(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 …
Ubiquitination of mGlu7 in heterologous cells and 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. …
(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 …
Mapping lysine residues of mGlu7 ubiquitination.
(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, …
Binding analysis of mGlu7, Nedd4, and beta-arrestins complex.
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 …
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 …
Binding affinity of Nedd4 and beta-arrestin 1 to mGlu7 ubiquitination site mutants.
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, …
Binding of Nedd4 to mGlu7 by beta-arrestins in neurons.
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 …
Ubiquitination of mGlu7 by beta-arrestins in neurons.
(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 …
(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 …
Endocytosis of mGlu7 by beta-arrestins and Nedd4.
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 …
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.
(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 …
ERK and JNK signaling of mGlu7 by beta-arrestins and Nedd4.
(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…
Degradation pathways of ubiquitinated mGlu7.
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 …
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 |