Differential trafficking of AMPA and NMDA receptors by SAP102 and PSD-95 underlies synapse development

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.     

The LGI1–ADAM22 protein complex directs synapse maturation through regulation of PSD-95 function

Synapse development is coordinated by a number of transmembrane and secreted proteins that come together to form synaptic organizing complexes. Whereas a variety of synaptogenic proteins have been characterized, much less is understood about the molecular networks that support the maintenance and functional maturation of nascent synapses. Here, we demonstrate that leucine-rich, glioma-inactivated protein 1 (LGI1), a secreted protein previously shown to modulate synaptic AMPA receptors, is a paracrine signal released from pre- and postsynaptic neurons that acts specifically through a disintegrin and metalloproteinase protein 22 (ADAM22) to set postsynaptic strength. We go on to describe a novel role for ADAM22 in maintaining excitatory synapses through PSD-95/Dlg1/zo-1 (PDZ) domain interactions. Finally, we show that in the absence of LGI1, the mature synapse scaffolding protein PSD-95, but not the immature synapse scaffolding protein SAP102, is unable to modulate synaptic transmission. These results indicate that LGI1 and ADAM22 form an essential synaptic organizing complex that coordinates the maturation of excitatory synapses by regulating the functional incorporation of PSD-95.Proper development of synapses involves recruitment of proteins that establish presynaptic release sites and postsynaptic densities (PSDs), and later coordinate maturation, maintenance, and plasticity of the synapse. In the last decade, a number of transmembrane synaptic adhesion proteins and secreted proteins that initiate and modulate excitatory synapses—termed synaptic organizing proteins—have been identified (1, 2). Whereas the synaptogenic properties of many synaptic organizing proteins have been described in detail (2–11), much less is known about how synaptic organizing proteins regulate synapse maintenance and maturation (1).A key component to the functional maturation of excitatory synapses is the recruitment of AMPA-type glutamate receptors (AMPARs) to the PSD, which is coordinated by PSD-95/Dlg1/zo-1 (PDZ) domain-containing scaffolding proteins (12). Specifically, one family of PDZ proteins, the membrane-associated guanylyl kinases (MAGUKs), is known to determine basal synaptic AMPAR content (13–16). Like synaptic AMPAR content, the expression of different MAGUKs is developmentally regulated; whereas synapse-associated protein 102 (SAP102) is expressed in the early postnatal period and is critical to the function of immature synapses, postsynaptic density proteins 93 and 95 (PSD-93 and PSD-95) are first expressed around postnatal day 10 (P10) and are required for proper function of mature synapses (14, 17). Mice lacking PSD-93 and PSD-95 have synapses with significantly reduced AMPAR content (14), indicating the shift in MAGUK expression is critical to synapse maturation. However, what guides the incorporation of PSD-93 and PSD-95 into developing synapses remains unknown.We recently identified an instructive role for leucine-rich, glioma-inactivated protein 1 (LGI1) in regulating synaptic AMPAR content—application of LGI1 increases and loss of LGI1 decreases synaptic AMPAR localization (18, 19). LGI1 is a secreted protein that is localized to synapses, where it binds to the extracellular domain of the transmembrane a disintegrin and metalloproteinase proteins 11, 22, and 23 (ADAM11, ADAM22, and ADAM23) (19). Notably, LGI1 and ADAM22 are found in complex with the mature MAGUKs, PSD-93 and PSD-95, in vivo (18, 19), and their expression levels follow a similar timeline (17, 18, 20). However, little is known about the neuronal function of ADAM11, ADAM22, and ADAM23, and it is not clear which mediates the function of LGI1 at the synapse. Moreover, the source and destination of secreted LGI1 remains unstudied.Here, we show that LGI1 released from pre- and postsynaptic cells acts in a paracrine fashion to regulate synaptic AMPAR content. We find that the function of LGI1 at the synapse is fully dependent on its interaction with ADAM22, the only ADAM in the LGI1 complex that contains a PDZ-binding motif. ADAM22, in turn, maintains excitatory synapses through PDZ domain interactions. Finally, we demonstrate that PSD-95, but not SAP102, requires LGI1 to function at synapses, indicating that the LGI1–ADAM22 complex directs synapse maturation by controlling the incorporation of the mature MAGUK PSD-95.     

