Nedd4 E3 ligase and beta-arrestins regulate ubiquitination, trafficking, and stability of the mGlu7 receptor
- Seoul National University College of Medicine, Republic of Korea
- Seoul National University Hospital, Republic of Korea
Figures
Figure 1 with 1 supplement
mGlu7 is ubiquitinated by agonist treatment or Nedd4 expression in heterologous cells and neurons.
(A) mGlu7 is ubiquitinated in HEK 293 T cells. N-terminal c-myc epitope-tagged mGlu7a (myc-mGlu7) was co-transfected with HA epitope-tagged ubiquitin (HA-Ub) in HEK 293 T cells. Cell lysates were …
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Figure 1—source data 1
Ubiquitination of mGlu7 in heterologous cells and neurons.
- https://doi.org/10.7554/eLife.44502.004
Figure 1—figure supplement 1
Endogenous mGlu7 is ubiquitinated in cortical neurons.
After cortical neurons were treated with 800 μM L-AP4 for 5 min, the lysates were immunoprecipitated with rabbit anti-mGlu7 antibody. Rabbit anti-GFP antibody was used as a negative control. …
Figure 2
mGlu7 is ubiquitinated at lysine residues in both CT and iL2 domains.
(A) A schematic diagram showing amino acid sequences of mGlu7 intracellular loop regions (iL1‒3) and the cytoplasmic C-terminus tail (CT) of rat mGlu7a. The twelve lysine (K) residues in the CT and …
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Figure 2—source data 1
Mapping lysine residues of mGlu7 ubiquitination.
- https://doi.org/10.7554/eLife.44502.006
Figure 3 with 4 supplements
β-arrestin 1 (β-arr1), β-arrestin 2 (β-arr2), and Nedd4 are recruited to mGlu7 by agonist stimulation within 5 min.
(A–F) Time-course interaction among mGlu7, Nedd4, and β-arrestins. HEK 293 T cells were co-transfected with FLAG-mGlu7, HA-Nedd4, and β-arrestin 1-GFP (A) or β-arrestin 2-GFP (B). In panel E, …
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Figure 3—source data 1
Binding analysis of mGlu7, Nedd4, and beta-arrestins complex.
- https://doi.org/10.7554/eLife.44502.015
Figure 3—figure supplement 1
Binding of β-arrestins to mGlu7.
GFP-tagged β-arrestin 1 or 2 was co-transfected with either FLAG-tagged mGlu7 or FLAG-tagged β2AR in HEK 293 T cells. The lysates were immunoprecipitated with anti-FLAG antibody and western blotting …
Figure 3—figure supplement 2
The binding affinity of Nedd4 and β-arrestin 1 to mGlu7 is not altered by ubiquitination site mutations of mGlu7.
mGlu7 WT, iL 4K4R, Ct 8K8R, or 12K12R was expressed on cultured cortical neurons via lentivirus-mediated transduction. After treatment with 400 μM L-AP4 for 5 min, co-immunoprecipitation assay was …
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Figure 3—figure supplement 2—source data 1
Binding affinity of Nedd4 and beta-arrestin 1 to mGlu7 ubiquitination site mutants.
- https://doi.org/10.7554/eLife.44502.010
Figure 3—figure supplement 3
Co-immunoprecipitation assay to supplement Figures 3K and L.
Bar graph represents mean ± SEM showing quantification of binding affinity between Nedd4 and mGlu7 normalized to Ctl KD (β-arr1/2 KD, 0.84 ± 0.23; Ctl KD + L-AP4, 1.73 ± 0.14; β-arr1/2 KD + L-AP4, …
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Figure 3—figure supplement 3—source data 1
Binding of Nedd4 to mGlu7 by beta-arrestins in neurons.
- https://doi.org/10.7554/eLife.44502.012
Figure 3—figure supplement 4
Ubiquitination assay to supplement Figures 3M and N.
Bar graph represents mean ± SEM showing mGlu7 ubiquitination levels normalized to Ctl KD (β-arr1/2 KD, 1.15 ± 0.09; Ctl KD + L-AP4, 1.42 ± 0.07; β-arr1/2 KD + L-AP4, 1.06 ± 0.09; n = 3, *p<0.05 …
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Figure 3—figure supplement 4—source data 1
Ubiquitination of mGlu7 by beta-arrestins in neurons.
- https://doi.org/10.7554/eLife.44502.014
Figure 4
Mapping of binding domains between mGlu7 and Nedd4 or β-arrestins.
