SAP102 mediates synaptic clearance of NMDA receptors.
ABSTRACT: Membrane-associated guanylate kinases (MAGUKs) are the major family of scaffolding proteins at the postsynaptic density. The PSD-MAGUK subfamily, which includes PSD-95, PSD-93, SAP97, and SAP102, is well accepted to be primarily involved in the synaptic anchoring of numerous proteins, including N-methyl-D-aspartate receptors (NMDARs). Notably, the synaptic targeting of NMDARs depends on the binding of the PDZ ligand on the GluN2B subunit to MAGUK PDZ domains, as disruption of this interaction dramatically decreases NMDAR surface and synaptic expression. We recently reported a secondary interaction between SAP102 and GluN2B, in addition to the PDZ interaction. Here, we identify two critical residues on GluN2B responsible for the non-PDZ binding to SAP102. Strikingly, either mutation of these critical residues or knockdown of endogenous SAP102 can rescue the defective surface expression and synaptic localization of PDZ binding-deficient GluN2B. These data reveal an unexpected, nonscaffolding role for SAP102 in the synaptic clearance of GluN2B-containing NMDARs.
Project description: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.
Project description:Membrane-associated guanylate kinases (MAGUKs) are major components of the postsynaptic density and play important roles in synaptic organization and plasticity. Most excitatory synapses are located on dendritic spines, which are dynamic structures that undergo morphological changes during synapse formation and plasticity. Synapse-associated protein 102 (SAP102) is a MAGUK that is highly expressed early in development and mediates receptor trafficking during synaptogenesis. Mutations in human SAP102 cause mental retardation, which is often accompanied with abnormalities in dendritic spines. However, little is known about the role of SAP102 in regulating synapse formation or spine morphology. We now find that SAP102 contains a novel NMDA receptor binding site in the N-terminal domain, which is specific for the NR2B subunit. The interaction between SAP102 and NR2B is PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain independent and is regulated by alternative splicing of SAP102. We show that SAP102 that possesses an N-terminal insert is developmentally regulated at both mRNA and protein levels. In addition, expression of SAP102 increases synapse formation. Furthermore, the alternative splicing of SAP102 regulates dendritic spine morphology. SAP102 containing the N-terminal insert promotes lengthening of dendritic spines and preferentially promotes the formation of synapses at long spines, whereas a short hairpin RNA knockdown of the same SAP102 splice variant causes spine shrinkage. Finally, blocking NMDA receptor activity prevents the spine lengthening induced by the N-terminal splice variant of SAP102. Thus, our data provide the first evidence that SAP102 links NMDA receptor activation to alterations in spine morphology.
Project description:The development of glutamatergic synapses involves changes in the number and type of receptors present at the postsynaptic density. To elucidate molecular mechanisms underlying these changes, we combine in utero electroporation of constructs that alter the molecular composition of developing synapses with dual whole-cell electrophysiology to examine synaptic transmission during two distinct developmental stages. We find that SAP102 mediates synaptic trafficking of AMPA and NMDA receptors during synaptogenesis. Surprisingly, after synaptogenesis, PSD-95 assumes the functions of SAP102 and is necessary for two aspects of synapse maturation: the developmental increase in AMPA receptor transmission and replacement of NR2B-NMDARs with NR2A-NMDARs. In PSD-95/PSD-93 double-KO mice, the maturational replacement of NR2B- with NR2A-NMDARs fails to occur, and PSD-95 expression fully rescues this deficit. This study demonstrates that SAP102 and PSD-95 regulate the synaptic trafficking of distinct glutamate receptor subtypes at different developmental stages, thereby playing necessary roles in excitatory synapse development.
Project description:The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-d-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre- or postsynaptic membrane, and depletes the number of membrane-associated PSD-95-like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membrane-associated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD.
Project description:The corticotropin-releasing hormone receptor type 1 (CRHR1) plays an important role in orchestrating neuroendocrine, behavioral, and autonomic responses to stress. To identify molecules capable of directly modulating CRHR1 signaling, we performed a yeast-two-hybrid screen using the C-terminal intracellular tail of the receptor as bait. We identified several members of the membrane-associated guanylate kinase (MAGUK) family: postsynaptic density protein 95 (PSD95), synapse-associated protein 97 (SAP97), SAP102 and membrane associated guanylate kinase, WW and PDZ domain containing 2 (MAGI2). CRHR1 is co-expressed with the identified MAGUKs and with the additionally investigated PSD93 in neurons of the adult mouse brain and in primary hippocampal neurons, supporting the probability of a physiological interaction in vivo. The C-terminal PDZ (PSD-95, discs large, zona occludens 1) binding motif of CRHR1 is essential for its physical interaction with MAGUKs, as revealed by the CRHR1-STAVA mutant, which harbors a functionally impaired PDZ binding motif. The imitation of a phosphorylation at Thr413 within the PDZ binding motif also disrupted the interaction with MAGUKs. In contrast, distinct PDZ domains within the identified MAGUKs are involved in the interactions. Expression of CRHR1 in primary neurons demonstrated its localization throughout the neuronal plasma membrane, including the excitatory post synapse, where the receptor co-localized with PSD95 and SAP97. The co-expression of CRHR1 and respective interacting MAGUKs in HEK293 cells resulted in a clustered subcellular co-localization which required an intact PDZ binding motif. In conclusion, our study characterized the PDZ binding motif-mediated interaction of CRHR1 with multiple MAGUKs, which directly affects receptor function.