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

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.The postsynaptic density (PSD) at excitatory glutamatergic synapses, appearing in electron micrographs as a prominent electron-dense thickening lining the postsynaptic membrane (1) is a complex macromolecular machine positioned across from synaptic vesicle release sites at the presynaptic active zone. The PSD clusters and organizes neurotransmitter receptors and signaling molecules at the postsynaptic membrane, transmits and processes synaptic signals, and can undergo structural changes to encode and store information (2–5). Two types of ionotropic glutamate receptors, AMPA receptors (AMPARs) and NMDA receptors (NMDARs), present at PSDs of excitatory synapses (6–10) mediate almost all synaptic transmission in the brain (11). Biochemistry and mass spectrometry of the detergent-extracted cellular fraction of PSDs have additionally identified many proteins associated with AMPAR and NMDAR complexes (12, 13).The membrane-associated guanylate kinases (MAGUKs), a class of abundant scaffold proteins consisting of PSD-95, PSD-93, synapse-associated protein (SAP)102, and SAP97, interact directly with NMDARs (14–18). These MAGUK proteins share conserved modular structures consisting of three PDZ domains (19, 20) and one SH3-GK supermodule (21). PDZ domains of MAGUKs bind to a conserved motif at the extreme C-terminal region of GluN2 subunits of NMDARs (16, 22). PSD-95 controls the number of AMPARs at the PSD through interactions with auxiliary proteins, such as Stargazin/TARPs in complex with AMPARs (23–25). Single-particle tracking of AMPARs provides evidence that AMPAR/Stargazin complexes are stabilized by PSD-95 at the membrane (26), where PSD-95 is thought to provide hot spots for accumulating AMPARs at synapses (27, 28). Germ-line knockout of PSD-95 reduces AMPAR transmission with little effects on NMDARs (29), whereas acute loss of single members of the MAGUK family decreases primarily AMPAR-mediated synaptic transmission (30–32), and removal of multiple MAGUKs results in greater losses of transmission mediated by both AMPARs and NMDARs (30).PSD-95 and PSD-93 include N-terminal palmitoylation sites that enable PSD-95 and PSD-93 to associate with membrane lipids. N-terminal palmitoylation of PSD-95 is necessary for its synaptic localization, clustering of receptors (33–35), and stability at the PSD (36). PSD-95 palmitoylation regulates synaptic strength by controlling the accumulation of AMPARs at the PSD (35). Consistent with these results, a recent immunogold electron microscopy (immuno-EM) mapping of the positions of the two ends of the PSD-95 molecule at the PSD shows that its N terminus is located at the membrane, whereas its C terminus is farther away from the membrane in a relatively extended configuration, where it is vertically oriented with respect to the membrane (3, 4, 37). In contrast, neither SAP102 nor SAP97 has palmitoylation sites. SAP97 contains a L27 domain at the N terminus (31, 38), which might be involved in self-association, and has a role in sorting and trafficking of AMPARs and NMDARs (39) but is not required for basal synaptic transmission (40).The MAGUK family proteins interact with a host of other proteins in the PSD, such as GKAP (41, 42), which binds to the GK domain of the MAGUKs, whereas GKAPs in turn bind Shank and Homer (43–45). Both Shank and Homer can interact with actin-associated proteins, thus indirectly linking the core PSD structure to the actin system prevalent in the cytoplasm of dendritic spines (45). MAGUKs interact with signaling complexes such as AKAPs (46, 47), K channels (48), and postsynaptic adhesion molecules such as neuroligin (49, 50). With an average density of 300–400 molecules per PSD (51, 52), the MAGUKs outnumber glutamate receptors by a significant margin. With so many potential binding partners, the MAGUKs are positioned to play an important role in organizing glutamate receptors as well as other scaffolding and signaling molecules at the PSD (53).We have examined the consequences of knocking down three key MAGUKs on excitatory synaptic transmission and found an ∼80% reduction in both AMPAR and NMDAR synaptic responses (54). Interestingly, despite the rather ubiquitous distribution of MAGUKs at excitatory synapses, the reduction in synaptic AMPAR-mediated transmission appeared to be attributable primarily to an all-or-none loss of functional synapses. We present evidence that after the knockdown, there is an initial uniform decrease in AMPARs across all synapses, but over a 4-d period, a consolidation process in which a “winner-take-all” phenomenon occurs (54).Here, we have used EM tomography (3, 4) to study the structural effects of knocking down the three key MAGUKs at the PSD to develop a molecular model of the organization of the core PSD structure in intact hippocampal spine synapses. PSDs in intact synapses show numerous regularly spaced and membrane-associated vertical filaments containing PSD-95 in extended conformation connecting with NMDAR and AMPAR-type complexes. These vertical structures in turn contact horizontal elements, resulting in a molecular scaffold supporting a core PSD structure (3, 4, 37). Thus, vertical filaments appear to be of crucial importance in sustaining the core PSD structure. Here, we show that knocking down three key synaptic MAGUKs results in a profound loss of vertical filaments and the electron-dense materials manifested by the PSD. The loss of MAGUKs is accompanied by a dramatic loss of both NMDAR- and AMPAR-type structures at the PSD.     

