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 associated proteins including the PSD-95 family of Membrane-Associated Guanylate Kinases (MAGUKs) and the Synaptic Adhesion-Like Molecule (SALM) family of adhesion molecules. MAGUKs interact directly with NR2 subunits of the NMDA receptor through their cytoplasmic C-termini. 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. 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 reported in 2000 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. Last year we reported 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. This past year we investigated the role of the C-terminus in the ER trafficking of the NMDA receptor in the context of the whole receptor complex. Our previous studies, and those of others, were done using chimeras of the C-termini of NR1 and a single transmembrane protein. Our experiments showed that two independent ER retention motifs in the C1 cassette, KKK and RRR, are the signals mediating ER retention of the full-length NR1 subunits and that the C2 cassette has an additional inhibitory effect on the forward trafficking of NR1 subunits. On the other hand, C0 and C2 cassettes had an enhancing effect on the trafficking of NR1subunits to the cell surface. Our observations identify the unique roles of C-terminal cassettes in the trafficking of full-length NR1 subunits. This work was published earlier this year. In addition to being present at the postsynaptic membrane, the NMDA receptor also is 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 investigated extrasynaptic NMDA receptors by localizing them at the ultrastructural level in both cultured neurons and intact tissue. Our results show that at least some extrasynaptic receptors may be present in specific areas where non-synaptic contacts are made with other neurons or glia. Adhesion molecules are also present at these sites. These results suggest that some extrasynaptic receptors are not highly mobile and may be functionally important and localized at distinct sites. This work was recently submitted for publication. A group of proteins essential to the trafficking of NMDA receptors is the PSD-95 family of MAGUKs, which, in addition to PSD-95, includes PSD-93, SAP102 and SAP97. Our study focused on the distribution and synaptic turnover of SAP102, a major protein in neurons, but the least characterized of the MAGUKs. PSD-95 is known to be highly concentrated at the synapse due to lipid attachments to the plasma membrane while SAP102 lacks such attachments. We find, however, that SAP102 is enriched at the synapse by association with other proteins through its SH3/GK domains. We also find that SAP102 is highly mobile at the synapse, while PSD-95 is largely immobile. Therefore, the properties of SAP102 are very different from those of PSD-95, suggesting that SAP102 may play a distinct role in synaptic plasticity. This work was recently submitted for publication. We identified flotillin-1 as a binding partner of the NR2 C-terminal domain of the NMDA receptor. We investigated 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. This work was published earlier this year. In addition to being present at the postsynaptic membrane, NMDA receptors are present at the presynaptic membrane of some neurons. We are studying presynaptic receptors in cultured hippocampal neurons using functional and immunocytochemical approaches. Our results show that presynaptic NMDA receptors are expressed transiently in developing hippocampal neurons. We have been studying a new family of adhesion proteins, SALMs that we identified and first reported in 2006. 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 based on immunoprecipitation studies using brain detergent extracts. This past year we focused on 1) proteins that interact with the SALMs, and 2) the forward trafficking of the SALMs. Our earlier 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 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. We were unable to find reticulon 3 expressed on the surface of either heterologous cells or neurons suggesting that its interaction occurs at an intracellular site, possibly the ER. A manuscript describing this work is now in press. 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. Using heterologous cells we investigated the mechanism underlying the behavior of SALM1 after removal of its PDZ BD and found that its intracellular retention is caused by a dileucine signal in its cytoplasmic C-terminus. Mutation of the dileucine motif in the PDZ BD mutant resulted in recovery of surface expression. If the dileucine motif is mutated in the WT construct, there is a significant increase in surface label. Therefore, these two motifs, one of which enhances surface expression and the other which reduces surface expression, together regulate surface expression of SALM1 in heterologous cells. This study is currently being expanded to a study of trafficking in neurons. 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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National Institute on Deafness and Other Communication Disorders
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Petralia, Ronald S (2012) Distribution of extrasynaptic NMDA receptors on neurons. ScientificWorldJournal 2012:267120
Szarama, Katherine B; Gavara, NĂºria; Petralia, Ronald S et al. (2012) Cytoskeletal changes in actin and microtubules underlie the developing surface mechanical properties of sensory and supporting cells in the mouse cochlea. Development 139:2187-97
Yang, Ying; Shu, Xiaogang; Liu, Dan et al. (2012) EPAC null mutation impairs learning and social interactions via aberrant regulation of miR-124 and Zif268 translation. Neuron 73:774-88
Seabold, Gail K; Wang, Philip Y; Petralia, Ronald S et al. (2012) Dileucine and PDZ-binding motifs mediate synaptic adhesion-like molecule 1 (SALM1) trafficking in hippocampal neurons. J Biol Chem 287:4470-84
Petralia, Ronald S; Wang, Ya-Xian; Mattson, Mark P et al. (2012) Subcellular distribution of patched and smoothened in the cerebellar neurons. Cerebellum 11:972-81
Mitchell, Nicholas; Petralia, Ronald S; Currier, Duane G et al. (2012) Sonic hedgehog regulates presynaptic terminal size, ultrastructure and function in hippocampal neurons. J Cell Sci 125:4207-13
Standley, Steve; Petralia, Ronald S; Gravell, Manneth et al. (2012) Trafficking of the NMDAR2B receptor subunit distal cytoplasmic tail from endoplasmic reticulum to the synapse. PLoS One 7:e39585
Hu, Jia-Hua; Yang, Linlin; Kammermeier, Paul J et al. (2012) Preso1 dynamically regulates group I metabotropic glutamate receptors. Nat Neurosci 15:836-44
Zheng, Chan-Ying; Wang, Ya-Xia; Kachar, Bechara et al. (2011) Differential localization of SAP102 and PSD-95 is revealed in hippocampal spines using super-resolution light microscopy. Commun Integr Biol 4:104-5
Wu, Jing; Petralia, Ronald S; Kurushima, Hideaki et al. (2011) Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent ?-amyloid generation. Cell 147:615-28

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