Original Article | Published:

Molecular Pharmacology

Phorbol-induced surface expression of NR2A subunit homologues in HEK293 cells



N-methyl-D-aspartate receptors (NMDAR) are heteromeric complexes primarily assembled from NR1 and NR2 subunits. In normal conditions, NR2 sub-units assemble into homodimers in the endoplasmic reticulum (ER). These homodimers remain in the ER until they coassemble with NR1 dimers and are trafficked to the cell surface. However, it still remains unclear whether functional homomeric NMDAR exist in physiological or pathological conditions.


We transfected GFP-NR2A alone into HEK293 cells, treated the cells with PKC activator 12-myristate-13 acetate (PMA), and then detected surface NR2A sub-units with a live cell immunostaining method. We also used a series of NR2A mutants with a partial deletion of its C-terminus to identify the regions that are involved in the PMA-mediated surface expression of NR2A subunits.


NR2A subunits were expressed on the cell membrane after incubation with PMA (200 nmol/L, 30 min), although no functional NMDA channels were detected after PMA-induced membrane trafficking. Immunostaining with an ER marker also revealed that NR2A subunits were exported from the ER after PMA treatment. Furthermore, the deletion of amino acids between 1149-1347 or 1354-1464 of NR2A inhibited PMA-induced surface expression of NR2A subunits.


First, our data suggests that PMA treatment can induce the surface expression of homomeric NR2A subunits. Furthermore, this process is probably mediated by the NR2A C-terminal region between positions 1149 and 1464.


  1. 1

    Monyer H, Sprengel R, Schoepfer R, Herb A, Higuchi M, Lomeli H, et al. Heteromeric NMDA receptors: molecular and functional distinction of subtypes. Science 1992; 256: 1217–21.

  2. 2

    Luo J, Wang Y, Yasuda RP, Dunah AW, Wolfe BB . The majority of N-methyl-D-aspartate receptor complexes in adult rat cerebral cortex contain at least three different subunits (NR1/NR2A/NR2B). Mol Pharmacol 1997; 51: 79–86.

  3. 3

    Huggins DJ, Grant GH . The function of the amino terminal domain in NMDA receptor modulation. J Mol Graph Model 2005; 23: 381–8.

  4. 4

    Moriyoshi K, Masu M, Ishii T, Shigemoto R, Mizuno N, Nakanishi S . Molecular cloning and characterization of the rat NMDA receptor. Nature 1991; 354: 31–7.

  5. 5

    Karp SJ, Masu M, Eki T, Ozawa K, Nakanishi S . Molecular cloning and chromosomal localization of the key subunit of the human N-methyl-D-aspartate receptor. J Biol Chem 1993; 268: 3728–33.

  6. 6

    Yamazaki M, Mori H, Araki K, Mori KJ, Mishina M . Cloning, expression and modulation of a mouse NMDA receptor subunit. FEBS Lett 1992; 300: 39–45.

  7. 7

    Qiu S, Hua YL, Yang F, Chen YZ, Luo JH . Subunit assembly of N-methyl-D-aspartate receptors analyzed by fluorescence resonance energy transfer. J Biol Chem 2005; 280: 24 923–30.

  8. 8

    Hawkins LM, Prybylowski K, Chang K, Moussan C, Stephenson FA, Wenthold RJ . Export from the endoplasmic reticulum of assembled N-methyl-D-aspartic acid receptors is controlled by a motif in the C terminus of the NR2 subunit. J Biol Chem 2004; 279: 28 903–10.

  9. 9

    Kutsuwada T, Kashiwabuchi N, Mori H, Sakimura K, Kushiya E, Araki K, et al. Molecular diversity of the NMDA receptor channel. Nature 1992; 358: 36–41.

  10. 10

    Meguro H, Mori H, Araki K, Kushiya E, Kutsuwada T, Yamazaki M, et al. Functional characterization of a heteromeric NMDA receptor channel expressed from cloned cDNAs. Nature 1992; 357: 70–4.

