Fig. 8.

Overexpression of GluR1 and GluR2 C-terminal decoys disrupted the BDNF-mediated increase in AMPA receptors. Neocortical neurons were cultured for 4 days and then incubated with (+) or without (−) BDNF for the last 24 h before harvesting. The shorter BDNF treatment was still able to mimic a similar magnitude of the protein increase in AMPA receptors. During the incubation with BDNF, neurons were infected with the Sindbis viruses coding for EGFP alone, EGFP-R1 (A), or for EGFP-R2 (B) (Iwakura et al., 2001) to study the consequences of disrupting AMPA receptors’ interaction with their associated PDZ proteins. At the viral titre used (> 1 × 10⁷), more than 90% of the cells were infected, as monitored by EGFP fluorescence (Iwakura et al., 2001). The control EGFP construct was essentially the same as the EGFP-R1 and EGFP-R2 constructs but lacked the decoy peptides. (A and B) nsp, nonstructural proteins; psg, subgenomic promoter; and poly A; poly-adenylated transcript tail. Hatched boxes fused to the EGFP represent the C-terminal decoys of GluR1 and GluR2 (short splice variant). (C) Probing with anti-C-terminal GluR1 revealed the native GluR1 band as well as a lower band corresponding to the expected size of EGFP-R1 fusion protein (28 kDa). (D) Similarly, the BDNF-induced increase in GluR2/3 protein was challenged by the overexpression of EGFP-R2 fusion protein. Statistical analysis of the GluR1 (E) and GluR2/3 (F) immunoreactivities between pairs of BDNF-plus and BDNF-minus cultures (n = 3). GluR1 or GluR2/3 immunoreactivity in virus-free control culture was set as 100%. Student’s t-test was applied to comparison between BDNF-plus and BDNF-minus cultures (*P < 0.05; **P < 0.01) or between the cultures transfected with EGFP-R2 and EGFP alone (⁺P < 0.05). (G) The overexpression of EGFP-R1 and EGFP-R2 decoys reduced the BDNF-stimulated interaction between GluR1 and SAP97 and between GluR2/3 and GRIP1, respectively, compared with that in the EGFP overexpression. Cell lysates were prepared from infected neocortical cultures and treated with anti-N-terminal GluR1 antibody or anti-N-terminal GluR2 antibody to immunoprecipiate the complexes of AMPA receptors and PDZ protein(s).