietic cells, suggesting a possible link between the opioid system and hematopoietic development. The PU.1 protein binds to the 34-bp element of the MOR gene in a sequence-specific manner confirmed by electrophoretic mobility shift assay and supershift assays. We have also determined endogenous PU.1 interactions with the 34-bp element of MOR promoter by chromatin immunoprecipitation assays. In co-transfection studies PU.1 represses MOR promoter reporter constructs through its PU.1 binding site. When the PU.1 gene is disrupted as in PU.1 knock-out mice and using small interfering RNA-based strategy in RAW264.7 cells, the transcription of the endogenous target MOR gene is increased significantly. This increase is probably mediated through modification of the chromatin structure, as suggested by the reversal of the PU.1-mediated repression of MOR promoter activity after trichostatin A treatment in neuroblastoma NMB cells. Our results suggest that PU.1 may be an important regulator of the MOR gene, particularly in brain and immune cells. [ABSTRACT FROM AUTHOR]

Explores the mechanism by which morphine promotes the incidence of HIV infection. Evaluation of the regulatory role of morphine on the interferon-gamma promoter in activated T cells from wild type and micro-opioid receptor knockout mice; Role of cyclic adenylic acid in morphine's modulatory function.

Identifies transcription factors that bind to specific sequences in a 5'-distal promoter regulatory sequences of the mouse mu opioid receptor promoter using the yeast one-hybrid system. Use of the sequence between -746 and -707 in more distal promoter as the bait; Confirmation of the presence of a binding site for SOX proteins in the region using electrophoretic mobility shift assays.

It is generally accepted that the internalization and desensitization of mu-opioid receptor (MOR) involves receptor phosphorylation and beta-arrestin recruitment. However, a mutant MOR, which is truncated after the amino acid residue Ser363 (MOR363D), was found to undergo phosphorylation-independent internalization and desensitization. As expected, MOR363D, missing the putative agonist-induced phosphorylation sites, did not exhibit detectable agonist-induced phosphorylation. MOR363D underwent slower internalization as reflected in the attenuation of membrane translocation of beta-arrestin 2 when compared with wild type MOR, but the level of receptor being internalized was similar to that of wild type MOR after 4 h of etorphine treatment. Furthermore, MOR363D was observed to desensitize faster than that of wild type MOR upon agonist activation. Surface biotinylation assay demonstrated that the wild type receptors recycled back to membrane after agonist-induced internalization, which contributed to the receptor resensitization and thus partially reversed the receptor desensitization. On the contrary, MOR363D did not recycle after internalization. Hence, MOR desensitization is controlled by the receptor internalization and the recycling of internalized receptor to cell surface in an active state. Taken together, our data indicated that receptor phosphorylation is not absolutely required in the internalization, but receptor phosphorylation and subsequent beta-arrestin recruitment play important roles in the resensitization of internalized receptors.