Protein kinase Cγ associates directly with the GluR4 α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit -: Effect on receptor phosphorylation

Ionotropic glutamate receptors mediate the majority of excitatory synaptic transmission in the brain and are thought to be involved in learning and memory formation. The activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors can be regulated by direct phosphorylation of their subunits, which affects the electrophysiological properties of the receptor, and the receptor association with numerous proteins that modulate membrane traffic and synaptic targeting of the receptor. In the present study we investigated the association of protein kinase C (PKC) gamma isoform with the GluR4 AMPA receptor subunit. PKCgamma was co-immunoprecipitated with GluR4 AMPA receptor subunit in rat cerebellum and in cultured chick retina cell extracts, and immunocytochemistry experiments showed co-localization of GluR4 and PKCgamma in cultured chick retinal neurons. Pull-down assays showed that native PKCgamma binds the GIuR4 C-terminal membrane-proximal region, and recombinant PKCgamma was retained by GST-GluR4 C-terminal fusion protein, suggesting that the kinase binds directly to GluR4. Furthermore, GST-GluR4 C-terminal protein was phosphorylated on GluR4 Ser-482 by bound kinases, retained by the fusion protein, including PKCgamma. The GluR4 C-terminal segment that interacts with PKCgamma, which lacks the PKC phosphorylation sites, inhibited histone H1 phosphorylation by PKC, to the same extent as the PKC pseudosubstrate peptide 19-31, indicating that PKCgamma bound to GluR4 preferentially phosphorylates GluR4 to the detriment of other substrates. Additionally, PKCgamma expression in GluR4 transfected human embryonic kidney 293T cells increased the amount of plasma membrane-associated GluR4. Our results suggest that PKCgamma binds directly to GluR4, thereby modulating the function of GluR4-containing AMPA receptors.