  11. 11

    Scott DB, Blanpied TA, Swanson GT, Zhang C, Ehlers MD . An NMDA receptor ER retention signal regulated by phosphorylation and alternative splicing. J Neurosci 2001; 21: 3063–72.

  12. 12

    Lan JY, Skeberdis VA, Jover T, Grooms SY, Lin Y, Araneda RC, et al. Protein kinase C modulates NMDA receptor trafficking and gating. Nat Neurosci 2001; 4: 382–90.

  13. 13

    Grant ER, Guttmann RP, Seifert KM, Lynch DR . A region of the rat N-methyl-D-aspartate receptor 2A subunit that is sufficient for potentiation by phorbol esters. Neurosci Lett 2001; 310: 9–12.

  14. 14

    Ali DW, Salter MW . NMDA receptor regulation by Src kinase signalling in excitatory synaptic transmission and plasticity. Curr Opin Neurobiol 2001; 11: 336–42.

  15. 15

    Liu WS, Heckman CA . The sevenfold way of PKC regulation. Cell Signal 1998; 10: 529–42.

  16. 16

    Zheng X, Zhang L, Wang AP, Bennett MV, Zukin RS . Protein kinase C potentiation of N-methyl-D-aspartate receptor activity is not mediated by phosphorylation of N-methyl-D-aspartate receptor subunits. Proc Natl Acad Sci USA 1999; 96: 15262–7.

  17. 17

    Jones ML, Leonard JP . PKC site mutations reveal differential modulation by insulin of NMDA receptors containing NR2A or NR2B subunits. J Neurochem 2005; 92: 1431–8.

  18. 18

    Zheng F, Gingrich MB, Traynelis SF, Conn PJ . Tyrosine kinase potentiates NMDA receptor currents by reducing tonic zinc inhibition. Nat Neurosci 1998; 1: 185–91.

  19. 19

    Luo JH, Fu ZY, Losi G, Kim BG, Prybylowski K, Vissel B, et al. Functional expression of distinct NMDA channel subunits tagged with green fluorescent protein in hippocampal neurons in culture. Neuropharmacology 2002; 42: 306–18.

  20. 20

    Zheng CY, Luo JH, Fu T, Yang W, Shen HQ . Surface expression of NMDA receptors composed of NR1 subunit and NR2A subunit mutants with partially deleted C-terminus in HEK293 cells. J Zhejiang Univ Med Sci 2003; 32: 475–9. Chinese.

  21. 21

    Grant ER, Bacskai BJ, Anegawa NJ, Pleasure DE, Lynch DR . Opposing contributions of NR1 and NR2 to protein kinase C modulation of NMDA receptors. J Neurochem 1998; 71: 1471–81.

  22. 22

    Wenthold RJ, Prybylowski K, Standley S, Sans N, Petralia RS . Trafficking of NMDA receptors. Annu Rev Pharmacol Toxicol 2003; 43: 335–58.

  23. 23

    Zannat MT, Locatelli F, Rybak J, Menzel R, Leboulle G . Identification and localisation of the NR1 subunit homologue of the NMDA glutamate receptor in the honeybee brain. Neurosci Lett 2006; 398: 274–9.

  24. 24

    Ishmael JE, Franklin PH, Murray TF, Leid M . High level expression of the NMDAR1 glutamate receptor subunit in electroporated COS cells. J Neurochem 1996; 67: 1500–10.

  25. 25

    Liao GY, Wagner DA, Hsu MH, Leonard JP . Evidence for direct protein kinase-C mediated modulation of N-methyl-D-aspartate receptor current. Mol Pharmacol 2001; 59: 960–4.

Download references

Author information

Correspondence to Jian-hong Luo.

Additional information

Project supported by the National Basic Research Program of China (G2002CB713808), the National Natural Science Foundation of China (No 30270436 and 30470547) and by the Outstanding Cross-century Faculty Grant from the Ministry of Education of China.

Rights and permissions

Reprints and Permissions

About this article


  • NMDA receptor
  • NR2A subunit
  • PMA
  • surface expression
  • HEK293 cells

Further reading