Novel Approach to Probe Subunit-specific Contributions to NMDAR Trafficking

1. Novel Approach to Probe Subunit-specific Contributions toNMDAR Trafficking

2. Introduction • NR2A sorts predominantly into the degradation pathway, and NR2B preferentially traffics through the recycling pathway • NR2A/NR2B heterodimers traffic preferentially through the recycling pathway • NR2A and NR2Bintracellular C-terminal domains contain trafficking motifs that regulate NMDAR endocytosis and intracellular trafficking

3. Background Information MHC Class-II complex is formed by two chains, one α and one β (each one with two domains: α1 and α2, β1 and β2.Both chains are confronted, with domains 1 and 2, in the extracellular space. HLA-DR is a specific MHC class II cell surface receptor we use in the experiments.

4. Background Information Rab9&Rab11 • Rab11: locate in recycling endosomes. • Rab9: locate in late endosomes.

5. The key point of the approach Engineer chimeras between the α- and β-chains of MHC II with the NR2A and NR2B C-terminal domains. C-terminal of NR2B /NR2A/(NR2B+NR2A)

6. Experimental materials

8. Early stage of experiment

9. MHC II α - and β -chains must dimerize to reachthe plasma membrane Immunostaining for Total Protein Expression in HeLa Cells Label with Alexa Fluor 568-conjugated anti-mouse IgG2A secondary antibody

10. MHC II αβ-heterodimers would not internalize by it self Immunofluorescent Internalization Assay in HeLa Cells

11. MHC II αβ-heterodimers would not internalize by it self We observed robust steady-state surface expression of tailless -dimers (stack images) with negligible endocytosis (single-slice images) at both 0 and 30minof internalization

12. Main experiments

13. αβ -NR2A or NR2B Homodimers Undergo Internalization 75% reduction of NR2A homodimer expression after 30 min of internalization, whereas NR2B homodimer expression is only reduced 25% after internalization. These data are consistent with NR2A predominantly trafficking through the degradation pathway whereas NR2B preferentially sorts through recycling endosomes and traffics back to the cell surface.

14. NR2A Homodimers Traffic to Late Endosomes, NR2B Homodimers Recycle to Plasma Membrane • Cells expressing αNR2A-βNR2A together with GFP-Rab9 or GFP-Rab11 were labeled with MHC II-dimer (L243) antibody. After 30min’ internalization the cells were fixed and permeabilized, and the immunoreactivity was visualized with Alexa Fluor 568-conjugated (red)secondary antibody.

15. NR2A Homodimers Traffic to Late Endosomes, NR2B Homodimers Recycle to Plasma Membrane αNR2A-βNR2A was colocalized extensively with GFP-Rab9 in perinuclear late endosomes , whereas αNR2B- β NR2B homodimers were colocalized with GFP-Rab11 in recycling endosomes at or near the cell membrane .

16. αNR2A-βNR2b Heterodimers Preferentially Sort to RecyclingEndosomes

17. It should be noted that the overlap coefficient of α NR2B-βNR2B homodimers (Fig. 2C) and α NR2A-βNR2B heterodimers (Fig. 3B) with Rab11 are not significantly different from each other (0.59 versus 0.56). Thus, NR2B is dominant over the lysosomal sorting of NR2A in heteromeric complexes.

18. examine heterodimer recyclingdirectly • Recycling Assay in HeLa Cells: Ratio of recycling: red/(red+green) • Permeabilize. • incubated with Alexa Fluor 488-conjugated secondary antibody to label internalized receptors that had not recycled back to the plasma membrane (green)

19. examine heterodimer recyclingdirectly These results are consistent with our colocalization analyses demonstrating that NR2B-containing homodimers and heterodimers preferentially sort to recycling endosomes and reveal NR2B-dependent recycling of NR2 heteromers.

20. Experiments in hippocampus neurons

21. Sorting of αNR2A-βNR2A, αNR2B-βNR2B, and αNR2A-βNR2B Dimers in Hippocampus Neurons

22. Recycling of NR2B Is Unaffected in NR2A knock-out Hippocampal Neurons • To demonstrate conclusively that the NR2B sorting motif is dominant for NMDAR trafficking, we analyzed NMDAR recycling in NR2A-deficient neurons. • We expressed full-length NR2B containing an N-terminal GFP epitope tag in hippocampal neurons derived either from wild-type (WT) or NR2A knock-out mice

23. Recycling of NR2B Is Unaffected in NR2A knock-out Hippocampal Neurons

24. Significance of nmdars’ different endocytosis pathway

25. At this stage of development, endogenous NMDARs contain NR2B, and the dynamic recycling of endogenous NMDARs early in development fits very well with the presence of dominant endocytic and recycling motifs in NR2B present in NMDAR heteromers. • Later in development endogenous NMDARs become more stable on the plasma membrane. This stabilization corresponds with the increased expression of NR2A.