(A) GST pull-down experiments to identify the interaction domains of mGlu7 with β-arrestin 1 or Nedd4. Total rat brain extract was subjected to a pull-down assay using immobilized GST fusion …
Figure 5 with 2 supplements
β-arrestins and Nedd4 regulate endocytosis of mGlu7 in neurons.
(A) The endocytosis of mGlu7 was analyzed by an antibody uptake internalization assay. myc-mGlu7 was co-transfected with Nedd4 WT, C867S mutant, or vector control (Vec) in cultured hippocampal …
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Figure 5—source data 1
Endocytosis of mGlu7 by beta-arrestins and Nedd4.
- https://doi.org/10.7554/eLife.44502.020
Figure 5—figure supplement 1
Ubiquitination is required for endocytosis of endogenous mGlu7.
Cortical neurons were treated with 0.1 μM MLN7243 for 18 hr to inhibit ubiquitination. After treatment with 800 μM L-AP4 for 15 min, surface biotinylation assay was performed. Treatment of MLN7243 …
Figure 5—figure supplement 2
Separated images that supplement the merged images shown in Figure 5.
Surface-expressed and internalized mGlu7 signals in Figures 5A, C and E were separated, and individual images were presented in A, B, and C, respectively.
Figure 6
β-arrestins and Nedd4 regulate ERK and JNK signaling of mGlu7 in neurons.
(A) Cultured hippocampal neurons were infected with control (Ctl KD), β-arrestin 1 (β-arr1 KD), β-arrestin 2 (β-arr2 KD), or Nedd4 KD shRNA lentiviruses for 7 days. At DIV 14, neurons were treated …
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Figure 6—source data 1
ERK and JNK signaling of mGlu7 by beta-arrestins and Nedd4.
- https://doi.org/10.7554/eLife.44502.022
Figure 7
Nedd4-mediated or agonist-stimulated mGlu7 degradation occurs via both the proteasomal and lysosomal degradation pathways.
(A) myc-mGlu7 and FLAG-Nedd4 WT or C867S were co-transfected in HEK 293 T cells. Thirty-six hours after transfection, cell lysates were analyzed by western blotting using the indicated antibodies. (B…
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Figure 7—source data 1
Degradation pathways of ubiquitinated mGlu7.
- https://doi.org/10.7554/eLife.44502.024
Author response image 1
Surface expression levels of mGlu7 ubiquitination site mutants by cell surface biotinylation assay. mGlu7 WT, iL 4K4R, Ct 8K8R, 12K12R, Δ857, or Δ857/iL K688/689R mutants were expressed in cultured cortical neurons for 7 days via lentivirus-mediated transduction.
At DIV14, after cell surface biotinylation with membrane impermeable Sulfo-NHS-LC-Biotin, surface proteins were isolated by Streptavidin-agarose beads. Western blotting was performed with the …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Recombinant DNA reagent | pRK5-myc-mGlu7 WT | Dr. Katherine W. Roche (NIH) | ||
| Recombinant DNA reagent | pRK5-FLAG-mGlu7 WT | This study | ||
| Recombinant DNA reagent | pRK5-myc-mGlu7 Δ857 | This study | ||
| Recombinant DNA reagent | pRK5-myc-mGlu7 Δ860 | This study | ||
| Recombinant DNA reagent | pRK5-myc-mGlu7 Δ879 | This study | ||
| Recombinant DNA reagent | pRK5-myc-mGlu7 Δ893 | This study | ||
| Recombinant DNA reagent | pRK5-myc-mGlu7 CT 8K8R | This study | ||
| Recombinant DNA reagent | pRK5-myc-mGlu7 iL 4K4R | This study | ||
| Recombinant DNA reagent | pRK5-myc-mGlu7 12K12R | This study | ||
| Recombinant DNA reagent | pRK5-myc-mGlu7 iL K688/689R | This study | ||
