Abstract
Ca(2+) influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor leads to activation and postsynaptic accumulation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and ultimately to long term potentiation, which is thought to be the physiological correlate of learning and memory. The NMDA receptor also serves as a CaMKII docking site in dendritic spines with high affinity binding sites located on its NR1 and NR2B subunits. We demonstrate that high affinity binding of CaMKII to NR1 requires autophosphorylation of Thr(286). This autophosphorylation reduces the off rate to a level (t(12) = approximately 23 min) that is similar to that observed for dissociation of the T286D mutant CaMKII (t(12) = approximately 30 min) from spines after its glutamate-induced accumulation (Shen, K., Teruel, M. N., Connor, J. H., Shenolikar, S., and Meyer, T. (2000) Nat. Neurosci. 3, 881-886). CaMKII as well as the previously identified NR1 binding partners calmodulin and alpha-actinin bind to the short C-terminal portion of the C0 region of NR1. Like Ca(2+)/calmodulin, autophosphorylated CaMKII competes with alpha-actinin-2 for binding to NR1. We conclude that the NR1 C0 region is a key site for recruiting CaMKII to the postsynaptic site, where it may act in concert with calmodulin to modulate the stimulatory role of alpha-actinin interaction with the NMDA receptor.
Highlights
Long term potentiation (LTP)[1] in the hippocampus is a longlasting increase in synaptic transmission, which likely constitutes a physiological correlate of learning and memory (1, 2)
To evaluate the binding partners and molecular mechanisms of calmodulin-dependent protein kinase II (CaMKII) recruitment and retention at postsynaptic sites, we determined the effect of Ca2ϩ/calmodulin and the effect of autophosphorylation of CaMKII on its binding to the three high affinity interaction sites NR1, NR2B/P, and NR2B/C (Fig. 1)
Previous studies indicate that the NR1, NR2A, and NR2B subunits of the NMDA receptor complex serve as postsynaptic anchor sites for CaMKII and that these interactions are directly regulated by the autophosphorylation state of the kinase (11–13, 21, 34)
Summary
Long term potentiation (LTP)[1] in the hippocampus is a longlasting increase in synaptic transmission, which likely constitutes a physiological correlate of learning and memory (1, 2). CaMKII and ␣-Actinin Binding and Competition Assays with NMDA Receptor Fusion Proteins—Recombinant CaMKII␣ (1–2 g) was preincubated for 5 min at 0 °C (to promote Thr[286] autophosphorylation in selected samples) in basic phosphorylation buffer (50 mM Hepes-NaOH, pH 7.4, 10 mM MgCl2) containing, if indicated, 500 M CaCl2, 1 M calmodulin, and 500 M ATP. Preincubation of CaMKII with Ca2ϩ/calmodulin and ATP under conditions that result in Thr[286] phosphorylation was necessary to induce high affinity binding of CaMKII to NR1 and
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