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. 2010 Mar 9:11:33.
doi: 10.1186/1471-2202-11-33.

Interaction of the mu-opioid receptor with GPR177 (Wntless) inhibits Wnt secretion: potential implications for opioid dependence

Jay Jin  1 , Saranya Kittanakom, Victoria Wong, Beverly A S Reyes, Elisabeth J Van Bockstaele, Igor Stagljar, Wade Berrettini, Robert Levenson
Affiliations

Interaction of the mu-opioid receptor with GPR177 (Wntless) inhibits Wnt secretion: potential implications for opioid dependence

Jay Jin et al. BMC Neurosci. .

Abstract

Background: Opioid agonist drugs produce analgesia. However, long-term exposure to opioid agonists may lead to opioid dependence. The analgesic and addictive properties of opioid agonist drugs are mediated primarily via the mu-opioid receptor (MOR). Opioid agonists appear to alter neuronal morphology in key brain regions implicated in the development of opioid dependence. However, the precise role of the MOR in the development of these neuronal alterations remains elusive. We hypothesize that identifying and characterizing novel MOR interacting proteins (MORIPs) may help to elucidate the underlying mechanisms involved in the development of opioid dependence.

Results: GPR177, the mammalian ortholog of Drosophila Wntless/Evi/Sprinter, was identified as a MORIP in a modified split ubiquitin yeast two-hybrid screen. GPR177 is an evolutionarily conserved protein that plays a critical role in mediating Wnt protein secretion from Wnt producing cells. The MOR/GPR177 interaction was validated in pulldown, coimmunoprecipitation, and colocalization studies using mammalian tissue culture cells. The interaction was also observed in rodent brain, where MOR and GPR177 were coexpressed in close spatial proximity within striatal neurons. At the cellular level, morphine treatment caused a shift in the distribution of GPR177 from cytosol to the cell surface, leading to enhanced MOR/GPR177 complex formation at the cell periphery and the inhibition of Wnt protein secretion.

Conclusions: It is known that chronic morphine treatment decreases dendritic arborization and hippocampal neurogenesis, and Wnt proteins are essential for these processes. We therefore propose that the morphine-mediated MOR/GPR177 interaction may result in decreased Wnt secretion in the CNS, resulting in atrophy of dendritic arbors and decreased neurogenesis. Our results demonstrate a previously unrecognized role for GPR177 in regulating cellular response to opioid drugs.

