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 this past year was 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. NMDA receptors are present at nearly all glutamatergic synapses in the central nervous system. ? ? We have been studying the role of a PDZ protein, GIPC (GAIP interacting protein, C-terminus), in the trafficking of the NMDA receptor as presented in last years report. We have extended this study significantly by showing a) that NMDA receptors containing either 2A or 2B are similarly affected by over-expression of GIPC or disrupting the expression of GIPC using a dominant/negative construct or siRNA. b) that GIPC expression also regulates functional endogenous NMDA receptors. The latter study was done on both cerebellar granule cells and hippocampal neurons. These data collectively show GIPC to be a key molecule regulating the trafficking of NMDA receptors.? ? As presented in last years report, we have been studying 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 five members (we initially identified 4, and another group identified a 5th member), and all have PDZ-binding domains 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. SALMs regulate both neurite outgrowth and synapse formation. SALMs form both homomeric and heteromeric complexes; however, in brain SALMs 4 and 5 appear to be mostly in homomeric complexes. This past year we investigated the extracellular binding partners of the SALMs in two ways. First, we are following up on a candidate partner that we identified through a yeast two-hybrid screen. Second, we are investigating if SALMs can form trans interactions. Surprisingly, we found that two SALMs, 4 and 5, form trans homomeric interactions when expressed in HeLa cells while the other SALMs do not. SALMs 4 and 5 do not form trans heteromeric interactions with each other or with the other SALM members. SALMs are highly homologous in their extracellular domains, but not in their intracellular domains suggesting that different properties of the SALMs may rest in their intracellular domains. However, by making chimeras with SALM2, we showed that the ability of SALM4 to form trans interactions is due to its N-terminus. We have also extended our characterization of the role of SALM proteins in neurite outgrowth. We show 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 increase in neurites from the cell body seen with SALM4 over-expression was due to the N-terminal domain.? ? The NR2 subunit of the NMDA receptor is the main determinant of the functional properties of the receptor. In the forebrain there are two major NR2 subunits, NR2A and NR2B, which differ in their developmental patterns, and in their distribution patterns with NR2A being more synaptic and NR2B being more extrasynaptic. We determined the nature of the NMDA receptor complexes present in the hippocampus at various stages of development. At P42 we found that the receptors are distributed nearly equally among complexes of NR1/NR2A, NR1/NR2B, and NR1/NR2A/NR2B. Interestingly, at P7, when there is a limited amount of NR2A produced, there is a significant amount of NR1/NR2A present, indicating that the subunit composition of receptors is not due only to subunit availability. As part of this study, we asked if the four MAGUKs of the PSD-95 family interacted with NR1/NR2A and NR1/NR2B. Contrary to some other reports, we found that SAP102 and PSD-95 interacted to a similar extent with these two populations of receptors. Finally, we used mass spectrometry analysis to identify unknown interacting proteins of NMDA receptors. We identified collapsing response mediator protein 2 to preferentially interact with NR1/NR2B receptors. These findings were recently published. ? ? 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 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. The NR2 subunit has at least two retention signals, one in its C-terminus and one in the remaining part of the molecule. We found also, that the NR1 subunit has additional retention signals since removal of the C-terminus results in a molecule that largely ER retained; however, this truncated form of NR1 can assemble with NR2 to from functional receptors. In this study we investigated the ER retention due to signals before the C-terminus. Using a series of deletion constructs and chimeras with cell surface assays, we found 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.? ? We identified flotillin-1 as a binding partner of the NR2 C-terminal domain. We are investigating how the interaction with flotillin-1 plays a role in the trafficking of the NMDA receptor.
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