Cyclic AMP-dependent Protein Kinase and Protein Kinase C Phosphorylate N-Methyl-d-aspartate Receptors at Different Sites

Abstract

Ca2؉ influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors plays a pivotal role in synaptic plasticity during brain development as well as in mature brain. Cyclic AMP-dependent protein kinase (PKA) and members of the protein kinase C (PKC) family are also essential for various forms of synaptic plasticity and regulate the activity of different ion channels including NMDA and non-NMDA receptors. We now demonstrate that PKA and various PKC isoforms phosphorylate the NMDA receptor in vitro. Ionotropic glutamate receptors mediate fast synaptic transmission by glutamate, the prevailing excitatory neurotransmitter in the mammalian brain (1, 2). These glutamate receptors can be divided into two major families, N-methyl-D-aspartate (NMDA) 1 receptors and non-NMDA receptors, based on their pharmacological and electrophysiological properties as well as sequence identity (1-3). NMDA receptors are key participants in brain development (4, 5) and synaptic plasticity, which may underlie learning and memory (6 -8).Analogous to other ligand-gated ion channels of the nicotinic acetylcholine receptor superfamily, glutamate receptors are thought to consist of five different subunits that are homologous to each other (3, 9, 10). NMDA receptors are formed by NR1 subunits in various combinations with NR2A-NR2D subunits (11-13). The apparent mass, as determined by immunoblotting, is 110 -120 kDa for NR1 and 160 -190 kDa for NR2A-D (14 -19). Deglycosylation in vitro reduces the apparent M r of glutamate receptor subunits, usually by 10 -20 kDa. Immunoprecipitation experiments suggest that native NMDA receptors are pentamers made of one or two NR1 and two or three NR2A-D subunits (15,19,20). Functional NMDA receptors are formed when NR1 is expressed alone or in combination with NR2 subunits, but not when NR2 subunits are expressed without NR1 (11,12). Targeted disruption of the NR1 gene results in a lethal effect (21), while the disruption of the NR2A gene has been implicated in reduced hippocampal long term potentiation (LTP) in mice (22). As expected from their role in synaptic transmission, biochemical (23) and immunohistochemical studies demonstrate that glutamate receptors are specifically localized at postsynaptic sites of excitatory, but not inhibitory, synapses (17,18,24,25).Hydrophobicity plots identified four hydrophobic regions in NMDA and non-NMDA receptors termed M1-M4. By comparison with nicotinic acetylcholine receptors, these regions were originally thought to form four transmembrane segments so that the NH 3 ϩ -and COO Ϫ -terminal ends would both be located extracellularly. Recent studies on non-NMDA receptor subunits, however, demonstrated that only the NH 3 ϩ -terminal end is extracellular, followed by the transmembrane region M1; the M2 region, which loops only partially into the plasma membrane and back into the cytosol; the transmembrane regions M3 and M4; and finally the cytosolic COO Ϫ terminus (26 -28). NMDA receptor subunits probably adopt a similar membrane topology (16).NMDA receptor-mediated cu...

Postsynaptic Application of a Peptide Inhibitor of cAMP-Dependent Protein Kinase Blocks Expression of Long-Lasting Synaptic Potentiation in Hippocampal Neurons

“…The list of potential candidates is long, but PKA-mediated phosphorylation of glutamatergic receptor subunit 1 (GluR1) at serine-845 is important for the bidirectional regulation of hippocampal synaptic plasticity (Banke et al, 2000;Lee et al, 2000) [for GluR4 data, see Carvalho et al (1999)], and PKA activators can increase EPSC amplitudes by promoting PKA-mediated phosphorylation of AMPA receptors (Wang et al, 1991). NMDA receptor-mediated currents are also increased by PKA-mediated phosphorylation (Raman et al, 1996;Leonard and Hell, 1997). PKA-mediated phosphorylation of a protein phosphatase inhibitor, I-1, could allow for the suppression of protein phosphatase-1, and the subsequent disinhibition of CaMKII may gate the expression of long-lasting LTP at postsynaptic sites (Blitzer et al, 1995(Blitzer et al, , 1998Brown et al, 2000).…”

Increased phosphorylation of the neuronal L-type Ca 2+ channel Ca v 1.2 during aging

“…The antibody anti-CH1923-1932P, which specifically binds to ␣ 1 1.2 when phosphorylated at serine 1928, was raised against a phosphopeptide consisting of residues 1923-1932 (25). Antibodies against NMDA receptor NR1 were from R. Jahn (Max Planck Institute for Biophysical Chemistry, Goettingen, Germany); antibodies against NR2A and NR2B were from R. J. Wenthold (National Institute on Deafness and other Communication Disorders, National Institutes of Health, Bethesda) (32,33); antibodies against PKA C␣ were from C. S. Rubin (Albert Einstein College of Medicine, Bronx, NY) (26); and antibodies against protein phosphatase-1 inhibitors 1 and 2 were from A. C. Nairn (Yale University, New Haven, CT). Anti-PSD-95 (34) and anti-SAP102 correspond, respectively, to JH62092 and JH62514 in ref.…”

“…20 and 21) before immunoblotting with anti-CH1923-1932P and subsequently with anti-CNC1 (26,36). For quantification of PSD-95 and SAP102, 50 mg of hippocampal tissue was homogenized in 0.5 ml of 1% deoxycholate buffer before ultracentrifugation (32,37). Supernatants were used either directly for immunoblotting (20 l) or after immunoprecipitation (500 l) with a mixture of anti-NR2A and anti-NR2B antibodies (32,33,37).…”

cAMP-Dependent Protein Kinase Mediates Activity-Regulated Synaptic Targeting of NMDA Receptors

“…NMDA receptor subunits NR1, NR2A, and NR2B can all be phosphorylated by PKA and show some basal phosphorylation in hippocampal tissue (Leonard and Hell, 1997;Tingley et al, 1997). However, the consequences of these individual phosphorylation events on receptor trafficking or anchoring or interaction with binding proteins is not known.…”