| Annotated protein: | Transferrin receptor protein 1 (TR) (TfR) (TfR1) (Trfr) (CD antigen CD71). Gene symbol: TFRC. Taxonomy: Mus musculus (Mouse). Uniprot ID: Q62351 |
| antibody wiki: | |
| SynGO gene info: | SynGO data @ TFRC |
| Ontology domain: | Biological Process |
| SynGO term: | regulation of postsynaptic membrane neurotransmitter receptor levels (GO:0099072) |
| Synapse type(s): | hippocampus |
| Annotated paper: | Liu K, et al. "Transferrin Receptor Controls AMPA Receptor Trafficking Efficiency and Synaptic Plasticity" Sci Rep. 2016 Feb 16;6:21019 PMID:26880306 |
| Figure(s): | Fig 2, 4, 5 and movieS3 |
| Annotation description: | Fig2A-B: Different synaptic protein expression in whole brain lysates and P2 fractions from both P20 WT and KO mice were analysed. AMPA receptor subunits GluR1 and GluR2 were both increased in whole brain lysates and in the P2 fraction of TFR KO mice brain versus WT littermates, accompanied by unchanged protein expression of GRIP, PICK1, α-CamKII, PSD95 and synaptophysin. The other excitatory glutamate receptor (NMDA receptor) expression level in TFR KO mice remained unchanged. Fig2C-F: In cultured low-density, TFR KO hippocampal neurons, immunofluorescence staining with anti-N-terminal GluR1 or GluR2 antibodies revealed an increase in surface GluR1 and GluR2 compared to WT neurons. Fig2G-H: Surface AMPA receptor in both WT and KO 14 div neurons by surface protein biotinylation method also showed that surface GluR1 and GluR2 were increased in TFR KO neurons compared to WT. Fig2I-J: To confirm this result in vivo, antibodies against GluR2 or GluR2/3/4 were used to label surface and intracellular AMPA receptor pools in mice hippocampus lysates by Bis (sulphosuccinimidyl) substrate (BS3) crosslinking assay. The normalized surface GluR2 and surface GluR2/3/4 intensity was dramatically increased in TFR KO hippocampus compared with that in WT littermates. Fig4A-B: GluR1 and GluR2 internalization efficiency in steady-status and activity-dependent conditions were examined in TFR KO and WT mice using the antibody feeding assay. 14 div cultured hippocampal neurons were incubated with an anti-GluR1 or anti-GluR2 N-terminal antibody followed by treatment with AMPA (100 μM) or NMDA (50 μM) and CNQX (50 μM) for 10 min to trigger AMPA receptor endocytosis. Internalization index was measured as the internal to surface AMPA receptor ratio. Fig4B-D: In non-stimulation cases, we observed no difference of GluR1 or GluR2 internalization index between WT and KO neurons. Upon stimulation with either AMPA or NMDA, KO neurons displayed a robust decrease in GluR1 and GluR2 internalization index compared to WT neurons, suggesting regulatory endocytosis of AMPA receptor is reduced in TFR KO neurons. Fig4F: Internalization efficiency was measured by quantifying internalized AMPA receptors. AMPA receptors were biotinylated following stimulation by NMDA, then protein biotinylation at the cell surface was quenched by salt wash and the biotinylated receptors left inside cells were used for calculation. Internalized GluR1 and GluR2 were significantly increased upon NMDA stimulation in WT neurons but not in KO neurons. Fig5 & Movie S3: To directly visualize activity-dependent AMPA receptor internalization with higher spatiotemporal resolution, pH-sensitive GFP variant super ecliptic pHluorin (SEP) fused at the N-terminal extracellular domain of GluR1 or GluR2 (SEP-GluR1, SEP-GluR2) was used as a fluorescent reporter in live imaging experiment. The reporter acts as an indicator of AMPA receptor distribution by fluorescing at pH 7.4 at the plasma membrane and becoming non-fluorescent in intracellular endosomes where the pH is less than 6.0. Both WT and KO neurons can internalize surface SEP-GluR1 and SEP-GluR2 upon NMDA stimulation and recycle receptors back to the cell surface during washout perfusion. However, a decreased maximum internalization amplitude and an increased half recycling time of SEP-GluR1 or SEP-GluR2 were observed in TFR KO neurons versus WT neurons suggesting that TFR is required for both AMPA receptor internalization and membrane re-insertion. |
| Evidence tracking, Biological System: | Cultured neurons |
| Evidence tracking, Protein Targeting: | Genetic transformation (eg; knockout, knockin, mutations) Antibody (detection) |
| Evidence tracking, Experiment Assay: | Confocal Western blot |
| Annotator(s): | Dnyanada Sahasrabudhe (ORCID:0000-0003-2916-7616) Guus Smit (ORCID:0000-0002-2286-1587) Matthijs Verhage (ORCID:0000-0002-2514-0216) |
| Lab: | Department of Functional Genomics, Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands |
| SynGO annotation ID: | 4119 |
| Dataset release (version): | 20231201 |
| View annotation as GO-CAM model: |  |