Identification of interactors that differentially bind to the carboxyl-terminal SH3GK modules of MPP2 and/or PSD-95.

(a) Schematic domain structures of PSD-95 and MPP2 drawn to scale and aligned by their central PDZ domain. Both proteins contain two N-terminal domains (PDZ1+PDZ2 for PSD-95 and two L27 domains for MPP2) in addition to the carboxyl-terminal “MAGUK core” domains PDZ-SH3-GK. Note the differences in the length of the “linker” between PDZ and SH3 domain and of the “hook” between SH3 and GK domains. (b) Schematic representation of the quantitative LC–MS/MS experiment using ¹⁶O/¹⁸O-labelling to identify differential interactors from adult rat brain crude synaptosomal preparations by GST pull-down of bacterially expressed GST-MPP2-SH3GK or GST-PSD-95-SH3GK. (c) GST pull-downs were performed in duplicates with inverted labelling and 188 interacting proteins were identified and quantified by mass spectrometry passing our threshold settings. PSD-95/MPP2 protein ratios from both replicates A and B (normalised by the ratio of GST) are plotted against each other. Proteins in the first quadrant indicate preferential enrichment to PSD-95 (ratios >> 1), while proteins in the third quadrant indicate preferential enrichment to MPP2 (ratios << 1). Proteins with ratios of approximately 1 show no preferential binding and thus reflect equal binding to both baits or background proteins that were not fully removed by the washing steps. Selected novel potential interaction partners were validated by co-immunoprecipitation (yellow, see also S6 Fig). The most significantly enriched proteins to the GST-SH3GK construct of MPP2 (green cluster) are 7 different GABA_A receptor subunits, of which α1, α2, and β3 have been validated for direct interaction with MPP2 (see also Fig 4). For further details, see also Table 1 and S1 Data. GABA, γ-aminobutyric acid; LC–MS/MS, liquid chromatography with tandem mass spectrometry; MAGUK, membrane-associated guanylate kinase; MPP2, membrane protein palmitoylated 2; PSD, postsynaptic density.