Brain receptors are involved in synaptic changes underlying learning and memory in central nervous system. Although receptor subunits have been linked to memory formation, data on the involvement of the receptor complexes is limited. In addition, post-translation modifications account for a number of activities such as gene expression, activation /deactivation of enzymatic activity, protein stability or disruption, mediation of protein-protein interactions, and modulation of protein cellular function involved in signalling and regulatory mechanisms. Recent studies have addressed the involvement of post-translational modifications (PTMs) in cognitive processes. It is well documented that post-translation modifications such as phosphorylation of serine, threonine and tyrosine play a role in the synaptic plasticity of the brain, neurotransmitter release, and vesicle trafficking. The aim of our study was to investigate whether learning paradigms change the composition of NMDA- and AMPA- receptor complexes in mice. Subsequently, identification of receptor complexes, their post- translational modifications and their binding partners are proposed.
Therefore, in order to study the involvement of native receptor complexes, mice were trained and compared to untrained mice in MTM (Multiple T maze) and BM (Barnes maze). After the completion of training, mice were euthanized, hippocampal samples were taken, proteins were extracted and run on native polyacrylamide gels, followed by immunoblotting with specific antibodies against mouse brain receptors.
To identify the brain receptors, their post-translational modifications and their compositions, immunoprecipitation with specific antibodies against receptors from hippocampi was carried out and samples were run under denature condition. Spots were picked, destained, treated with trypsin and chymotrypsin, and peptides were identified by tandem mass spectrometry (Nano-LC-ESI-MS/MS). We found that the level GluN1- and GluA1-containing complexes increased in trained mice and GluA2-containing complexes decreased in trained mice. Furthermore, The NMDA receptor subunits (GluN1 and GluN2A-D), AMPA receptor subunits, five novel phosphorylation sites on NMDAR (Serine S511 on GluN2A and S886, S917, S1303 and S1323 on GluN2B), and two protein kinases (Protein kinase C gamma and Calcium/calmodulin-dependent protein kinase type 2 subunit beta), which are associated with NMDA receptor, were identified by mass spectrometry. Herein, we apply native polyacrylamide gels to determine and study hippocampal receptor complexes in learning and memory. The results extend our knowledge on NMDA- and AMPA- receptor complexes. In addition, five novel phosphorylation sites on NMDA receptor are detected. This new discovery is useful for further studies in terms of receptor's PTM quantification in learning / memory and disease.