Functional analysis of mutant G266E-FMRP. (a) Constitutive AMPA receptor assay showing that G266E-FMRP is unable to rescue exaggerated AMPA receptor internalization in KO neurons. Hippocampal neurons from Fmr1 KO mice were cultured for 18 days, infected with either WT or G266E-FMRP, and the percentage of internalized to total AMPA receptors was calculated from individual dendrites. G266E-FMRP-infected neurons were statistically different from WT-FMRP-infected neurons (one-way ANOVA; n=30; F=609.92, P<0.001, Tukey post hoc analysis: ***P<0.001 for all pairwise comparisons except WT versus KO+WT-FMRP P=0.42). As the variance in uninfected KO neurons was so low, G266E-infected neurons were still statistically different from KO neurons even though the mutant protein clearly does not rescue AMPA receptor internalization like WT-FMRP. Data are represented as boxplot with whiskers from minimum to maximum. (b) Polyribosome assay showing that G266E-FMRP does not associate with polyribosome fractions. The top graph is a representative A254 absorbance profile from Fmr1 KO MEF cells infected with either WT or G266E-FMRP, and the monosome (80S) and polyribosome peaks are indicated. Below is the distribution of FMRP by western blot analysis for each fraction corresponding to the same region of the linear sucrose gradient above. S6 ribosomal protein is also shown to verify sample loading in each well. These are representative blots from n=3 experiments. (c) RNA co-immunoprecipitation showing that G266E-FMRP does not bind three well-validated FMRP targets using qPCR analysis of the relative mRNA enrichment of Map1B, PSD95, and CamKII mRNAs after FMRP immunoprecipitation. Cortical neurons from Fmr1 KO mice were cultured for 10 days, infected with GFP, WT-FMRP, or G266E-FMRP lentivirus, and then processed for FMRP-RNA co-immunoprecipitation. The relative mRNA level for each primer set was normalized to each sample's β-actin mRNA and also relative FMRP expression level as determined by western blot densitometry. When mRNA enrichment (IP:input) for WT-FMRP is set to equal 1.0, G266E-FMRP mRNA enrichment drops by twofold to the same levels as GFP-infected neurons (paired Student's t-test; n=4; t=16.92, ***P<0.001 for Map1B; t=12.544, **P=0.001 for PSD95; t=12.919, **P=0.001 for CamKII). Data are represented as mean±SD.