Regulation of AMPA receptor phosphorylation by the neuropeptide PACAP38

Dynamic changes in synaptic strength are thought to be critical for higher brain function such as learning and memory. Alterations in synaptic strength can result from modulation of AMPA receptor (AMPAR) function and trafficking to synaptic sites. The phosphorylation state of AMPAR subunits is one mechanism by which cells regulate receptor function and trafficking. Receptor phosphorylation is in turn regulated by extracellular signals; these include neuronal activity, neuropeptides, and neuromodulators such as dopamine and norepinephrine (NE). Although numerous studies have reported that the neuropeptide pituitary adenylate cyclase activating polypeptide 38 (PACAP38) alters hippocampal CA1 synaptic strength and GluA1 synaptic localization, its effect on AMPAR phosphorylation state has not been explored. We determined that PACAP38 stimulation of hippocampal cultures increased phosphorylation of S845, and decreased phosphorylation of T840 on the GluA1 AMPAR subunit. Increases in GluA1 S845 phosphorylation primarily occurred via PAC1 and VPAC2 receptor activation, whereas a reduction in GluA1 T840 phosphorylation was largely driven by PAC1 receptor activation and to a lesser extent by VPAC1 and VPAC2 receptor activation. GluA1 S845 phosphorylation could be blocked by a PKA inhibitor, and GluA1 T840 dephosphorylation could be blocked by a protein phosphatase 1/2A (PP1/PP2A) inhibitor and was partly blocked by a NMDA receptor (NMDAR) antagonist. These results demonstrate that the neuropeptide PACAP38 inversely regulates the phosphorylation of two distinct sites on GluA1 and may play an important role modulating AMPAR function and synaptic plasticity in the brain.AMPA-type glutamate receptors (AMPARs) are a tetrameric assembly composed of the GluA1, 2, 3, or 4 subunits. Within the adult hippocampus, receptors consist of primarily GluA1/2 and GluA2/3 complexes (1). Because AMPARs conduct the majority of excitatory transmission in the brain, modulation of AMPAR synaptic transmission is a powerful tool by which the cell can regulate synaptic strength and cell firing. Furthermore, it is hypothesized that complex behaviors such as learning, memory, and drug addiction involve alterations in synaptic strength (2, 3).The cell can regulate synaptic strength through changes in AMPAR conductance, trafficking, and tethering at synaptic sites. Such changes can be achieved through alterations in AMPAR expression, binding partners, and posttranslational modifications (4). A number of GluA1 and GluA2 phosphorylation sites have been proposed to play a role in AMPAR trafficking and synaptic plasticity. GluA1 S845 and T840 are two phosphorylation sites particularly relevant to this study. GluA1 S845 is phosphorylated by PKA and cGMP-dependent protein kinase II (5, 6). Its phosphorylation levels are regulated by NMDA receptors (NMDARs) (7), β-adrenergic receptors (8, 9), and muscarinic cholinergic receptors (9), and during homeostatic scaling (10), long-term depression (LTD) (11), and emotionally stressful conditions (8). Likewise, GluA1 S845 phospho-mutants show GluA1 trafficking and LTD deficits (12–14). In contrast, the GluA1 T840 site is less well characterized. PKC, calcium/calmodulin-dependent protein kinase II, protein phosphatase 1/2A (PP1/PP2A), and NMDAR activity have been reported to regulate GluA1 T840 phosphorylation (15–17). GluA1 T840 phosphorylation has also been found to enhance channel conductance (18).PACAP38 (pituitary adenylate cyclase activating polypeptide 38) is a neuropeptide that has been shown to regulate hippocampal CA1 synaptic strength (19–22). PACAP38 can bind to and activate three different G protein coupled receptors, the PAC1, VPAC1, and VPAC2 receptors, which can lead to elevated cyclic AMP and Ca²⁺ levels, and activation of phospholipase C and phospholipase D (23). In the hippocampus, PACAP38 stimulation has been shown to alter synaptic strength (19–22) and AMPAR excitatory postsynaptic currents (EPSCs) (24) and to reduce GluA1 synaptic localization (25). PACAP knockout mice are impaired in contextual fear conditioning and novel object recognition (26), and PAC1 receptor knockouts exhibit impaired contextual fear conditioning (27). Given the ability of PACAP38 to regulate basal synaptic transmission and AMPAR EPSCs (24), we hypothesized that PACAP38 stimulation could alter AMPAR phosphorylation levels.We found that PACAP38 stimulation led to increased GluA1 S845 phosphorylation and decreased GluA1 T840 phosphorylation. We also demonstrated that unique signaling pathways were used to drive these phosphorylation changes. Although activation of the PAC1 and VPAC2 receptor elicited a robust increase in GluA1 S845 phosphorylation, only PAC1 receptor activity could elicit a robust decrease in GluA1 T840 phosphorylation. In addition, a PKA inhibitor blocked the increase in S845 phosphorylation, while a PP1/PP2A inhibitor blocked the decrease in T840 phosphorylation and a NMDAR antagonist partially blocked the decrease in T840 phosphorylation.