A Study Of Regulation Of GluN2B Containing NMDA Receptor By Phosphorylation

Glutamate is the most important excitatory transmitter in mammalian central nervous system. There are two types of receptor for the ligand of glutamate in vivo. One is metabolic glutamate receptor (mGluR), the other is ionotropic glutamate receptor (iGluR). Among the iGluRs, based on their affinity of agonist and the properties of pharmacology and structure, they can be divided into three groups:the first one is N-methyl-D-aspartate receptor (NMDAR), the second one is a-amino-3-hydroxy-5-methylisoxazole-4-pro pionic acid receptor (AMPAR), the third one is2-carboxy-3-carboxymethyl-4-isopropenylpyrrolidine receptor(kainate receptor, KAR). NMDAR has its unique character among these three iGluRs. In general, NMDAR is a voltage and ligand gated cation channel, while it has more calcium permeability than sodium and potassium. In central nervous system, NMDAR is thought to be open only when agonist glutamate and co-agonist glycine or serine are bound to the receptor under proper voltage. NMDAR is demonstrated to form functional receptor as a tetramer, constituting two essential subunits GluNl, two identical or different subunits GluN2(A-D), occasionally with inhibitory subunit GluN3(A-B). Growing evidences indicate GluN2subunits endow NMDAR with diverse properties.GluN2A and GluN2B are extensively studied for its function importance and dominant expression profile in brain. As for GluN2B, its expression begins in early development and maintains in adulthood. Previous study has shown that GluN2B is the predominant tyrosine-phosphorylated protein in the postsynaptic density (PSD). In the past two decades, with advancing of the techniques of proteomics, a lot of phosphorylation sites of GluN2B have been identified and studied, leading us to in-depth understanding the function of the phosphorylation site itself and its regulator role in receptor. Acompanying with the identify of the phosphorylation sites, their corresponding kinases are acknowledged in the same time. For instance, up to date, the most primary tyrosine kinase for GluN2B is Src family kinase. All of the identified tyrosine phosphorylation sites in GluN2B, are demonstrated to be substrates of Src family kinase, including our focus of Y1070in the present study and other previous reported tyrosine sites, Y1252, Y1336, Y1472. As known, Src family kinase is profoundly involved in regulation of receptor function via phosphorylating the receptors. Previously, there are reports that Src family kinase regulates NMDAR through phosphorylating GluN2B subunit. For example, Src family kinase can phosphorylate Y1472located on the extreme distal of GluN2B. With phosphorylation occurring, the event impairs the ability of GluN2B associated with clathrin adaptor protein AP-2, thus eliciting the imbalance of surface delivery and endocytosis of GluN2B, finally leading the consequence of enhanced surface expression of GluN2B.In the present study, we mainly focused on two novel phosphorylation sites in GluN2B. They are tyrosine1070(Y1070) and serine1284(S1284), respectively. With combination of molecular cloning, cell imaging, biochemistry and electrophysiology, we explored the function of the phosphorylation site. By kinase inhibitor and overexpression in heterogenous cells, we found that Y1070was phosphorylated by Fyn, a member of Src family kinase. Using biotinylation assay and immuocytochemistry, we observed that phosphorylated GluN2B at Y1070was enriched in PSD and cell surface. We made mutation of GluN2B at Y1070to phenylalanine, whose mutation is a phosphorylation deficient for the mutant being not able to be phosphorylated by kinase. The results indicated that phosphorylation deficient mutation of GluN2B at Y1070caused decreased surface expression of GluN2B containing NMDAR in heterogenous cells as well as neurons. We further addressed the mechanism why such phosphorylation deficient mutation reduced its surface expression. We found that tyrosine to phenylalanine mutation at1070of GluN2B, disrupting the association of GluN2B with Fyn, subsequently resulting in reduction of phosphorylation level of Y1472, which is function importance for surface expression of GluN2B, finally bringing about decreased surface number of GluN2B containing NMDAR. We also found that cLTD reduced the phosphorylation level of S1284, while cLTP caused no significant changes of the phosphorylation level of S1284. In HEK293cells, phosphorylation deficient mutation (S1284A) and phosphorylation mimic mutation (S1284E) did not result in any differences in GluN2B’s association with PSD95.Taken together, we identified and explored two novel phosphorylation sites of GluN2B. The results suggested that the tyrosine phosphorylation site was much relevant to surface expression of GluN2B and the binding of GluN2B and Fyn. Further efftors are needed for exploration of the functional roles of the two phophorylation sites of GluN2B in physiology as well as pathology process.