At all levels the correct functioning of the nervous system requires communication among individual neurons at sites of contact known as synapses. Information originating at sensory structures such as the inner ear is transferred through neurons with multiple synaptic contact points to the cerebral cortex where additional processing using synaptic contacts occurs. A major effort of our laboratory in recent years has been directed at characterizing the trafficking of key proteins in neurons of the central nervous system. We have focused on NMDA receptors and their associated proteins. Since the NMDA receptor performs a critical function at the synapse and is a key player in synaptic plasticity, 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 are interested in characterizing this population of receptors and their regulation, as well as well as the relationship between synaptic and extrasynaptic receptors. NMDA receptors are present at nearly all glutamatergic synapses in the central nervous system. ? ? The functional NMDA receptor is formed by the assembly of two NR1 and two NR2 subunits into a tetrameric complex. The NR2 subunits and some of the NR1 splice variants are retained in the endoplasmic reticulum (ER) until they are assembled. We have shown previously that ER export of NR1 splice variants is controlled by two signals in their C-termini, a retention signal and an export signal, which can override the retention signal. This past year we completed and published a study showing that the transmembrane domain 3 (TM3) plays a key role in the ER retention of both NR1 and NR2. Our results would suggest that the assembly of NR1 and NR2 subunits negates the ER retention of these motifs, perhaps by a direct interaction of the TM3 domains of the two subunits. Previous studies on the NR1 C-terminus were limited to using chimeras of the C-terminus and tac. We are now studying the retention motifs of NR1 using the full-length molecule. In the chimera studies, a RXR motif was found to be responsible for ER retention of the NR1-1 C-terminus. In the full-length molecule, however, it appears that a KKK motif also plays a role in ER retention. ? ? In addition to being present at the postsynaptic membrane, the NMDA receptor also appears to be located at extrasynaptic sites. These extrasynaptic receptors may simply represent receptors awaiting addition to the synapse or recently removed from the synapse. Alternatively, they may be functionally important. We are investigating extrasynaptic NMDA receptors by localizing them at the ultrastructural level in both cultured neurons and intact tissue. Our preliminary results suggest that at least some extrasynaptic receptors may be present in specific areas where non-synaptic contacts are made with other neurons or glia. These results would suggest that some extrasynaptic receptors are not highly mobile and may be functionally important and localized at distinct sites.? ? A group of proteins essential to the trafficking of NMDA receptors are the MAGUKs, which include PSD-95, PSD-93, SAP102 and SAP97. These are PDZ proteins that associate with NMDA receptors through their C-terminal PDZ binding domains (BDs). We are investigating the distribution and synaptic turnover of two of these proteins, PSD-95 and SAP102. PSD-95 is known to be highly concentrated at the synapse due to lipid attachments, while SAP102 lacks such attachments. We find, however, that SAP102 is enriched at the synapse, and this may be due to association with other proteins through its SH3/GK domains.? ? We identified flotillin-1 as a binding partner of the NR2 C-terminal domain of the NMDA receptor. We are investigating how the interaction with flotillin-1 plays a role in the trafficking of the NMDA receptor. Flotillin-1 appears to be associated with a relatively small subset of receptors and may not be responsible for the lipid raft association of NMDA receptors. ? ? We completed our project on GIPC (GAIP interacting protein, C-terminus) and its role in the trafficking of the NMDA receptor and published the results. ? ? We have been studying a new family of adhesion proteins, SALMs (Synaptic Adhesion-like Molecules) that we identified through yeast two-hybrid screening. This family consists of five members, and all have PDZ BDs except SALM4 and SALM5. They have a single transmembrane domain and their extracellular domains contain leucine-rich motifs, an Ig domain and a FNIII domain. SALMs interact directly and/or indirectly with NMDA receptors. This past year we focused on three aspects of the SALMs: their role in neurite outgrowth, their biochemical properties and interactions with other proteins, and their processing in the ER and other intracellular organelles. We found that all five SALMS promote neurite outgrowth, although with somewhat different phenotypes. Most apparent was that over-expression of SALM4 increased the number of small neurites extending from the cell body while SALM2 over-expression resulted in increased neurite length. Using the SALM2/4 chimera discussed above, we found that characteristic increases in neurites from the cell body seen with SALM4 over-expression was due to the N-terminal domain. Neurite outgrowth could be blocked using antibodies to extracellular domains or RNAi to knock down expression of SALMs. Our biochemical studies showed that SALMs form homomeric and heteromeric complexes in heterologous cells and in brain and likely exist as dimers or higher order multimers. We also investigated the formation of cis and trans interactions. We found that SALMs 4 and 5 form homomeric trans interactions, while no trans interactions were detected for SALMs 1-3. Since most SALMs do not form trans interactions with themselves or other SALMs, we have been searching for other binding partners. We found that reticulon 3 binds to the extracellular domain of the SALMs. Immunoprecipitation studies from brain showed that only a subset of reticulon 3 isoforms interacted with the SALMs. Our results suggest that reticulon 3 interacts with SALMs in the ER. While investigating the processing of SALMs in heterologous cells, we found that deletion of the PDZ BD of SALM1 greatly decreased its surface expression. SALMs 2 and 3 were not affected by removal of their PDZ BDs, while SALMs 4 and 5, which do not have PDZ BDs, readily go to the cell surface. We investigated the mechanism underlying the behavior of SALM1 after removal of its PDZ BD and found that intracellular retention is caused by a discrete signal in its C-terminus. It is interesting that only SALM1 is affected by removal of its PDZ BD and raises interesting questions about the functional significance of the PDZ BD in the intracellular trafficking of SALM1.
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