A. 99, 5710 -5715). Here, we identified the signaling cascade by which RACK1 is released from the NMDA receptor complex and identified the consequences of the dissociation. We found that activation of the cAMP/protein kinase A pathway in hippocampal slices induced the release of RACK1 from NR2B and Fyn. This resulted in the induction of NR2B phosphorylation and the enhancement of NMDA receptor-mediated activity via Fyn. We identified the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP(1-38)), as a ligand that induced phosphorylation of NR2B and enhanced NMDA receptor potentials. Finally, we found that activation of the cAMP/protein kinase A pathway induced the movement of RACK1 to the nuclear compartment in dissociated hippocampal neurons. Nuclear RACK1 in turn was found to regulate the expression of brain-derived neurotrophic factor induced by PACAP(1-38). Taken together our results suggest that activation of adenylate cyclase by PACAP(1-38) results in the release of RACK1 from the NMDA receptor and Fyn. This in turn leads to NMDA receptor phosphorylation, enhanced activity mediated by Fyn, and to the induction of brain-derived neurotrophic factor expression by RACK1.
The ionotropic glutamate receptor subtype, N-methyl-D-as-1 plays an essential role in neuronal development, excitotoxicity, synaptic plasticity, and learning and memory (2). The ligand-gated NMDA receptor channel is a heteromer comprised of NR1 and at least one of four NR2 subunits (A-D) (3). The cytoplasmic tail of the NR2B subunit is phosphorylated on tyrosine residues (4) and is the most abundant tyrosine-phosphorylated protein in the postsynaptic density (5). Phosphorylation of NMDA receptor subunits regulates the activity of the channel (6). Specifically, application of a tyrosine kinase inhibitor causes a progressive decrease in NMDA receptor-mediated currents, and conversely, inhibition of protein-tyrosine phosphatases results in an increase in NMDA receptor-mediated currents (7). Subsequent studies have identified the Src family of protein-tyrosine kinases (PTKs) as enzymes that phosphorylate the NR2 subunits, regulating NMDA receptor activities (6 -8). Hence, modulation of NMDA receptor phosphorylation by Src family protein-tyrosine kinases is likely to play an important role in modulating glutamate-mediated pathways. In the recent years it has become increasingly apparent that the formation of localized signaling complexes, which include receptors, kinases, phosphatases and their substrates, are highly important for the regulation of signal transduction cascades (9, 10). Scaffolding proteins play a major role in the assembly of such signaling complexes (11). Recently, we identified the scaffolding protein RACK1 as a novel regulator of the phosphorylation state and function of the NMDA receptor. We found that RACK1 interacts with both the cytoplasmic tail of the NR2B subunit and Fyn and inhibits the ability of Fyn to phosphorylate the NR2B subunit and consequently inhibits NMDA receptor-mediated excitatory postsyna...