Faculty Bibliography

Long-term changes in excitatory synapse strength are thought to reflect changes in synaptic abundance of AMPA receptors mediated by receptor trafficking. AMPA receptor-binding protein (ABP) and glutamate receptor-interacting protein (GRIP) are two similar PDZ (postsynaptic density 95/Discs large/zona occludens 1) proteins that interact with glutamate receptors 2 and 3 (GluR2 and GluR3) subunits. Both proteins have proposed roles during long-term potentiation and long-term depression in the delivery and anchorage of AMPA receptors at synapses. Here we report a variant of ABP-L (seven PDZ form of ABP) called pABP-L that is palmitoylated at a cysteine residue at position 11 within a novel 18 amino acid N-terminal leader sequence encoded through differential splicing. In cultured hippocampal neurons, nonpalmitoylated ABP-L localizes with internal GluR2 pools expressed from a Sindbis virus vector, whereas pABP-L is membrane targeted and associates with surface-localized GluR2 receptors at the plasma membrane in spines. Mutation of Cys-11 to alanine blocks the palmitoylation of pABP-L and targets the protein to intracellular clusters, confirming that targeting the protein to spines is dependent on palmitoylation. Non-palmitoylated GRIP is primarily intracellular, but a chimera with the pABP-L N-terminal palmitoylation sequence linked to the body of the GRIP protein is targeted to spines. We suggest that pABP-L and ABP-L provide, respectively, synaptic and intracellular sites for the anchorage of AMPA receptors during receptor trafficking to and from the synapse

AMPA receptor (AMPAR) trafficking is crucial for synaptic plasticity that may be important for learning and memory. NSF and PICK1 bind the AMPAR GluR2 subunit and are involved in trafficking of AMPARs. Here, we show that GluR2, PICK1, NSF, and alpha-/beta-SNAPs form a complex in the presence of ATPgammaS. Similar to SNARE complex disassembly, NSF ATPase activity disrupts PICK1-GluR2 interactions in this complex. Alpha- and beta-SNAP have differential effects on this reaction. SNAP overexpression in hippocampal neurons leads to corresponding changes in AMPAR trafficking by acting on GluR2-PICK1 complexes. This demonstrates that the previously reported synaptic stabilization of AMPARs by NSF involves disruption of GluR2-PICK1 interactions. Furthermore, we are reporting a non-SNARE substrate for NSF disassembly activity

Synaptophysin is a synaptic vesicle (SV) protein of unknown function. Here we show that a repeated sequence in the cytoplasmic tail of synaptophysin mediates the formation of a protein complex containing the GTPase dynamin. The formation of this complex requires a high Ca(2+) concentration, suggesting that it occurs preferentially at the sites of SV exocytosis. Coimmunoprecipitation of a dynamin-synaptophysin complex from brain extracts is promoted by dissociation of vesicle-associated membrane protein 2 from synaptophysin. This finding suggests that dynamin only associates with synaptophysin in vivo after vesicle-associated membrane protein 2 (VAMP2) enters the SNARE complex. GTP binding releases dynamin from synaptophysin, possibly serving to regulate dynamin selfassembly during endocytosis. Our results suggest that synaptophysin plays a role in SV recycling by recruiting dynamin to the vesicle membrane

AMPA-receptor (AMPAR) transport to synapses plays a critical role in the modulation of synaptic strength. We show that the functionally critical GluR2 subunit stably resides in an intracellular pool in the endoplasmic reticulum (ER). GluR2 in this pool is extensively complexed with GluR3 but not with GluR1, which is mainly confined to the cell surface. Mutagenesis revealed that elements in the C-terminus including the PDZ motif are required for GluR2 forward-transport from the ER. Surprisingly, ER-retention of GluR2 is controlled by Arg607 at the Q/R-editing site. Reversion to Gln (R607Q) resulted in rapid release from the pool and elevated surface expression of GluR2 in neurons. Therefore, Arg607 is a central regulator. In addition to channel gating, it also controls ER-exit, and may thereby ensure the availability of GluR2 for assembly into AMPARs

