We generated a polyclonal antibody, 12P3, specifically recognizing rat AMPA-type glutamate receptor (GluR) subunits phosphorylated at Ser-696 of GIuR2 or at the homologous sites in GIuRl, GIuR3, and GIuR4. Using 12P3, we demonstrate that a brief exposure of a rat cerebellar slice to AMPA leads to transient phosphorylation of the GluR subunits in Purkinje cell dendrites. Persistent phosphorylation over 30 min was obtained when exposure to AMPA was preceded by a 15 min perfusion of the slice with 8-bromo-cGMP, dibutyryl-cGMP, or calyculin A but not phorbol 12,13-diacetate. These results indicate that Ser-696 of GIuR2, or the corresponding sites in other AMPA receptor subunits, is a specific site at which phosphorylation takes place when AMPA-type GluRs are activated by agonists, especially under the influence of certain second messenger activities. © 1995.
