MAGUKs are proteins that act as key scaffolds in surface complexes containing receptors, adhesion proteins, and various signaling molecules. These complexes evolved prior to the appearance of multicellular animals and play key roles in cell-cell intercommunication. A major example of this is the neuronal synapse, which contains several presynaptic and postsynaptic MAGUKs including PSD-95, SAP102, SAP97, PSD-93, CASK, and MAGIs. Here, they play roles in both synaptic development and in later synaptic plasticity events. During development, MAGUKs help to organize the postsynaptic density via associations with other scaffolding proteins, such as Shank, and the actin cytoskeleton. They affect the clustering of glutamate receptors and other receptors, and these associations change with development. MAGUKs are involved in long-term potentiation and depression (e.g., via their phosphorylation by kinases and phosphorylation of other proteins associated with MAGUKs). Importantly, synapse development and function are dependent on the kind of MAGUK present. For example, SAP102 shows high mobility and is present in early synaptic development. Later, much of SAP102 is replaced by PSD-95, a more stable synaptic MAGUK; this is associated with changes in glutamate receptor types that are characteristic of synaptic maturation.
Membrane-associated guanylate kinases (MAGUKs), which are essential proteins in the postsynaptic density (PSD), cluster and anchor glutamate receptors and other proteins at synapses. The MAGUK family includes PSD-95, PSD-93, SAP102, and SAP97. Individual family members can compensate for one another in their ability to recruit and retain receptors at the postsynaptic membrane as shown through deletion and knock-down studies. SAP102 is highly expressed in both young and mature neurons; however, little is known about its localization and mobility at synapses. Here, we compared the distribution, mobility, and turnover times of SAP102 to the well studied MAGUK PSD-95. Using light and electron microscopy, we found that SAP102 shows a broader distribution as well as peak localization further away from the postsynaptic membrane than PSD-95. Using fluorescence recovery after photobleaching (FRAP), we found that 80% of SAP102 and 36% of PSD-95 are mobile in spines. Previous studies showed that PSD-95 was stabilized at the PSD by N-terminal palmitoylation. We found that stabilization of SAP102 at the PSD was dependent on its SH3/GK domains but not its PDZ interactions. Furthermore, we showed that stabilizing actin or blocking NMDA/AMPA receptors reduced the mobile pool of SAP102 but did not affect the mobile pool of PSD-95. Our results show significant differences in the localization, binding mechanism, and mobility of SAP102 and PSD-95. These differences and the compensatory properties of the MAGUKs point out an unrecognized versatility of the MAGUKs in their function in synaptic organization and plasticity.
FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares. Video LinkThe video component of this article can be found at https://www.jove.com/video/2568/ Protocol 1. Neuron transfection 1. Culture embryonic day 18 (E18) rat hippocampal neurons on poly-d-lysine-coated MatTek 35-mm glass-bottom dishes 1 . On 16-18 days in vitro(DIV), transfect neurons using the Clontech CalPhos Mammalian Transfection Kit. First, replace the culture medium with 1.5 ml Dulbecco's Modified Eagle Medium (DMEM) per 35-mm dish 0.5 hour prior to transfection. Save the original culture medium in a sterile 15 ml tube for later (step 1.6) use. 2. Mix 10 μg pEGFP-N1 plasmid DNA with sterile H 2 O (Clontech) and 12.4 μl 2 M calcium solution (Clontech) to a total volume of 100 μl. 3. Add the mixture from step 1.2) to 100 μl 2×HBS dropwise while vortexing 2×HBS at medium speed. 4. Let the mixture sit at room temperature for 20 minutes and then add the final mixture from step 1.3) into DMEM-incubated neurons. 5. Put the neurons back into the 37 °C incubator for 1-1.5 hours. 6. Remove the calcium phosphate-containing medium, then wash cells with DMEM three times. Before returning the culture dish to the incubator, exchange the DMEM medium with the original culture medium. FRAP experiment on a spine1. Neurons are used for the FRAP experiment two to four days after transfection. 2. Replace the culture medium from the 35-mm glass-bottom dish, by immediately adding pre-warmed Tyrode Solution, which contains (in mM) NaCl 145, KCl 5, HEPES 10, Glucose 10, Glycine 0.005, CaCl 2 2.6, and MgCl 2 1.3 (pH adjusted to 7.4 with NaOH). 3. A Zeiss LSM 710 confocal microscope is used for the FRAP experiment. The Zeiss TempModule system is used to control the temperature (37°C), the humidity and the CO 2 (5%) of the working system. Make sure that the CO 2 tank is connected and the water bottle, which is used for balancing humidity, is filled with water. 4. Find a transfected mature dendrite with the 100×objective (αplan-APOCHROMAT 100×/1.46 oil). If the transfected cells in the dish are sparse, search for a desired cell with the 40× objective (plan-NEOFLUAR ...
The cytoplasmic C-terminal domains of NR2 subunits have been proposed to modulate the assembly and trafficking of NMDA receptors. However, questions remain concerning which domains in the C terminus of NR2 subunits control the assembly of receptor complexes and how the assembled complexes are selectively trafficked through the various cellular compartments such as endoplasmic reticulum (ER) to the cell surface. In the present study, we found that the three amino acid tail after the TM4 region of NR2 subunits is necessary for surface expression of functional NMDA receptors, while truncations with only two amino acids following the TM4 region (NR2⌬2) completely eliminated surface expression of the NMDA receptor on co-expression with NR1-1a in HEK293 cells. FRET (fluorescence resonance energy transfer) analysis showed that these NR2⌬2 truncations are able to form homomers and heteromers on co-expression with NR1-1a. Furthermore, when NR2⌬2 subunits were cotransfected with either the NR1-4a or NR1-1a AAA mutant, lacking the ER retention motif (RRR), functional NMDA receptors were detected in the transfected HEK293 cells. Unexpectedly, we found that the replacement of five residues after TM4 with alanines gave results indistinguishable from those of NR2B⌬5 (EHLFY), demonstrating the short tail following the TM4 of NR2 subunits is not sequence-specific-dependent. Taken together, our results show that the C terminus of the NR2 subunits is not necessary for the assembly of NMDA receptor complexes, whereas a three amino acid long cytoplasmic tail following the TM4 of NR2 subunits is sufficient to overcome the ER retention existing in the C terminus of NR1, allowing the assembled NMDA receptors to reach the cell surface. N-methyl-D-aspartate (NMDA)3 receptors are heteromeric complexes primarily assembled from two subunit classes: NR1 and NR2. Co-assembly of NR1 and NR2 subunits is essential for formation of a functional channel, presumed to be a tetramer containing two NR1 and two NR2 subunits (1-2). NR1 is a single subunit with eight splicing variants, which have distinct trafficking and functional properties (3). NR2 subunits are coded by four separate genes, NR2A-D, each of which can endow the receptor channel with different properties (4). Thus, the subunit composition of NMDA receptors is a major determinant of NMDA receptor-mediated activity in the central nervous system. Although much is known about the physiological roles that NMDARs play in long term potentiation (LTP), learning and memory (5-7), much remains to be learned about the mechanisms by which these receptors are assembled, sorted, targeted, and anchored to the appropriate location.The C-terminal domains of the receptor subunits contain critical determinants of subcellular receptor localization. Regulation of receptor trafficking by these determinants ensures that only fully assembled multimeric receptors are expressed on the plasma membrane. When expressed alone in heterogeneous cells, the major NR1 isoform (NR1-1) is retained in the ER because of an RR...
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