Movement of AMPA receptors into and out of the postsynaptic membrane is correlated with LTP and LTD, respectively. The roles of specific AMPA receptor subunits, in particular their cytosolic C-termini, have been examined with respect to exocytosis of exogenous receptors in neurons. Contributions of AMPA receptor C-terminal sequences to endocytosis of exogenous receptor subunits, however, have not been examined in intact neurons.)We have employed mutagenesis to examine the role of protein binding to the GluR2 C-terminus in endocytic trafficking of exogenously expressed GluR2 subunits in cultured hippocampal neurons. Using immunocytochemistry we analyzed internalization of GluR2 mutants in which binding of proteins known to interact with the GluR2 C-terminus was abrogated. We also examined a mutation in which the juxtamembrane 10 amino acids of the GluR2 C-terminus were deleted. Mutation of the NSF binding site resulted in a modest but significant (26%) increase in receptor internalization. Deletion of the juxtamembrane region however resulted in a 96% increase in the rate of internalization of the receptor.)We show that internalization of exogenous GluR2 subunits takes place mainly in dendritic shafts and is largely absent in dendritic spines. Internalized GluR2 was highly colocalized with the early endosomal marker EEA1 when observed after 10 minutes of exposure to extracellular antibodies.)We will discuss models which could account for the contribution of the GluR2 juxtamembrane region in stabilizing AMPA receptors in the plasma membrane

We have studied the role and the control of neuronal nitric oxide synthase (nNOS) by NMDA receptors (NMDARs) in excitotoxicity. Phosphorylation of nNOS regulates its activity. NMDAR activation in neurons greatly reduces the constitutive phosphorylation of nNOS by a mechanism sensitive to the NMDAR antagonist, MK801. Okadaic acid, a general phosphatase inhibitor and cyclosporine, a specific inhibitor of the Ca2+-activated phosphatase calcineurin, reduce the NMDAR-induced dephosphorylation of nNOS. We demonstrated that in brain nNOS is phosphorylated at two different phosphorylation sites by Western blotting and immunocytochemistry in brain, retina, and cortical cultured neurons using phospho-specific antibodies. NMDAR-induced death in these experiments is apoptotic since cell death is blocked by the baculovirus-encoded caspase inhibitor protein, p35, and is accompanied by caspase-3 activation. FK506 was neuroprotective since it reduced NMDAR-dependent nitrotyrosine formation and TUNEL positivity. This suggests that NMDA-induced cell death of neurons involves nNOS activation via dephosphorylation leading to caspase activation and apoptosis

Regulated trafficking of AMPA receptors (AMPAR) contributes to the plasticity of excitatory synapses. AMPAR consisting of GluR2/3 subunit heteromers can cycle constitutively between intracellular and synaptic membranes. The multi-PDZ protein AMPA receptor Binding Protein (ABP) binds to GluR2/3 and may function as an AMPAR membrane anchor. ABP synaptic membrane association depends upon palmitoylation of the N-terminal variable exon of the pABP-L isoform. Here we identify an ABP subdomain, LI-SII, consisting of PDZ4-6 plus the N-terminally neighboring linker region that targets non-palmitoylated ABP (ABP-L) to intracellular membranes, where it clusters GluR2. We show that following endocytosis from the plasma membrane, GluR2 trafficks to intracellular membranes where it localizes with ABP-L via LI-SII. Phosphorylation of GluR2 at serine 880, which releases AMPAR for traffficking, is blocked by association of GluR2 with LI-SII. These data indicate that the ABP region LI-SII functions as an intracellular GluR2 tether that can stabilize the receptor against transport by inhibiting S880 phosphorylation

The PICK1 protein interacts in neurons with the AMPA-type glutamate receptor subunit 2 (GluR2) and with several other membrane receptors via its single PDZ domain. We show that PICK1 also binds in neurons and in heterologous cells to protein kinase Calpha (PKCalpha) and that the interaction is highly dependent on the activation of the kinase. The formation of PICK1-PKCalpha complexes is strongly induced by TPA, and PICK1-PKCalpha complexes are cotargeted with PICK1-GluR2 complexes to spines, where GluR2 is found to be phosphorylated by PKC on serine 880. PICK1 also reduces the plasma membrane levels of the GluR2 subunit, consistent with a targeting function of PICK1 and a PKC-facilitated release of GluR2 from the synaptic anchoring proteins ABP and GRIP. This work indicates that PICK1 functions as a targeting and transport protein that directs the activated form of PKCalpha to GluR2 in spines, leading to the activity-dependent release of GluR2 from synaptic anchor proteins and the PICK1-dependent transport of GluR2 from the synaptic membrane