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Dopamine D1 receptor-mediated NMDA receptor insertion depends on Fyn but not Src kinase pathway in prefrontal cortical neurons.

Hu JL, Liu G, Li YC, Gao WJ, Huang YQ - Mol Brain (2010)

Bottom Line: Activation of D1 receptor by selective agonist SKF-81297 significantly increased the expression of NR2B subunits.Under control conditions, neither Fyn nor Src knockdown exhibited significant effect on basal NR2B expression.D1 stimulation significantly enhanced NR2B insertion into plasma membrane in cultured PFC neurons, a process obstructed by Fyn, but not Src, knockdown.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA 19129, USA. jh356@drexel.edu

ABSTRACT

Background: Interactions between dopamine and glutamate in the prefrontal cortex are essential for cognitive functions such as working memory. Modulation of N-methyl-D-aspartic acid (NMDA) receptor functions by dopamine D1 receptor is believed to play a critical role in these functions. The aim of the work reported here is to explore the signaling pathway underlying D1 receptor-mediated trafficking of NMDA receptors in cultured rat prefrontal cortical neurons.

Results: Activation of D1 receptor by selective agonist SKF-81297 significantly increased the expression of NR2B subunits. This effect was completely blocked by small interfering RNA knockdown of Fyn, but not Src. Under control conditions, neither Fyn nor Src knockdown exhibited significant effect on basal NR2B expression. D1 stimulation significantly enhanced NR2B insertion into plasma membrane in cultured PFC neurons, a process obstructed by Fyn, but not Src, knockdown.

Conclusions: Dopamine D1 receptor-mediated increase of NMDA receptors is thus Fyn kinase dependent. Targeting this signaling pathway may be useful in treating drug addiction and schizophrenia.

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Related in: MedlinePlus

Fyn, but not Src, affects the surface NR2B expression after D1 receptor stimulation. (A and B) Surface biotinylation of NMDA receptors in high-density PFC neurons at 14 DIV. PFC neurons at12 DIV were transfected with Fyn siRNA for 48 h. The PFC neurons at DIV 14 were treated with DMSO (0.1%, lanes 1 and 2); SKF-81297 (10 μM, lanes 3 and 4); Fyn (A) or Src (B) knockdown PFC neurons treated with DMSO (0.1%, lanes 5 and 6); or Fyn (A) or Src (B) knockdown neurons treated with SKF-81297 (10 μM, lanes 7 and 8). After surface receptor biotinylation, 20% of the lysis supernatant was used to detect the total (T) proteins. The remaining 80% of the supernatant was incubated with NeurAvidin Agarose beads and purified as surface (S) proteins. After SDS-PAGE the proteins were immunoblotted for NR2B and β-tubulin. (C and D) Quantification of surface NR2B expression. Surface NR2B was corrected by total NR2B to calculate the surface/total ratio and the control group was set to 100% for normalization. Fyn, but not Src, knockdown appeared to be effective in blocking the surface insertion of NR2B mediated by D1 stimulation (* p < 0.05, ** p < 0.01).
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Figure 7: Fyn, but not Src, affects the surface NR2B expression after D1 receptor stimulation. (A and B) Surface biotinylation of NMDA receptors in high-density PFC neurons at 14 DIV. PFC neurons at12 DIV were transfected with Fyn siRNA for 48 h. The PFC neurons at DIV 14 were treated with DMSO (0.1%, lanes 1 and 2); SKF-81297 (10 μM, lanes 3 and 4); Fyn (A) or Src (B) knockdown PFC neurons treated with DMSO (0.1%, lanes 5 and 6); or Fyn (A) or Src (B) knockdown neurons treated with SKF-81297 (10 μM, lanes 7 and 8). After surface receptor biotinylation, 20% of the lysis supernatant was used to detect the total (T) proteins. The remaining 80% of the supernatant was incubated with NeurAvidin Agarose beads and purified as surface (S) proteins. After SDS-PAGE the proteins were immunoblotted for NR2B and β-tubulin. (C and D) Quantification of surface NR2B expression. Surface NR2B was corrected by total NR2B to calculate the surface/total ratio and the control group was set to 100% for normalization. Fyn, but not Src, knockdown appeared to be effective in blocking the surface insertion of NR2B mediated by D1 stimulation (* p < 0.05, ** p < 0.01).

Mentions: Finally, we used a surface biotinylation assay to examine whether Fyn and Src affect NR2B trafficking under this condition. We found that treatment of high-density PFC cultured neurons with D1 receptor agonist SKF-81297 significantly increased the ratio of surface/total NR2B expression compared with the control group (n = 3, p < 0.01; Figure 7). As Figure 4 shows, neither Fyn nor Src knockdown affected total protein levels of NR2B expression (Fyn knockdown: n = 3, p = 0.45; Src knockdown: n = 3, p = 0.10). In contrast to the almost unaltered surface NR2B expression under conditions of activation of D1 agonist SKF-81297 after Src knockdown (n = 3, p < 0.05; Figure 7B and 7D), however, the D1 effects on the surface NR2B expression were completely blocked by Fyn knockdown (n = 3, p = 0.448; Figure 7A and 7C). Taken together, these results demonstrate that D1 receptor activation specifically increases surface NR2B expression by a Fyn-dependent signaling pathway, whereas Src is not involved.


