Nedd4 E3 ligase and beta-arrestins regulate ubiquitination, trafficking, and stability of the mGlu7 receptor

  1. Sanghyeon Lee
  2. Sunha Park
  3. Hyojin Lee
  4. Seulki Han
  5. Jae-man Song
  6. Dohyun Han
  7. Young Ho Suh  Is a corresponding author
  1. Seoul National University College of Medicine, Republic of Korea
  2. Seoul National University Hospital, Republic of Korea
8 figures, 1 table and 1 additional file

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 …

https://doi.org/10.7554/eLife.44502.002
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. …

https://doi.org/10.7554/eLife.44502.003
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 …

https://doi.org/10.7554/eLife.44502.005
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, …

https://doi.org/10.7554/eLife.44502.007
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 …

https://doi.org/10.7554/eLife.44502.008
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 …

https://doi.org/10.7554/eLife.44502.009
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, …

https://doi.org/10.7554/eLife.44502.011
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 …

https://doi.org/10.7554/eLife.44502.013
Figure 3—figure supplement 4—source data 1

Ubiquitination of mGlu7 by beta-arrestins in neurons.

https://doi.org/10.7554/eLife.44502.014
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 …

https://doi.org/10.7554/eLife.44502.016
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 …

https://doi.org/10.7554/eLife.44502.017
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 …

https://doi.org/10.7554/eLife.44502.018
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.

https://doi.org/10.7554/eLife.44502.019
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 …

https://doi.org/10.7554/eLife.44502.021
Figure 6—source data 1

ERK and JNK signaling of mGlu7 by beta-arrestins and Nedd4.

https://doi.org/10.7554/eLife.44502.022
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