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Figures

Figure 1
Figure 1
Directed Y2H screen. The carboxyl-terminus of GPR177 (residues 451-541) was used as bait in a directed Y2H assay for interaction with each of the intracellular loops (IL) and carboxyl-terminus (C-tail) of the mu-opioid receptor (MOR) and the second intracellular loop of the D2 dopamine receptor (D2IC2). Full-length CAPS (calcium activated protein for secretion), a known interactor of the D2IC2 was used as a positive control, and empty bait vector (pACT2) was used as a negative control. A positive interaction is indicated by the production of a blue colony in the β-gal assay.
Figure 2
Figure 2
GPR177 interacts with MOR. (A) GST Pulldown. A MORIL2-GST fusion protein was used to pull down S-tagged GPR177 C-terminal fragment from a bacterial lysate. The 18 kDa S-tagged GPR177 fragment produced in bacteria is shown in the lysate lane, while the 18 kDa S-tagged pull-down product is shown in the pulldown lane. No bands were detectable using beads alone or GST-coated beads. (B) Coimmunoprecipitation. The MOR was immunoprecipitated from 293-MOR cells overexpressing FLAG/6× His-tagged GPR177 using an anti-MOR antibody. Immunocomplexes were probed for the presence of GPR177 using chicken anti-GPR177 antibodies. An immunoreactive band of ~50 kDa was detected in the lysate and IP lanes, but not in immunocomplexes from either wild-type HEK293 cells (-MOR lane), 293-MOR cells that were not transfected with GPR177 (-GPR lane), or in IPs in which no anti-MOR antibodies were added (MOCK lane). (C) Specificity of MOR/GPR177 interaction. MOR was immunoprecipitated from 293-MOR cells, and the D2 dopamine receptor immunoprecipitated from 293-D2R cells, using anti-FLAG antibodies. Immunocomplexes were probed for the presence of GPR177 using chicken anti-GPR177 antibodies. For all three panels, molecular weight markers (kDa) are shown at the left. Data shown is representative of at least five separate experiments.
Figure 3
Figure 3
Interaction of MOR and GPR177 in PC12 cells and rat brain. (A) Lysates prepared from PC12 cells were immunoprecipitated with anti-MOR antibodies. (B) Lysates were prepared from total rat brain and immunoprecipitated with anti-MOR antibodies. In A and B, immunocomplexes were separated by SDS-PAGE, transferred to a PVDF filter, and filters probed with anti-GPR177 antibodies. Molecular weight markers (kDa) are shown at the left. The upper band in rat brain lysates is non-specific. Data shown is representative of at least three separate experiments.
Figure 4
Figure 4
MOR and GPR177 colocalize within mouse striatum. Region of the striatum selected for immunohistochemical analysis is shown in the inset of Panel A. (A-C) Confocal images of immunolabeling of GPR177 (red) and MOR (green) within striatum. Arrowheads in A indicate GPR177 immunolabeling; arrowheads in B indicate MOR immunolabeling; arrowheads in C (merged image) indicate colocalization of the two proteins. Thin arrows (A and C) indicate a cell expressing only GPR177, whereas thick arrows (B and C) indicate a cell expressing only MOR. Arrows in lower left corner of panel A indicate dorsal (D) and lateral (L) orientations. Scale bar = 0.05 μm. (D) Electron photomicrograph showing immunogold-silver (GPR177; arrowheads) and immunoperoxidase (MOR; arrows) labeling within a dendritic process in mouse striatum. An unlabeled terminal (ut) forms a synaptic contact with a dendrite double-labeled for GPR177 and MOR (MOR-d). Scale bar = 50 μm. (E-F) Electron photomicrographs showing dual immunogold-silver-labeled GPR177 (arrowheads) and MOR (arrows) within a perikaryon (E) and dendrite (F) within mouse striatum. Scale bar = 0.5 μm. er: Endoplasmic reticulum; Nu: Nucleus; m: Mitochondria.
Figure 5
Figure 5
Effect of opioid agonists on MOR/GPR177 interaction. (A) 293-MOR cells were grown either in the absence of drugs (upper panels), or in the presence of 10 μM morphine (middle panels) or 10 μM DAMGO (lower panels) for one hour. Cells were stained with anti-MOR (red) and anti-GPR177 (green) antibodies. Merged images are shown in the right-hand panels. (B) 293-MOR cells were treated with agonist as in A. Cell lysates were immunoprecipitated with anti-MOR antibodies, and immunocomplexes probed with anti-GPR177 antibodies. The bands were analyzed by densitometry and quantitated using ImageJ software. Open bars represent untreated cells, black bars represent morphine-treated cells, and grey bars represent DAMGO-treated cells. Data was analyzed using paired Student's t-test (expressed as mean ± SEM; n = 3, *p < 0.01).
Figure 6
Figure 6
Morphine inhibits Wnt secretion. Wild-type HEK293 (A) and 293-MOR cells (B) were transfected with myc-tagged Wnt2 cDNA. 24 hours after transfection, cells were treated with either 10 μM morphine or 10 μM DAMGO for an additional 24 hours. In A and B, open bars represent non-drug-treated controls, black bars represent morphine-treated cells, and grey bars represent cells treated with DAMGO. (C) 293-D2R cells were transfected and processed as in A and B, but were cultured in the presence of 10 μM dopamine instead of morphine. Media was collected, proteins separated by SDS-PAGE, then transferred to a PVDF filter. Filters were probed with anti-myc antibodies. Open bars represent non-drug-treated controls, black bars represent drug-treated cells. (D) Overexpression of GPR177 rescues Wnt secretion. 293-MOR cells were transfected with myc-tagged Wnt2 and GPR177 cDNAs. Cells were processed and proteins visualized as described above. Open bars represent no drug treatment, black bars represent morphine-treated cells, and grey bars represent cells overexpressing GPR177. Bands were analyzed by densitometry and quantitated using ImageJ software. Data was analyzed using a two-sided Student's t-test (expressed as mean ± SEM; n= 4-5; *p < 0.05; **p < 0.01).
Figure 7
Figure 7
Effect of MOR antagonists on Wnt secretion. 293-MOR cells were transfected with myc-tagged Wnt2 cDNA. 24 hours after transfection, cells were treated with either (A) 10 μM CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) or (B) 10 μM naloxone for one hour followed by an additional 23 hours of treatment with 1 μM morphine. Media was collected, proteins separated by SDS-PAGE, then transferred to a PVDF filter. Filters were probed with anti-myc antibodies and immunoreactive bands scanned by laser densitometry and quantitated using ImageJ software. In A and B, open bars represent untreated 293-MOR cells, black bars represent 293-MOR cells treated with morphine, and grey bars represent 293-MOR cells treated with either CTAP plus morphine or naloxone plus morphine. Data was analyzed using a two-sided Student's t-test (expressed as mean ± SEM; n= 5-6; *p < 0.05).
Figure 8
Figure 8
Proposed model for effect of MOR/GPR177interaction on Wnt secretion. (A) Under basal conditions, MOR and GPR177 are associated at the plasma membrane (PM) and in recycling vesicles (V). However, GPR177 is located primarily in vesicles in association with Wnts, whereas MOR is predominantly plasma membrane-associated. Recycling of GPR177 from PM to Golgi (G) is mediated via retromer, and is required for Wnt secretion. (B) In the presence of morphine, MORs are inefficiently internalized which leads to an increase in the association between MOR and GPR177 near the plasma membrane. This results in reduced retromer-mediated GPR177 recycling and functional sequestration of GPR177 activity. Reduced recycling of GPR177 leads to inhibition of Wnt secretion. (C) In the presence of DAMGO, MORs are efficiently internalized which allows for internalization of GPR177 proteins associated with MORs. Once internalized, GPR177 is recycled to the Golgi apparatus via retromer, thereby allowing GPR177 to associate with Wnt proteins and functionally support Wnt protein secretion.
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