Figure 2.
LRRTM2 Regulates GluR1 Surface Expression in Hippocampal Neurons and Interacts with Postsynaptic Proteins
(A) Hippocampal neurons were electroporated with the sh-vector (sh-vec), sh-LRRTM2, and sh-LRRTM2 with human myc-LRRTM2 (sh-LRRTM2 + hLRRTM2), surface labeled for GluR1, and immunostained at DIV 15 for GFP and VGlut1. (B) Quantification of the density of synaptic surface GluR1 puncta (VGlut1/GluR1 colocalization) per length of dendrite normalized to sh-vec control neurons (**p < 0.001). (C) Quantification of the GluR1 surface puncta per length of dendrite (**p < 0.01). (D) Quantification of the VGlut1 puncta per 10 μm dendrite sh-vec 0.68 ± 0.03 (mean ± SEM); sh-LRRTM2 0.59 ± 0.02; sh-LRRTM2 + hLRRTM2 0.70 ± 0.04. Bar graphs show mean ± SEM (difference not significant by ANOVA; p = 0.02 by Student’s t test for control versus sh-LRRTM2). (E–G) 293T cells were cotransfected with myc-LRRTM2 mutant constructs and GluR1-GFP (E) or GluR2-GFP (F). Cell lysates were immunoprecipitated with myc antibodies and were analyzed by western blot using anti-GFP antibodies. EphB2-YFP was used as a negative control. A representative blot showing input samples probed with myc antibody is shown in (G). High and low molecular weight bands for LRRTM2 on western blot are probably due to glycosylation of LRRTM2. (H) Coexpression of PSD-95-mCherry with myc-LRRTM2 in 293T cells induces translocation to the cell membrane of PSD-95, which is diffusely distributed throughout the cytosol in GFP-expressing control cells. (I) Immunoprecipitation of myc-LRRTM2 deletion mutants lacking the cytoplasmic domain (LRRTM2 ΔC) or the C-terminal ECEV motif (LRRTM2 ΔECEV) coexpressed with PSD-95-mCherry in 293T cells. LRRTM2 interacts with PSD-95-mCherry via the ECEV motif. The high molecular weight band is shown for LRRTM2. Molecular weight markers in kDa indicated on the right. Scale bar in (A) and (H), 10 μm.