| Recombinant DNA reagent | pRK5-myc-mGlu7 Δ857/iL 4K4R | This study | ||
| Recombinant DNA reagent | pRK5-myc-mGlu7 Δ857/IL K688/689R | This study | ||
| Recombinant DNA reagent | FLAG-ß2AR | Addgene | RRID:Addgene_14697 | |
| Recombinant DNA reagent | pRK5-HA-Ub WT | Addgene | RRID:Addgene_17608 | |
| Recombinant DNA reagent | pRK5-HA-Ub K48 | Addgene | RRID:Addgene_17605 | |
| Recombinant DNA reagent | pRK5-HA-Ub K63 | Addgene | RRID:Addgene_17606 | |
| Recombinant DNA reagent | pGFP-N3-ß-arrestin1-GFP | Dr. Katherine W. Roche (NIH) | ||
| Recombinant DNA reagent | pGFP-N3-ß-arrestin2-GFP | Dr. Katherine W. Roche (NIH) | ||
| Recombinant DNA reagent | pcDNA3-ß-arrestin1-FLAG | Addgene | RRID:Addgene_14687 | |
| Recombinant DNA reagent | pCI-HA-Nedd4 WT | Addgene | RRID:Addgene_27002 | |
| Recombinant DNA reagent | pCI-HA-Nedd4 C867S | This study | ||
| Recombinant DNA reagent | FLAG-Nedd4 WT | This study | ||
| Recombinant DNA reagent | FLAG-Nedd4 C867S | This study | ||
| Recombinant DNA reagent | FLAG-Nedd4 ΔC2 | This study | ||
| Recombinant DNA reagent | FLAG-Nedd4 ΔC2-WW1/2 | This study | ||
| Recombinant DNA reagent | FLAG-Nedd4 ΔWW3 | This study | ||
| Recombinant DNA reagent | FLAG-Nedd4 ΔWW4 | This study | ||
| Recombinant DNA reagent | FLAG-Nedd4 ΔHECT | This study | ||
| Recombinant DNA reagent | pSuper-rat Nedd4 KD | This study | ||
| Recombinant DNA reagent | pSuper-human ß-arrestin1 KD | This study | ||
| Recombinant DNA reagent | pSuper-human ß-arrestin2 KD | This study | ||
| Recombinant DNA reagent | pSuper-rat ß-arrestin1 KD | This study | ||
| Recombinant DNA reagent | pSuper-rat ß-arrestin2 KD | This study | ||
| Recombinant DNA reagent | pSuper-non-related target KD | This study | ||
| Recombinant DNA reagent | FHUGW-rat ß-arrestin1 KD | This study | ||
| Recombinant DNA reagent | FHUGW-rat ß-arrestin2 KD | This study | ||
| Recombinant DNA reagent | FHUGW-non-related target KD | This study | ||
| Recombinant DNA reagent | FHUGW-myc-mGlu7 WT | This study | ||
| Recombinant DNA reagent | FHUGW-myc-mGlu7 CT 8K8R | This study | ||
| Recombinant DNA reagent | FHUGW-myc-mGlu7 iL 4K4R | This study | ||
| Recombinant DNA reagent | FHUGW-myc-mGlu7 12K12R | This study | ||
| Recombinant DNA reagent | FHUGW-FLAG-Nedd4 WT | This study | ||
| Recombinant DNA reagent | FHUGW-FLAG-Nedd4 C867S | This study | ||
| Recombinant DNA reagent | pGEX-4T-1-mGlu7 iL1 | This study | ||
| Recombinant DNA reagent | pGEX-4T-1-mGlu7 iL2 | This study | ||
| Recombinant DNA reagent | pGEX-4T-1-mGlu7 iL3 | This study | ||
| Recombinant DNA reagent | pGEX-4T-1-mGlu7a CT | This study | ||
| Recombinant DNA reagent | pGEX-4T-1-mGlu7b CT | This study | ||
| Recombinant DNA reagent | pET28a-Nedd4 | This study | ||
| Recombinant DNA reagent | pET28b-Barr1 | This study | ||
| Recombinant DNA reagent | pET28b-Barr2 | This study | ||
| Antibody | HA (clone 16B12) | BioLegend | cat# 901501 RRID:AB_2565006 | 1:1000, western blotting (WB) |
| Antibody | c-myc (clone 9E10) | SIGMA | cat# M5546 RRID:AB_260581 | 1:1000, WB; 1:500, immunofluorescence staining (IF) |
| Antibody | FLAG (clone M2) | SIGMA | cat# F1804 RRID:AB_262044 | 1:1000, WB |
| Antibody | FLAG | SIGMA | cat# F7425 RRID:AB_439687 | 1:1000, WB |
| Antibody | mGlu7a | EMD Millipore | cat# 07–239 RRID:AB_310459 | 1:2000, WB |
| Antibody | Ub (clone FK2) | EMD Millipore | cat# 04–263 RRID:AB_612093 | 1:1000, WB |
