FIGURE 6.
NMDA-induced GluA1 turnover is altered in LRP1-deficient neurons. A, primary cultured cortical neurons were prepared from E15 NesCreLRP1lox/lox (KO) and E15 LRP1wt/lox (Control) mouse embryos. Internal and surface GluA1 were analyzed under NMDA treatment. To measure internal GluA1, on DIV 10–12, cell surface proteins were biotinylated, and then neurons were treated with NMDA (20 μm, 3 min). Neurons were washed and incubated in fresh medium for the time indicated to allow endocytosis of GluA1. After stripping off cell surface biotin, whole cell lysates were prepared, and biotinylated proteins were precipitated with Neutravidin. To measure surface GluA1, on DIV 10–12, neurons were first treated with NMDA (20 μm, 3 min). Then neurons were washed and incubated in fresh medium for the time indicated. Subsequently, cell surface proteins were biotinylated. Whole cell lysates were prepared, and biotinylated proteins were precipitated with Neutravidin. Aliquots from the lysates and supernatants were analyzed by immunoblotting with anti-LRP1 and anti-GluA1 antibodies. Actin served as a loading control and as an indicator of purity for surface samples. Shown are representative Western blots of total and internalized or surface GluA1, respectively (a), and quantitative analysis of total (for internalized analysis) GluA1 (b), internalized GluA1 (b), total (for surface analysis) GluA1 (b), and surface GluA1 (b). All values are normalized to actin from total lysate. Bars, means ± S.E. (error bars) (Student's t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001, n = 4 for internal GluA1, n = 3 for surface GluA1). B, detergent-soluble synaptosomal membrane fractions from DIV 10–12 wild type cortical neurons were prepared and subjected to immunoprecipitation with an anti-GluA1 antibody or rabbit-IgG as a negative control. Precipitated proteins were examined with an antibody against LRP1 by immunoblotting. Representative blots of five independent experiments are shown.