https://doi.org/10.7554/eLife.44502.023
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
DesignationSource or referenceIdentifiersAdditional
information
Recombinant DNA reagentpRK5-myc-mGlu7 WTDr. Katherine W. Roche (NIH)
Recombinant DNA reagentpRK5-FLAG-mGlu7 WTThis study
Recombinant DNA reagentpRK5-myc-mGlu7 Δ857This study
Recombinant DNA reagentpRK5-myc-mGlu7 Δ860This study
Recombinant DNA reagentpRK5-myc-mGlu7 Δ879This study
Recombinant DNA reagentpRK5-myc-mGlu7 Δ893This study
Recombinant DNA reagentpRK5-myc-mGlu7 CT 8K8RThis study
Recombinant DNA reagentpRK5-myc-mGlu7 iL 4K4RThis study
Recombinant DNA reagentpRK5-myc-mGlu7 12K12RThis study
Recombinant DNA reagentpRK5-myc-mGlu7 iL K688/689RThis study
Recombinant DNA reagentpRK5-myc-mGlu7 Δ857/iL 4K4RThis study
Recombinant DNA reagentpRK5-myc-mGlu7 Δ857/IL K688/689RThis study
Recombinant DNA reagentFLAG-ß2ARAddgeneRRID:Addgene_14697
Recombinant DNA reagentpRK5-HA-Ub WTAddgeneRRID:Addgene_17608
Recombinant DNA reagentpRK5-HA-Ub K48AddgeneRRID:Addgene_17605
Recombinant DNA reagentpRK5-HA-Ub K63AddgeneRRID:Addgene_17606
Recombinant DNA reagentpGFP-N3-ß-arrestin1-GFPDr. Katherine W. Roche (NIH)
Recombinant DNA reagentpGFP-N3-ß-arrestin2-GFPDr. Katherine W. Roche (NIH)
Recombinant DNA reagentpcDNA3-ß-arrestin1-FLAGAddgeneRRID:Addgene_14687
Recombinant DNA reagentpCI-HA-Nedd4 WTAddgeneRRID:Addgene_27002
Recombinant DNA reagentpCI-HA-Nedd4 C867SThis study
Recombinant DNA reagentFLAG-Nedd4 WTThis study
Recombinant DNA reagentFLAG-Nedd4 C867SThis study
Recombinant DNA reagentFLAG-Nedd4 ΔC2This study
Recombinant DNA reagentFLAG-Nedd4 ΔC2-WW1/2This study
Recombinant DNA reagentFLAG-Nedd4 ΔWW3This study
Recombinant DNA reagentFLAG-Nedd4 ΔWW4This study
Recombinant DNA reagentFLAG-Nedd4 ΔHECTThis study
Recombinant DNA reagentpSuper-rat Nedd4 KDThis study
Recombinant DNA reagentpSuper-human ß-arrestin1 KDThis study
Recombinant DNA reagentpSuper-human ß-arrestin2 KDThis study
Recombinant DNA reagentpSuper-rat ß-arrestin1 KDThis study
Recombinant DNA reagentpSuper-rat ß-arrestin2 KDThis study
Recombinant DNA reagentpSuper-non-related target KDThis study
Recombinant DNA reagentFHUGW-rat ß-arrestin1 KDThis study
Recombinant DNA reagentFHUGW-rat ß-arrestin2 KDThis study
Recombinant DNA reagentFHUGW-non-related target KDThis study
Recombinant DNA reagentFHUGW-myc-mGlu7 WTThis study
Recombinant DNA reagentFHUGW-myc-mGlu7 CT 8K8RThis study
Recombinant DNA reagentFHUGW-myc-mGlu7 iL 4K4RThis study
Recombinant DNA reagentFHUGW-myc-mGlu7 12K12RThis study
Recombinant DNA reagentFHUGW-FLAG-Nedd4 WTThis study
Recombinant DNA reagentFHUGW-FLAG-Nedd4 C867SThis study
Recombinant DNA reagentpGEX-4T-1-mGlu7 iL1This study
Recombinant DNA reagentpGEX-4T-1-mGlu7 iL2This study
Recombinant DNA reagentpGEX-4T-1-mGlu7 iL3This study
Recombinant DNA reagentpGEX-4T-1-mGlu7a CTThis study
Recombinant DNA reagentpGEX-4T-1-mGlu7b CTThis study
Recombinant DNA reagentpET28a-Nedd4This study
Recombinant DNA reagentpET28b-Barr1This study
Recombinant DNA reagentpET28b-Barr2This study
AntibodyHA (clone 16B12)BioLegendcat# 901501
RRID:AB_2565006
1:1000, western blotting (WB)
Antibodyc-myc (clone 9E10)SIGMAcat# M5546
RRID:AB_260581
1:1000, WB; 1:500, immunofluorescence staining (IF)
AntibodyFLAG (clone M2)SIGMAcat# F1804
RRID:AB_262044
1:1000, WB
AntibodyFLAGSIGMAcat# F7425
RRID:AB_439687
1:1000, WB
AntibodymGlu7aEMD Milliporecat# 07–239
RRID:AB_310459
1:2000, WB
AntibodyUb (clone FK2)EMD Milliporecat# 04–263
RRID:AB_612093
1:1000, WB
AntibodyUb (clone P4D1)Santa Cruz Biotechnologycat# sc-8017
RRID:AB_2762364
1:1000, WB
AntibodyNedd4EMD Milliporecat# 07–049
RRID:AB_310351
1:10000, WB
AntibodyNedd4R&D SYSTEMScat# MAB6218
RRID:AB_10920762
1:1000, WB
AntibodyBeta-arrestin 1 (clone E274)Abcamcat# ab32099
RRID:AB_722896
1:1000, WB
AntibodyBeta-arrestin 2 (clone C16D9)Cell Signaling Technologycat# 3857
RRID:AB_2258681
1:1000, WB
AntibodyGFP (clone B2)Santa Cruz Biotechnologycat# sc-9996
RRID:AB_627695
1:500, WB
AntibodyGFPThermo Fischer Scientificcat# A11122
RRID:AB_221569
1:2000, WB
Antibodyɑ-tubulinSIGMAcat# T6199
RRID:AB_477583
1:5000, WB
AntibodyPP1γ1EMD Milliporecat# 07–1218
RRID:AB_1977432
1:500, WB
Antibodyphospho-ERKSanta Cruz Biotechnologycat# sc-7383
RRID:AB_627545
1:1000, WB
AntibodyERKSanta Cruz Biotechnologycat# sc-94
RRID:AB_2140110
1:2000, WB
Antibodyphospho-JNKCell Signaling
Technology
cat# 4671
RRID:AB_331338
1:1000, WB
AntibodyJNKCell Signaling Technologycat# 9252
RRID:AB_2250373
1:1000, WB
AntibodyGoat anti-Mouse IgG (H+L) antibody, Alexa Fluor 568Thermo Fischer Scientificcat# A11031
RRID:AB_144696
1:500, IF
AntibodyGoat anti-Mouse IgG (H+L) antibody, Alexa Fluor 488Thermo Fischer Scientificcat# A11029
RRID:AB_138404
1:500, IF
Chemical compoundNormal goat serum blocking solutionVECTOR LABORATORIEScat# S-1000
RRID:AB_2336615
Chemical compoundParaformaldehydeSIGMAcat# 158127
Chemical compoundProLong Antifade KitThermo Fischer Scientificcat# P7481
Chemical compoundL-Glutamic acidTOCRIScat# 0218
Chemical compoundL-AP4TOCRIScat# 0103
Chemical compoundN-Ethylmaleimide (NEM)SIGMAcat# 04259
Chemical compoundMG132TOCRIScat# 1748
Chemical
compound
Leupeptin hemisulfateTOCRIScat# 1167
Chemical compoundCycloheximideSIGMAcat# C7698
Chemical compoundMLN7243CHEMIETEKcat# CT-M7243
Chemical
compound
Protein G Sepharose 4 Fast FlowGE Healthcarecat# 17061801
Chemical compoundProtein A Sepharose, from Staphylococcus aureusSIGMAcat# P3391
Chemical compoundGlutathione Sepharose 4B GST-tagged resinGE Healthcarecat# 17075601
Chemical compoundEZ-Link Sulfo-NHS-SS-BiotinThermo Fischer Scientificcat# 21331
Chemical compoundPierce Streptavidin AgaroseThermo Fischer Scientificcat# 20347
Chemical compoundshRNA to rat Nedd4Schwarz et al., 2010Oligonucleotides
Chemical compoundshRNA to rat beta-arrestin 1Simard et al., 2013Oligonucleotides
Chemical compoundshRNA to rat beta-arrestin 2Molteni et al., 2009Oligonucleotides
Chemical compoundshRNA to human beta-arrestin 1Shenoy et al., 2008Oligonucleotides
Chemical compoundshRNA to human beta-arrestin 2Skånland et al., 2009Oligonucleotides
Chemical compoundshRNA to non-related targetGenScriptOligonucleotides
Cell line (human)HEK293TATCCcat# CRL-3216
RRID:CVCL_0063
Rat (Rattus norvegicus)Primary cultured neuronsORIENT BIORGD Cat# 734476
RRID:RGD_734476)
SD rat

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