Fig 2: MPP2 binds directly to the synaptic adhesion molecule SynCAM1.(a) Targeted identification of putative MPP2 PDZ domain interactors: The 10 C-terminal amino acids of known synaptic PDZ-ligand proteins (see table on the left) were fused to the monomeric mCherry tag and tested for interaction with MYC-tagged MPP2 in coimmunoprecipitation experiments. Pulldown control (MYC-MPP2) and coprecipitated proteins (mCherry-PDZ-Ligand) were detected by western blot (upper panels); input controls are shown below. For uncropped blots, see Supplementary Fig. S3. (b) Isothermal titration calorimetry measurements with 350 μM mCherry-SynCAM1 and SynCAM1mut, respectively, injected to 55 μM MPP2-PSG module revealed a robust binding of the SynCAM1 C-terminus with a Kd of 3.1 ± 0.239 μM (left panels). Results for the mutated SynCAM1 C-terminus control are shown on the right. (c) Full-length HA-tagged SynCAM1 (HA-SynCAM1) and MYC-tagged MPP2 (MYC-MPP2), HA-tagged mutant SynCAM1 (HA-SynCAM1mut) and MYC-MPP2, or HA-SynCAM1 and MYC-tagged MPP2 SH3-GK (MYC-MPP2-SH3GK) were coexpressed in COS7 cells and immunoprecipitated with αHA (mHA IP) or mouse IgGs (mIgG IP) as a negative control. Pulldown controls are shown (HA-SynCAM); coprecipitated proteins are detected by western blot with αMYC antibody below. For uncropped blots, see Supplementary Fig. S3. (d) Immunoprecipitation of SynCAM1 (using a chicken SynCAM1-Biotin antibody or chicken IgYs as a negative control) from a crude synaptosome preparation of adult rat brain coprecipitates MPP2, as detected by western blot with antibodies to MPP2 (coIP) or SynCAM1 (IP control).(e) Cultured rat hippocampal neurons (E18) were fixed at DIV21 and stained for endogenous SynCAM1, MPP2 and MAP2 (depicted together with merged image on the right). Region selected for detailed image is indicated; scale bars: overview = 10 μm, detail = 5 μm.

Fig 3: MPP2 is a component of postsynaptic receptor complexes.(a) The AMPA receptor auxiliary subunit Stargazin/Tarpγ2 was immunoprecipitated from crude synaptosomal preparations of adult rat brain with a monoclonal mouse antibody and precipitates were analysed by western blot, with antibodies to synaptic proteins, as indicated. A pull-down with mouse IgGs (mIgG IP) was performed as negative control (a non-specific IgG background band is marked with a*). For uncropped blots, see Supplementary Fig. S2. (b) Cultured rat hippocampal neurons (E18) were fixed at DIV28 and stained for MPP2 (left panels) together with the microtubule-associated protein 2 (MAP2, dendritic marker), the postsynaptic density protein 95 (PSD-95, postsynaptic marker) or the vesicular glutamate transporter 1 (vGlut1, presynaptic marker) and respective secondary antibodies. Overlapping signals are visible in the merged images on the right, with the indicated regions selected for detailed images. Scale bars: overview = 10 μm, detail = 5 μm.

Fig 4: MPP2 interacts with the postsynaptic density proteins PSD-95 and GKAP, and it homomultimerises.(a) Structure of the murine membrane-associated guanylate kinase protein MPP2 and corresponding deletion constructs used in this study. L27 domains (green), PDZ domain (blue) and SH3-GK tandem domain (red, purple). (b) Immunoprecipitation of MPP2 (using a rabbit polyclonal MPP2 antibody or rabbit IgGs as a negative control) from a crude synaptosome preparation of adult rat brain coprecipitates PSD-95, as detected by western blot with antibodies to PSD-95 (coIP) or MPP2 (IP control). A background IgG signal is marked with a*. (c) MYC-tagged MPP2 (MPP2), MYC-tagged MPP2 PDZ-SH3GK (PSG), or MYC-tagged MPP2 SH3GK (SH3GK) were coexpressed in COS7 cells with recombinant PSD-95 and immunoprecipitated with αPSD-95 antibody (mPSD-95 IP) or with mouse IgGs (mIgG IP) as a negative control. Coprecipitated proteins were analysed by western blot with αMYC antibody, pulldown controls with a PSD-95 antibody. (d) MYC-tagged MPP2 (MPP2), MYC-tagged MPP2 PDZ-SH3GK (PSG), or MYC-tagged MPP2 SH3GK (SH3GK) were coexpressed with FLAG-tagged recombinant GKAP in COS7 cells and immunoprecipitated with αMYC antibody (mMYC IP) or with mouse IgGs (mIgG IP) as a negative control. Coprecipitated proteins were analysed by western blot with αFLAG antibody, pulldown controls with αMYC antibody. (e) FLAG- and MYC-tagged MPP2 proteins were coexpressed in COS7 cells. MYC-MPP2 was pulled down with αMYC antibody (mMYC IP) and coprecipitated FLAG-tagged MPP2 was analysed by western blot with αFLAG antibody. Mouse IgGs (mIgG IP) served as a negative control. For uncropped blots, see Supplementary Fig. S2.