PSD-95 clustering and its association with NMDA receptors are required for the PSD-95-mediated effect. HEK293 cells cotransfected with the NR1a/2A combination and wild-type (WT) PSD-95 or mutant PSD-95 (C3,5S) were treated with 100 μm glutamate and 100 μm glycine in the presence or absence of 100 μm MK-801 for 0 or 30 min and then labeled with NHS-biotin under nonlytic conditions. The extracellular and intracellular fragments were separated and subjected to Western blotting with an N-terminal antibody for NR2A (A-6475). The amount of immunoreactivity in the lysate (L), extracellular (E), and intracellular (I) fractions was quantified as a percentage of the MK-801 control condition. In cells transfected with PSD-95 (C3,5S), a representative Western blot and bar graph (A, B) demonstrate reduced levels of NR2A immunoreactivity after calpain activation in the intracellular fraction (47% decrease from the 0 min control) (n = 6; p = 0.021) and the extracellular fraction (34% decrease from the 0 min control) (n = 6; p = 0.0285), showing that the PSD-95 mutant (C3,5S) does not block calpain-mediated cleavage of NR2A in either the intracellular and extracellular fractions. An anti-actin antibody confirmed that actin remains primarily in the intracellular fraction. C, HEK293 cells cotransfected with the NR1a/2A combination and wild-type PSD-95 (WT) or mutant PSD-95 (C3,5S) were immunoprecipitated with an anti-NR2A antibody. Input, Total cell lysate; IPNR2A, immunoprecipitated fraction. The resulting blots were probed with both NR2A and PSD-95 antibodies. Representative Western blots show the disruption of the NR2A-PSD-95 interaction by transfection with PSD-95 (C3,5S). D, HEK293 cells cotransfected with the NR1a/2A combination and SAP102 were treated with 100 μm glutamate and 100 μm glycine in the presence or absence of 100 μm MK-801 for 0 or 30 min and analyzed by Western blotting. The amount of NR2A immunoreactivity was quantified as a percentage of the MK-801 control condition. Cotransfection with SAP102 did not protect NR2A from calpain-mediated cleavage (46% decrease from 0 min) (n = 5; p = 0.025). Data are expressed as mean ± SEM (error bars).