Project description:The mechanisms controlling synapse growth and maintenance are of critical importance for learning and memory. The MAGUK family of synaptic scaffolding proteins is abundantly expressed at glutamatergic central synapses, but their importance in controlling the synaptic content of glutamate receptors is poorly understood. Here, we use a chained RNAi-mediated knockdown approach to simultaneously remove PSD-93, PSD-95, and SAP102, the MAGUKs previously shown to be responsible for synaptic localization of glutamate receptors. We find that MAGUKs are specifically responsible for creating functional synapses after initial spine formation by filling functionally silent spines with glutamate receptors. Removal of the MAGUKs causes a transient reduction in AMPA receptor quantal size followed by synaptic consolidation resulting in a normalization of quantal size at the few remaining functional synapses. Consolidation requires signaling through L-type calcium channels, CaM kinase kinase, and the GluA2 AMPA receptor subunit, akin to a homeostatic process.
Project description:Proteins of the PSD-95-like membrane-associated guanylate kinase (PSD-MAGUK) family are vital for trafficking AMPA receptors (AMPARs) to synapses, a process necessary for both basal synaptic transmission and forms of synaptic plasticity. Synapse-associated protein 97 (SAP97) exhibits protein interactions, such as direct interaction with the GluA1 AMPAR subunit, and subcellular localization (synaptic, perisynaptic, and dendritic) unique within this protein family. Due in part to the lethality of the germline knockout of SAP97, this protein's role in synaptic transmission and plasticity is poorly understood. We found that overexpression of SAP97 during early development traffics AMPARs and NMDA receptors (NMDARs) to synapses, and that SAP97 rescues the deficits in AMPAR currents normally seen in PSD-93/-95 double-knockout neurons. Mature neurons that have experienced the overexpression of SAP97 throughout development exhibit enhanced AMPAR and NMDAR currents, as well as faster NMDAR current decay kinetics. In loss-of-function experiments using conditional SAP97 gene deletion, we recorded no deficits in glutamatergic transmission or long-term potentiation. These results support the hypothesis that SAP97 is part of the machinery that traffics glutamate receptors and compensates for other PSD-MAGUKs in knockout mouse models. However, due to functional redundancy, other PSD-MAGUKs can presumably compensate when SAP97 is conditionally deleted during development.
Project description:Transmembrane AMPA receptor (AMPAR) regulatory proteins (TARPs) modulate AMPAR synaptic trafficking and transmission via disc-large (DLG) subfamily of membrane-associated guanylate kinases (MAGUKs). Despite extensive studies, the molecular mechanism governing specific TARP/MAGUK interaction remains elusive. Using stargazin and PSD-95 as the representatives, we discover that the entire tail of stargazin (Stg_CT) is required for binding to PSD-95. The PDZ binding motif (PBM) and an Arg-rich motif upstream of PBM conserved in TARPs bind to multiple sites on PSD-95, thus resulting in a highly specific and multivalent stargazin/PSD-95 complex. Stargazin in complex with PSD-95 or PSD-95-assembled postsynaptic complexes form highly concentrated and dynamic condensates via phase separation, reminiscent of stargazin/PSD-95-mediated AMPAR synaptic clustering and trapping. Importantly, charge neutralization mutations in TARP_CT Arg-rich motif weakened TARP's condensation with PSD-95 and impaired TARP-mediated AMPAR synaptic transmission in mice hippocampal neurons. The TARP_CT/PSD-95 interaction mode may have implications for understanding clustering of other synaptic transmembrane proteins.
Project description:Membrane-associated guanylate kinases (MAGUKs) act as scaffolds to coordinate signaling events through their multiple domains at the plasma membrane. The MAGUK SH3 domain is noncanonical and its function remains unclear. To identify potential binding partners of MAGUK SH3, the synapse-associated protein 102 (SAP102) SH3 domain was used as bait in a yeast two-hybrid screen of a mouse embryonic cDNA library. A mouse homologue of the Drosophila discs large tumor suppressor (Dlg, also known as SAP97) bound preferentially to SAP102 SH3. The 4347bp cDNA sequence encoded an 893 amino acid protein with 94% identity to mouse SAP97. A deleted region (33-aa) strongly suggests this is a novel splice variant, which we call Embryonic-dlg/SAP97 (E-dlg). The interaction of SAP102 and E-dlg was confirmed in mammalian cells. E-dlg can also bind to potassium channel Kv1.4 in a pull-down assay. E-dlg was highly expressed in embryonic and some adult mouse tissues, such as brain, kidney, and ovary. Furthermore, in situ hybridization showed that E-dlg was mostly expressed in olfactory bulb and cerebellum.