PHOSPHORYLATION OF THE LPHA-AMINO-3-HYDROXY-5-METHYLISOXAZOLE-4-PROPIONIC ACID RECEPTOR GLUR1 SUBUNIT BY CALCIUM CALMODULIN-DEPENDENT KINASE-II/

Modulation of lpha-amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) receptors in the brain by protein phosphorylation may play a cr ucial role in the regulation of synaptic plasticity. Previous studies have demonstrated that calmodulin (CaM) kinase II can phosphorylate an d modulate AMPA receptors. However, the sites of CaM kinase phosphoryl ation have not been unequivocally identified. In the current study, we have generated two phosphorylation site-specific antibodies to analyz e the phosphorylation of the glutamate receptor GluR1 subunit. These a ntibodies recognize GluR1 only when it is phosphorylated on serine res idues 831 or 845. We have used these antibodies to demonstrate that se rine 831 is specifically phosphorylated by CaM kinase II in transfecte d cells expressing GluR1 as well as in hippocampal slice preparations, Two-dimensional phosphopeptide mapping experiments indicate that Ser- 831 is the major site of CaM kinase II phosphorylation on GluR1. In ad dition, treatment of hippocampal slice preparations with phorbol ester s and forskolin increase the phosphorylation of serine 831 and 845, re spectively, indicating that protein kinase C and protein kinase A phos phorylate these residues in hippocampal slices. These results identify the site of CaM kinase phosphorylation of the GluR1 subunit and demon strate that GluR1 is multiply phosphorylated by protein kinase A, prot ein kinase C, and CaM kinase II in situ.