The postsynaptic density protein 95 (PSD-95) takes a central role in the regulation of neuronal communication by engaging with membrane receptors, ion channels and cytosolic proteins via its three PSD-95/Discs large/ZO-1 (PDZ) domains. Therefore, PSD-95 is essential for sustaining the molecular organization of the postsynaptic density and misregulation is linked to neurological diseases. PDZ-mediated interactions are highly regulated by phosphorylation, either of the PSD-95 or the interacting C-terminal receptor ligand. The object of this studies is to map C-terminal binding partners of PSD-95 PDZ domains, as well as to examine how the plasticity of these interactions is regulated by phosphorylation and elucidate the functional consequences for the postsynaptic density.

As part of the PDZnet project, which is funded from the European Union’s H2020 framework program under the Marie Skłodowska-Curie Actions, we developed a next-generation inverted SPOT peptide array that is compatible with phosphorylated and unnatural amino acids, to map interaction partners of PSD-95 PDZ domains in a high-throughput manner. We applied the technology to map the PDZ-mediated interactome of PSD-95 and examined how plasticity of these interactions is regulated by phosphorylation. The identified regulations sides were validated by fluorescence polarization (FP) and isothermal isothermal titration calorimetry (ITC) assay. Furthermore, based on the acquired data of positive and negative regulation, we are performing pull-down experiments from neuron lysates to obtain fundamental insights into the dynamic regulation of PDZ-mediated interactions of PSD-95 by phosphorylation.