Bidirectional changes in the efficacy of neuronal synaptic transmission, such as hippocampal long-term potentiation (LTP) and long-term depression (LTD), are thought to be mechanisms for information storage in the brain. LTP and LTD may be mediated by the modulation of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazloe proprionic acid) receptor phosphorylation. Here we show that LTP and LTD reversibly modify the phosphorylation of the AMPA receptor GluR1 subunit. However, contrary to the hypothesis that LTP and LTD are the functional inverse of each other, we find that they are associated with phosphorylation and dephosphorylation, respectively, of distinct GluR1 phosphorylation sites. Moreover, the site modulated depends on the stimulation history of the synapse. LTD induction in naive synapses dephosphorylates the major cyclic-AMP-dependent protein kinase (PKA) site, whereas in potentiated synapses the major calcium/calmodulin-dependent protein kinase II (CaMKII) site is dephosphorylated. Conversely, LTP induction in naive synapses and depressed synapses increases phosphorylation of the CaMKII site and the PKA site, respectively. LTP is differentially sensitive to CaMKII and PKA inhibitors depending on the history of the synapse. These results indicate that AMPA receptor phosphorylation is critical for synaptic plasticity, and that identical stimulation conditions recruit different signal-transduction pathways depending on synaptic history.

Brief bath application of N-methyl-D-aspartate (NMDA) to hippocampal slices produces long-term synaptic depression (LTD) in CA1 that is (1) sensitive to postnatal age, (2) saturable, (3) induced postsynaptically, (4) reversible, and (5) not associated with a change in paired pulse facilitation. Chemically induced LTD (Chem-LTD) and homosynaptic LTD are mutually occluding, suggesting a common expression mechanism. Using phosphorylation site-specific antibodies, we found that induction of chem-LTD produces a persistent dephosphorylation of the GluR1 subunit of AMPA receptors at serine 845, a cAMP-dependent protein kinase (PKA) substrate, but not at serine 831, a substrate of protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase II (CaMKII). These results suggest that dephosphorylation of AMPA receptors is an expression mechanism for LTD and indicate an unexpected role of PKA in the postsynaptic modulation of excitatory synaptic transmission.

Modulation of N-methyl-D-aspartate receptors in the brain by protein phosphorylation may play a central role in the regulation of synaptic plasticity. To examine the phosphorylation of the NR1 subunit of N-methyl-Daspartate receptors in situ, we have generated several polyclonal antibodies that recognize the NR1 subunit only when specific serine residues are phosphorylated. Using these antibodies, we demonstrate that protein kinase C (PKC) phosphorylates serine residues 890 and 896 and cAMP-dependent protein kinase (PKA) phosphorylates serine residue 897 of the NR1 subunit. Activation of PKC and PKA together lead to the simultaneous phosphorylation of neighboring serine residues 896 and 897. Phosphorylation of serine 890 by PKC results in the dispersion of surface-associated clusters of the NR1 subunit expressed in fibroblasts, while phosphorylation of serine 896 and 897 has no effect on the subcellular 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 PKA and PKC phosphorylate distinct residues within a small region of the NR1 subunit and differentially affect the subcellular distribution of the NR1 subunit.Ionotropic glutamate receptors mediate most rapid excitatory transmission in the central nervous system and play important roles in synaptic plasticity, neuronal development, and neurological disorders (1-5). Glutamate receptors have been divided into NMDA 1 (N-methyl-D-aspartate) and non-NMDA (kainate or AMPA) receptors based on their pharmacological and physiological properties (1, 2). Non-NMDA glutamate receptors activate and desensitize rapidly and mediate excitatory synaptic transmission. NMDA receptors are more slowly activated and desensitized and have a high Ca 2ϩ permeability and a voltage-dependent Mg 2ϩ block, two properties thought to underlie use-dependent synaptic plasticity in the brain (1-3). Molecular cloning studies have recently identified the genes encoding subunits for the NMDA and non-NMDA receptors (1, 2). NMDA receptors consist of two families of homologous subunits, the NR1 and NR2A-D subunits (6 -9), and are thought to be pentameric or tetrameric complexes of the NR1 subunit with one or more of the NR2 subunits (10, 11). The differential expression of NR2 subunits in the various regions of the brain may account for the diversity of NMDA receptor subtypes (1). In addition, the NR1 subunit is highly alternatively spliced giving rise to at least seven forms of NR1 (NR1A-G) increasing the potential diversity of NMDA receptors in the brain (12-14).Protein phosphorylation has been recognized as a major mechanism for the regulation of glutamate receptor function (15). NMDA receptors appear to be regulated by a number of protein kinases and phosphatases. Activation of protein kinase C (PKC) by phorbol esters have been demonstrated to activate (16,17) or depress (18, 19) neuronal NMDA receptors. In addition, intracellular perfusion of purified ...

Modulation of ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) receptors in the brain by protein phosphorylation may play a crucial role in the regulation of synaptic plasticity. Previous studies have demonstrated that calmodulin (CaM) kinase II can phosphorylate and modulate AMPA receptors. However, the sites of CaM kinase phosphorylation have not been unequivocally identified. In the current study, we have generated two phosphorylation site-specific antibodies to analyze the phosphorylation of the glutamate receptor GluR1 subunit. These antibodies recognize GluR1 only when it is phosphorylated on serine residues 831 or 845. We have used these antibodies to demonstrate that serine 831 is specifically phosphorylated by CaM kinase II in transfected 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 addition, treatment of hippocampal slice preparations with phorbol esters and forskolin increase the phosphorylation of serine 831 and 845, respectively, indicating that protein kinase C and protein kinase A phosphorylate these residues in hippocampal slices. These results identify the site of CaM kinase phosphorylation of the GluR1 subunit and demonstrate that GluR1 is multiply phosphorylated by protein kinase A, protein kinase C, and CaM kinase II in situ.Long lasting changes in the efficiency of synaptic transmission are thought to underlie many forms of learning and memory. Two cellular models for learning and memory, long-term potentiation (LTP) 1 and long-term depression (LTD), have recently been the subject of intense investigation (1-3). LTP is the long-term increase in excitatory synaptic transmission between neurons after a brief high frequency stimulus. In contrast, LTD is the long-term decrease in excitatory synaptic transmission following long periods of low frequency stimulation. The modulation of excitatory synaptic transmission during LTP and LTD has been reported to be due to changes in the presynaptic release of the neurotransmitter glutamate (4 -6) or alternatively, to changes in the sensitivity of the postsynaptic glutamate receptors (7-9). Although the molecular mechanisms underlying LTP and LTD are not completely understood, postsynaptic calcium entry and protein phosphorylation and dephosphorylation have been shown to play essential roles in the induction and maintenance of LTP and LTD (1-3). For example, specific inhibitors of various protein kinases such as calcium/calmodulin-dependent (CaM) kinase II (10 -12), PKC (10, 13), and protein tyrosine kinases (14) have been shown to block the induction of LTP, while protein phosphatase inhibitors have been shown to block the induction of LTD (15, (17,20,21). However, the substrates for the various kinases and phosphatases that mediate changes in synaptic transmission have not been identified.Neurotransmitter receptors mediate signal transduction at the postsynaptic membrane of synapses i...