The agonists of -opioid receptor (OPRM1) induce extracellular signal-regulated kinase (ERK) phosphorylation through different pathways: morphine uses the protein kinase C (PKC)-pathway, whereas fentanyl functions in a ␤-arrestin2-dependent manner. In addition, the two pathways result in the different cellular location of phosphorylated ERK and the activation of different sets of transcriptional factors. In the current study, the influence of the two pathways on the expression of microRNAs (miRNAs) was investigated. After treating the primary culture of rat hippocampal neurons and the mouse hippocampi with morphine or fentanyl for 3 days, seven miRNAs regulated by one or two of the agonists were identified. One of the identified miRNAs, miR-190, was down-regulated by fentanyl but not by morphine. This down-regulation was attenuated by 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126), which blocks the phosphorylation of ERK. When fentanyl-induced but not morphine-induced ERK phosphorylation was blocked in the primary cultures from ␤-arrestin2(Ϫ/Ϫ) mouse, fentanyl did not decrease the expression of miR-190. However, a PKC inhibitor that blocked morphine-induced ERK phosphorylation specifically had no effect on the miR-190 down-regulation. Therefore the decrease in miR-190 expression resulted from the agonistselective ERK phosphorylation. In addition, the expressional changes in one of the miR-190 targets, neurogenic differentiation 1 (NeuroD), correlated with those in miR-190 expression, suggesting the OPRM1 could regulate the NeuroD pathways via the control of miR-190 expression.

The basis for agonist-selective signaling was investigated by using the -opioid receptor (MOR) as a model. In the absence of agonist, MOR located within the lipid raft domains, whereas etorphine, but not morphine, induced the translocation of MOR from lipid raft to nonraft domains, similar to the action of methyl-␤-cyclodextrin. The etorphine-induced MOR translocation required the dissociation of the receptor from G␣i2 first and then the binding of ␤-arrestin. In contrast, the low affinity of the morphine-MOR complex for ␤-arrestin and the rebinding of G␣i2 after GTP hydrolysis retained the complex within the lipid raft domains. Disruption of the MOR-G␣i2 interaction, either by deleting the 276 RRITR 280 sequence of MOR or knocking down the level of G␣i2, resulted in the translocation of MOR to the nonraft domains. In addition, lipid raft location of MOR was critical for G protein-dependent signaling, such as etorphine-and morphine-mediated inhibition of adenylyl cyclase activity and morphine-induced ERK phosphorylation, whereas ␤-arrestin-dependent, etorphine-induced ERK phosphorylation required MOR to translocate into the nonraft domains. Thus, agonist-selective signaling is regulated by the location of MOR, which is determined by interactions of MOR with G proteins and ␤-arrestin.lipid raft ͉ opioid A gonists possess different efficacies on different signaling pathways of particular receptors (1, 2). Understanding agonist-selective signaling will accelerate the development of pathway-selective drugs, which have higher efficacy and potency, but fewer side effects (2). Among the various observations of agonist-selective signaling, selectivity between G proteindependent and ␤-arrestin-dependent pathways of G proteincoupled receptor (GPCR) agonists has been well studied (2). For example, angiotensin II (angiotensin II receptor type 1A receptor agonist) uses both the G protein-dependent and ␤-arrestindependent pathways to induce ERK phosphorylation, whereas ICI118551 (␤2-adrenergic receptor agonist) and CCL19 (chemokine receptor CCR7 agonist) induce ERK phosphorylation completely via one of the two pathways (3-5). Classically, receptor-mediated activation of the G protein releases free G␤␥ subunits and induces GPCR kinase (GRK)-mediated receptor phosphorylation, which in turn increases the affinity of the receptor for ␤-arrestin (6). The binding of ␤-arrestin terminates the G protein-dependent pathway by uncoupling the G protein from the complex and activates signaling mediated by itself. Thus the activation of the ␤-arrestin-dependent pathway requires G protein activation. However, the existence of G proteinindependent ␤-arrestin signaling was observed with the GPCR mutants that were incapable of interacting with G proteins (7,8). How agonists select between the two pathways remains unclear.One probable mechanism is that the GPCR location within different membrane domains, such as lipid raft and nonraft domains, determines the agonist-selective signaling. The lipid raft domain is characterized as a dynamic plasma...

BackgroundA cholesterol-palmitoyl interaction has been reported to occur in the dimeric interface of the β2-adrenergic receptor crystal structure. We sought to investigate whether a similar phenomenon could be observed with μ-opioid receptor (OPRM1), and if so, to assess the role of cholesterol in this class of G protein-coupled receptor (GPCR) signaling.ResultsC3.55(170) was determined to be the palmitoylation site of OPRM1. Mutation of this Cys to Ala did not affect the binding of agonists, but attenuated receptor signaling and decreased cholesterol associated with the receptor signaling complex. In addition, both attenuation of receptor palmitoylation (by mutation of C3.55[170] to Ala) and inhibition of cholesterol synthesis (by treating the cells with simvastatin, a HMG-CoA reductase inhibitor) impaired receptor signaling, possibly by decreasing receptor homodimerization and Gαi2 coupling; this was demonstrated by co-immunoprecipitation, immunofluorescence colocalization and fluorescence resonance energy transfer (FRET) analyses. A computational model of the OPRM1 homodimer structure indicated that a specific cholesterol-palmitoyl interaction can facilitate OPRM1 homodimerization at the TMH4-TMH4 interface.ConclusionsWe demonstrate that C3.55(170) is the palmitoylation site of OPRM1 and identify a cholesterol-palmitoyl interaction in the OPRM1 complex. Our findings suggest that this interaction contributes to OPRM1 signaling by facilitating receptor homodimerization and G protein coupling. This conclusion is supported by computational modeling of the OPRM1 homodimer.

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 -opioid receptor (MOR) and the ponasterone A-induced expression of ␦-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.The ability of G protein-coupled receptors (GPCRs) 1 to homoor heterodimerize has implications in the functions of the receptors. Dimerization of the receptors has been reported for the class A GPCRs such as the adenosine (1), adrenergic (2-5), angiotensin (6), dopamine (7,8), muscarinic (9), vasopressin (2, 10), and opioid (11-15) receptors and the class C GPCRs such as the calcium-sensing (16), the metaboropic glutamate receptors (17), and the ␥-amino-n-butyric acid type B (GABA B ) receptors (18 -20). The homo-and heterodimerization of these receptors have been demonstrated by co-immunoprecipitation experiments (11, 21) and subsequently by the fluorescence resonance energy transfer or bioluminescence resonance energy transfer techniques (3,12,23). The heterodimerization of the GPCRs was shown to be selective, with formation of heterodimers with some but not all subtypes of the receptors (13, 24, 25). Most importantly, there are functional differences between the monomers and the homo-and heterodimers of the GPCRs. The classic example is the inability of individual GABA B1 and GABA B2 subunit to form a functional receptor (18 -20). Alteration in the GPCR function or expression was also observed with the heterodimerization of 5HT1B and -1D (26), dopamine D1 and adenosine A1 (27), muscarinic M2 and M3 (28), or dopamine and somatostatin (29) receptors. Heterooligomerization of the GPCRs with other receptor types, such as the ionotropic GABA A receptor, has been observed, resulting in the alteration ...