, 2000). To assess

the role of TfR-tail-AP-1 interactions in this axonal exclusion, we coexpressed TfR-GFP with mCherry-tagged μ1A-WT or μ1A-W408S, together with Tau-cyan fluorescent protein (CFP) to identify axons. Live-cell imaging (Movie S3; Figures 5C and 5D) and kymographs (Figures 5C and 5D) showed significant Onalespib increases in the number of TfR-GFP-containing particles moving in anterograde direction (lines with negative slopes in the kymographs) as well as stationary particles (vertical lines in the kymographs) in the axons of cells expressing μ1A-W408S versus μ1A-WT (Figure 5E). The number of retrograde TfR-GFP-containing particles (lines with positive slopes in the kymographs) was not significantly changed (Figure 5E), although their average intensity increased. Regardless of the conditions, particles moving along the axon exhibited average speeds of 1.0–1.2 μm/s, characteristic of axonal transport carriers. From these experiments, we concluded that disruption of the TfR-tail-AP-1 interaction resulted in misincorporation PD0325901 order of TfR into axonal carriers at the level of the TGN/RE in the neuronal soma. To assess whether AP-1 also plays a role in the somatodendritic sorting of neuron-specific proteins, we extended

our studies to various glutamate receptors that mediate excitatory synaptic transmission critical for learning and memory (Riedel et al., 2003). These receptors included the metabotropic glutamate receptor 1 (mGluR1), the NR2A and NR2B subunits of NMDA-type, and the GluR1 and GluR2 subunits of AMPA-type ionotropic glutamate receptors. Y2H assays showed that portions of the C-terminal cytosolic domains of mGluR1, NR2A, and NR2B interacted with μ1A in a manner dependent on μ1A-W408 (Figure 6A). We did not attempt the identification of the receptor sequences involved in these interactions because the cytosolic domains are very long relative to those of TfR and CAR. However, the requirement of μ1A-W408 for interactions suggests the involvement of YXXØ-type sequences. In line with these binding assays, GFP-tagged forms of mGluR1, NR2A,

and NR2B localized exclusively to the somatodendritic domain in DIV10 neurons overexpressing μ1A-WT (polarity indexes: 6.3 ± 2.2 to Phosphoprotein phosphatase 9.6 ± 2.6; Table 1) but appeared in the axon upon overexpression of μ1A-W408S (polarity indexes: 1.3 ± 0.3 to 1.6 ± 0.4; Table 1) (Figure 6B). In contrast, the cytosolic domains of GluR1 and GluR2 did not exhibit interactions with μ1A in Y2H assays (Figure 6A), and GFP- or superecliptic pHluorin (SEP)-tagged forms of these receptor proteins were restricted to the somatodendritic domain regardless of the overexpression of μ1A-WT (polarity indexes: 8.1 ± 2.0 and 7.0 ± 1.4, respectively; Table 1) or μ1A-W408S (polarity indexes: 7.8 ± 2.0 and 6.9 ± 2.8, respectively; Table 1) (Figure 6B). The exclusive axonal localization of transgenic neuron-glia cell adhesion molecule (NgCAM) (Sampo et al., 2003; Wisco et al.