A major effort of our laboratory this past year was directed at characterizing the trafficking of key proteins in neurons of the central nervous system and hair cells of the inner ear. In neurons, much of our research has focused on NMDA receptors and their associated proteins. Since the NMDA receptor performs a critical function at the synapse, it is important to understand how this receptor is delivered to the synapse and how the number and composition of receptors at the synapse are regulated. In addition to being at the synapse, some NMDA receptors are extrasynaptic where they may have functions distinct from those at the synapse. We have also carried out research on the trafficking of PMCA2, which is abundant in the stereocilia of hair cells and is important for the removal of calcium from the stereocilia. Mutations in this protein cause deafness. This past year we completed a study on LGN, the mammalian homologue of Drosophila Partner of Inscuteable (Pins), and its relationship to the trafficking of NMDA receptors in neurons. It has been shown previously that NMDA receptors can bind to several PDZ proteins through an interaction with the distal C-terminus of the NR2 subunit. Of particular importance is the PSD-95 family of PDZ proteins, which includes, in addition to PSD-95, PSD-93, SAP97 and SAP102. In our earlier studies, we found that SAP102, an interacting partner of the NMDA receptor, was present throughout the neuron while its companion PDZ protein, PSD-95, was much more restricted to the synapse. Since both associate with the NMDA receptor, this led to the suggestion that SAP102 may be associated with NMDA receptors that are being transported to and from the synapse. Addressing this hypothesis, we carried out a yeast two-hybrid screen using the PDZ domain of SAP102 as bait. We found that LGN interacts with SAP102 and a complex of LGN/SAP102/NMDA receptor is present in brain. LGN interacts with SH3/GK region of SAP102 through its linker domain. The properties of this interaction were investigated in heterologous cells and in cultured neurons. Expression of LGN in hippocampal neurons mediates trafficking of SAP102 and NMDA receptors to the cell surface. LGN expression also affects the number and size of dendritic spines. Using dominant/negative constructs and siRNA knock-down approaches, we show decreases in the number of receptors on the cell surface in neurons. Of particular interest is that LGN can interact with Gai raising the possibility that G-protein signaling could be coupled to NMDA receptor function through LGN and SAP102. Our studies show that GaI expression influences the number of NMDA receptors on the cell surface. This past year we completed a study investigating motifs within the NR2B C-terminus that are important for the synaptic localization of the NMDA receptor. The PDZ binding domain, -SDV, at the distal end of the C-terminus of the NR2B subunit is necessary for the synaptic localization of NR2B-containing NMDARs, but not for extrasynaptic expression. We find that a second domain, the clathrin adaptor protein (AP-2) binding motif, YEKL, also regulates the synaptic localization of receptors. Mutations in this motif can significantly increase the number of synaptic receptors. More importantly, mutations in the AP-2 binding motif allow the synaptic localization of NR2B subunits in the absence of a functional PDZ-binding domain. Using peptides that correspond to the AP-2 binding site of NR2B, which should block AP-2 internalization, we show that the number of synaptic NMDA receptors can be changed within minutes. This result dispels the idea that synaptic NMDA receptors are an inflexible, stable population of proteins. Fyn kinase can phosphorylate the NR2B subunit within the YEKL motif and potentially inhibit AP-2 binding. We find that expression of a constitutively active Fyn kinase increases the synaptic NMDA response and promotes the synaptic localization of NR2B subunits lacking PDZ-binding domains. Therefore, NR2B-containing NMDARs are regulated by a balance between PDZ-protein mediated stabilization and AP-2 mediated internalization. Our results also show that NR2A-containing NMDA receptors are regulated very differently. The PDZ binding domain of NR2A is not required for synaptic localization, and the putative AP-2 binding site that corresponds to the site on NR2B does not influence its synaptic expression. We completed a study in which we monitored the developmental expression of a number of key postsynaptic density proteins in neurons of the hippocampus. We used immunogold EM localization with specific antibodies on tissue obtained from animals at various stages of development. This study showed distinct changes during development for proteins of the postsynaptic density that are associated with glutamate receptors. We are characterizing the delivery to and removal from the stereocilia of PMCA2. A number of proteins, essential to the function of the hair cell, are located on the stereocilia, but little is known about their delivery and removal. Endo- and exocytotic vesicles are not found within the cytoplasm of the stereocilia indicating that addition and removal must occur at the base of the stereocilium. We chose PMCA2 as a model protein due to its relatively high abundance in stereocilia. We developed an antibody to its extracellular domain and used antibody-feeding to label and track PMCA2 molecules on the stereocilia. Tagged proteins are removed with a half-life estimated to be about 8 hours. In monitoring the removal of PMCA2, we found that labeled proteins were lost first from the apex of the stereocilia. This suggests an organized removal of PMCA2 from the stereocilia beginning with the base and ending with the apex, rather than a random removal. Antibody-tagged proteins are found in organelles within the hair and confined to the apical region of the cell. We investigated the role of alternatively spliced cassettes in the delivery of PMCA2 to the stereocilia. We find that the 2wa is the major splice form present in the organ of Corti, and a second splice form, 2za, is also present. Using a series of GFP-tagged constructs and transfection of cultured organs of Corti, we find the presence of the ?a? cassette is important to delivery to the stereocilia plasma membrane. This past year we completed the first study of a new family of proteins, SALMs (Synaptic Adhesion-like Molecules) that we identified through yeast two-hybrid screening using SAP97 as bait. This family consists of four members, and all have PDZ-binding domains except SALM4. They have a single transmembrane domain and their extracellular domains contain leucine-rich motifs, an Ig domain and a FNIII domain. Over-expression of SALM1 enhances neurite outgrowth of cultured hippocampal neurons. We also find the over-expression clusters PDZ proteins as well as NMDA receptors in cultured neurons. Its association with NMDA receptors in neurons appears to depend in interaction of both molecules with PDZ proteins, such as PSD-95. However, we have also found that SALM1 interacts directly with the NR1 subunit of the NMDA receptors through their extracellular domains, raising the interesting possibility that this interaction is important to the stabilization of one or both of these proteins on the cell surface. Additional studies are planned to investigate the function of this interesting family of novel proteins and their role in the localization and trafficking of NMDA receptors.
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