| Antibody | Ub (clone P4D1) | Santa Cruz Biotechnology | cat# sc-8017 RRID:AB_2762364 | 1:1000, WB |
| Antibody | Nedd4 | EMD Millipore | cat# 07–049 RRID:AB_310351 | 1:10000, WB |
| Antibody | Nedd4 | R&D SYSTEMS | cat# MAB6218 RRID:AB_10920762 | 1:1000, WB |
| Antibody | Beta-arrestin 1 (clone E274) | Abcam | cat# ab32099 RRID:AB_722896 | 1:1000, WB |
| Antibody | Beta-arrestin 2 (clone C16D9) | Cell Signaling Technology | cat# 3857 RRID:AB_2258681 | 1:1000, WB |
| Antibody | GFP (clone B2) | Santa Cruz Biotechnology | cat# sc-9996 RRID:AB_627695 | 1:500, WB |
| Antibody | GFP | Thermo Fischer Scientific | cat# A11122 RRID:AB_221569 | 1:2000, WB |
| Antibody | ɑ-tubulin | SIGMA | cat# T6199 RRID:AB_477583 | 1:5000, WB |
| Antibody | PP1γ1 | EMD Millipore | cat# 07–1218 RRID:AB_1977432 | 1:500, WB |
| Antibody | phospho-ERK | Santa Cruz Biotechnology | cat# sc-7383 RRID:AB_627545 | 1:1000, WB |
| Antibody | ERK | Santa Cruz Biotechnology | cat# sc-94 RRID:AB_2140110 | 1:2000, WB |
| Antibody | phospho-JNK | Cell Signaling Technology | cat# 4671 RRID:AB_331338 | 1:1000, WB |
| Antibody | JNK | Cell Signaling Technology | cat# 9252 RRID:AB_2250373 | 1:1000, WB |
| Antibody | Goat anti-Mouse IgG (H+L) antibody, Alexa Fluor 568 | Thermo Fischer Scientific | cat# A11031 RRID:AB_144696 | 1:500, IF |
| Antibody | Goat anti-Mouse IgG (H+L) antibody, Alexa Fluor 488 | Thermo Fischer Scientific | cat# A11029 RRID:AB_138404 | 1:500, IF |
| Chemical compound | Normal goat serum blocking solution | VECTOR LABORATORIES | cat# S-1000 RRID:AB_2336615 | |
| Chemical compound | Paraformaldehyde | SIGMA | cat# 158127 | |
| Chemical compound | ProLong Antifade Kit | Thermo Fischer Scientific | cat# P7481 | |
| Chemical compound | L-Glutamic acid | TOCRIS | cat# 0218 | |
| Chemical compound | L-AP4 | TOCRIS | cat# 0103 | |
| Chemical compound | N-Ethylmaleimide (NEM) | SIGMA | cat# 04259 | |
| Chemical compound | MG132 | TOCRIS | cat# 1748 | |
| Chemical compound | Leupeptin hemisulfate | TOCRIS | cat# 1167 | |
| Chemical compound | Cycloheximide | SIGMA | cat# C7698 | |
| Chemical compound | MLN7243 | CHEMIETEK | cat# CT-M7243 | |
| Chemical compound | Protein G Sepharose 4 Fast Flow | GE Healthcare | cat# 17061801 | |
| Chemical compound | Protein A Sepharose, from Staphylococcus aureus | SIGMA | cat# P3391 | |
| Chemical compound | Glutathione Sepharose 4B GST-tagged resin | GE Healthcare | cat# 17075601 | |
| Chemical compound | EZ-Link Sulfo-NHS-SS-Biotin | Thermo Fischer Scientific | cat# 21331 | |
| Chemical compound | Pierce Streptavidin Agarose | Thermo Fischer Scientific | cat# 20347 | |
| Chemical compound | shRNA to rat Nedd4 | Schwarz et al., 2010 | Oligonucleotides | |
| Chemical compound | shRNA to rat beta-arrestin 1 | Simard et al., 2013 | Oligonucleotides | |
| Chemical compound | shRNA to rat beta-arrestin 2 | Molteni et al., 2009 | Oligonucleotides | |
| Chemical compound | shRNA to human beta-arrestin 1 | Shenoy et al., 2008 | Oligonucleotides | |
| Chemical compound | shRNA to human beta-arrestin 2 | Skånland et al., 2009 | Oligonucleotides | |
| Chemical compound | shRNA to non-related target | GenScript | Oligonucleotides | |
| Cell line (human) | HEK293T | ATCC | cat# CRL-3216 RRID:CVCL_0063 | |
| Rat (Rattus norvegicus) | Primary cultured neurons | ORIENT BIO | RGD Cat# 734476 RRID:RGD_734476) | SD rat |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.44502.025
