CHARACTERIZATION OF PROTEIN-KINASE-A AND PROTEIN-KINASE-C PHOSPHORYLATION OF THE N-METHYL-D-ASPARTATE RECEPTOR NR1 SUBUNIT USING PHOSPHORYLATION SITE-SPECIFIC ANTIBODIES

Modulation of N-methyl-D-aspartate receptors in the brain by protein p hosphorylation may play a central role in the regulation of synaptic p lasticity, To examine the phosphorylation of the NR1 subunit of N-meth yl-D-aspartate receptors in situ, we have generated several polyclonal antibodies that recognize the NR1 subunit only when specific serine r esidues are phosphorylated. Using these antibodies, we demonstrate tha t protein kinase C (PKC) phosphorylates serine residues 890 and 896 an d cAMP-dependent protein kinase (PKA) phosphorylates serine residue 89 7 of the NR1 subunit. Activation of PKC and PKA together lead to the s imultaneous phosphorylation of neighboring serine residues 896 and 897 , Phosphorylation of serine 890 by PKC results in the dispersion of su rface associated clusters of the NR1 subunit expressed in fibroblasts, while phosphorylation of serine 896 and 897 has no effect on the subc ellular distribution of NR1. The PKC-induced redistribution of the NR1 subunit in cells occurs within minutes of serine 890 phosphorylation and reverses upon dephosphorylation. These results demonstrate that PK A and PKC phosphorylate distinct residues within a small region of the NR1 subunit and differentially affect the subcellular distribution of the NR1 subunit.