Dopamine D1 receptor-mediated NMDA receptor insertion depends on Fyn but not Src kinase pathway in prefrontal cortical neurons.

Hu JL, Liu G, Li YC, Gao WJ, Huang YQ - Mol Brain (2010)

Fyn, but not Src, affects the surface NR2B expression after D1 receptor stimulation. (A and B) Surface biotinylation of NMDA receptors in high-density PFC neurons at 14 DIV. PFC neurons at12 DIV were transfected with Fyn siRNA for 48 h. The PFC neurons at DIV 14 were treated with DMSO (0.1%, lanes 1 and 2); SKF-81297 (10 μM, lanes 3 and 4); Fyn (A) or Src (B) knockdown PFC neurons treated with DMSO (0.1%, lanes 5 and 6); or Fyn (A) or Src (B) knockdown neurons treated with SKF-81297 (10 μM, lanes 7 and 8). After surface receptor biotinylation, 20% of the lysis supernatant was used to detect the total (T) proteins. The remaining 80% of the supernatant was incubated with NeurAvidin Agarose beads and purified as surface (S) proteins. After SDS-PAGE the proteins were immunoblotted for NR2B and β-tubulin. (C and D) Quantification of surface NR2B expression. Surface NR2B was corrected by total NR2B to calculate the surface/total ratio and the control group was set to 100% for normalization. Fyn, but not Src, knockdown appeared to be effective in blocking the surface insertion of NR2B mediated by D1 stimulation (* p < 0.05, ** p < 0.01).
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Related In: Results  -  Collection

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Figure 7: Fyn, but not Src, affects the surface NR2B expression after D1 receptor stimulation. (A and B) Surface biotinylation of NMDA receptors in high-density PFC neurons at 14 DIV. PFC neurons at12 DIV were transfected with Fyn siRNA for 48 h. The PFC neurons at DIV 14 were treated with DMSO (0.1%, lanes 1 and 2); SKF-81297 (10 μM, lanes 3 and 4); Fyn (A) or Src (B) knockdown PFC neurons treated with DMSO (0.1%, lanes 5 and 6); or Fyn (A) or Src (B) knockdown neurons treated with SKF-81297 (10 μM, lanes 7 and 8). After surface receptor biotinylation, 20% of the lysis supernatant was used to detect the total (T) proteins. The remaining 80% of the supernatant was incubated with NeurAvidin Agarose beads and purified as surface (S) proteins. After SDS-PAGE the proteins were immunoblotted for NR2B and β-tubulin. (C and D) Quantification of surface NR2B expression. Surface NR2B was corrected by total NR2B to calculate the surface/total ratio and the control group was set to 100% for normalization. Fyn, but not Src, knockdown appeared to be effective in blocking the surface insertion of NR2B mediated by D1 stimulation (* p < 0.05, ** p < 0.01).
Mentions: Finally, we used a surface biotinylation assay to examine whether Fyn and Src affect NR2B trafficking under this condition. We found that treatment of high-density PFC cultured neurons with D1 receptor agonist SKF-81297 significantly increased the ratio of surface/total NR2B expression compared with the control group (n = 3, p < 0.01; Figure 7). As Figure 4 shows, neither Fyn nor Src knockdown affected total protein levels of NR2B expression (Fyn knockdown: n = 3, p = 0.45; Src knockdown: n = 3, p = 0.10). In contrast to the almost unaltered surface NR2B expression under conditions of activation of D1 agonist SKF-81297 after Src knockdown (n = 3, p < 0.05; Figure 7B and 7D), however, the D1 effects on the surface NR2B expression were completely blocked by Fyn knockdown (n = 3, p = 0.448; Figure 7A and 7C). Taken together, these results demonstrate that D1 receptor activation specifically increases surface NR2B expression by a Fyn-dependent signaling pathway, whereas Src is not involved.

Bottom Line: Activation of D1 receptor by selective agonist SKF-81297 significantly increased the expression of NR2B subunits.Under control conditions, neither Fyn nor Src knockdown exhibited significant effect on basal NR2B expression.D1 stimulation significantly enhanced NR2B insertion into plasma membrane in cultured PFC neurons, a process obstructed by Fyn, but not Src, knockdown.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA 19129, USA. jh356@drexel.edu

ABSTRACT

Background: Interactions between dopamine and glutamate in the prefrontal cortex are essential for cognitive functions such as working memory. Modulation of N-methyl-D-aspartic acid (NMDA) receptor functions by dopamine D1 receptor is believed to play a critical role in these functions. The aim of the work reported here is to explore the signaling pathway underlying D1 receptor-mediated trafficking of NMDA receptors in cultured rat prefrontal cortical neurons.

Results: Activation of D1 receptor by selective agonist SKF-81297 significantly increased the expression of NR2B subunits. This effect was completely blocked by small interfering RNA knockdown of Fyn, but not Src. Under control conditions, neither Fyn nor Src knockdown exhibited significant effect on basal NR2B expression. D1 stimulation significantly enhanced NR2B insertion into plasma membrane in cultured PFC neurons, a process obstructed by Fyn, but not Src, knockdown.

Conclusions: Dopamine D1 receptor-mediated increase of NMDA receptors is thus Fyn kinase dependent. Targeting this signaling pathway may be useful in treating drug addiction and schizophrenia.

Show MeSH
Related in: MedlinePlus