PSD-95 and PKC converge in regulating NMDA receptor trafficking and gating

Neuronal NMDA receptors (NMDARs) colocalize with postsynaptic density protein-95 (PSD-95), a putative NMDAR anchoring protein and core component of the PSD, at excitatory synapses. PKC activation and PSD-95 expression each enhance NMDAR channel opening rate and number of functional channels at the cell surface. Here we show in Xenopus oocytes that PSD-95 and PKC potentiate NMDA gating and trafficking in a nonadditive manner. PSD-95 and PKC each enhance NMDA channel activity, with no change in single-channel conductance, reversal potential or mean open time. PSD-95 and PKC each potentiate NMDA channel opening rate (k(beta)) and number of functional channels at the cell surface (N), as indicated by more rapid current decay and enhanced charge transfer in the presence of the open channel blocker MK-801. PSD-95 and PKC each increase NMDAR surface expression, as indicated by immunofluorescence. PKC potentiates NMDA channel function and NMDAR surface expression to the same final absolute values in the absence or presence of PSD-95. Thus, PSD-95 partially occludes PKC potentiation. We further show that Ser-1462, a putative phosphorylation target within the PDZ-binding motif of the NR2A subunit, is required for PSD-95-induced potentiation and partial occlusion of PKC potentiation. Coimmunoprecipitation experiments with cortical neurons in culture indicate that PKC activation promotes assembly of NR2 with NR1, and that the newly assembled NMDARs are not associated with PSD-95. These findings predict that synaptic scaffolding proteins and protein kinases convergently modulate NMDAR gating and trafficking at synaptic sites.     

PDZ-domain-mediated interaction of the Eph-related receptor tyrosine kinase EphB3 and the ras-binding protein AF6 depends on the kinase activity of the receptor

Eph-related receptor tyrosine kinases (RTKs) have been implicated in intercellular communication during embryonic development. To elucidate their signal transduction pathways, we applied the yeast two-hybrid system. We could demonstrate that the carboxyl termini of the Eph-related RTKs EphA7, EphB2, EphB3, EphB5, and EphB6 interact with the PDZ domain of the ras-binding protein AF6. A mutational analysis revealed that six C-terminal residues of the receptors are involved in binding to the PDZ domain of AF6 in a sequence-specific fashion. Moreover, this PDZ domain also interacts with C-terminal sequences derived from other transmembrane receptors such as neurexins and the Notch ligand Jagged. In contrast to the association of EphB3 to the PDZ domain of AF6, the interaction with full-length AF6 clearly depends on the kinase activity of EphB3, suggesting a regulated mechanism for the PDZ-domain-mediated interaction. These data gave rise to the idea that the binding of AF6 to EphB3 occurs in a cooperative fashion because of synergistic effects involving different epitopes of both proteins. Moreover, in NIH 3T3 and NG108 cells endogenous AF6 is phosphorylated specifically by EphB3 and EphB2 in a ligand-dependent fashion. Our observations add the PDZ domain to the group of conserved protein modules such as Src-homology-2 (SH2) and phosphotyrosine-binding (PTB) domains that regulate signal transduction through their ability to mediate the interaction with RTKs.     

MAGUKs are essential,but redundant,in long-term potentiation

This study presents evidence that the MAGUK family of synaptic scaffolding proteins plays an essential, but redundant, role in long-term potentiation (LTP). The action of PSD-95, but not that of SAP102, requires the binding to the transsynaptic adhesion protein ADAM22, which is required for nanocolumn stabilization. Based on these and previous results, we propose a two-step process in the recruitment of AMPARs during LTP. First, AMPARs, via TARPs, bind to exposed PSD-95 in the PSD. This alone is not adequate to enhance synaptic transmission. Second, the AMPAR/TARP/PSD-95 complex is stabilized in the nanocolumn by binding to ADAM22. A second, ADAM22-independent pathway is proposed for SAP102.

Discovered 50 y ago, long-term potentiation (LTP) remains the most compelling cellular model for learning and memory. It is generally agreed that NMDA receptor (NMDAR)-dependent LTP is mediated primarily by a postsynaptic modification involving the trafficking of the AMPA-type glutamate receptor (AMPAR) (1–4). During the past decade, effort has been focused on the molecular mechanisms underlying both the constitutive and activity-dependent trafficking of these receptors. A family of synaptic scaffolding proteins, referred to as membrane-associated guanylate kinases (MAGUKs) has featured prominently in these studies (5, 6). These proteins contain three PDZ domains, which are involved in protein–protein interactions. The PSD-95 family of synaptic MAGUKs include PSD-95, PSD-93, and SAP102 and are highly expressed at excitatory synapses (5, 7). In terms of AMPAR basal synaptic trafficking, all MAGUKs appear to play overlapping roles (8). Most research has focused on PSD-95. Overexpressing PSD-95 causes a roughly threefold enhancement in AMPAR excitatory postsynaptic currents (EPSCs) with no change in the NMDAR EPSC (9–13). The enhancement mimics LTP, especially with its selective effect on AMPAR EPSCs (as reviewed in ref. 3). Furthermore, PSD-95 occludes LTP, suggesting a common mechanism (9, 12). This suggests that PSD-95 is an essential step in LTP. However, LTP remains intact in cells lacking PSD-95 (14–16), raising the possibility that PSD-93 or SAP102 may play redundant roles.PSD-95 binds to many synaptic proteins (17, 18) including transsynaptic cell-adhesion proteins (e.g., neuroligins, LRRTMs) (19–24). Of particular interest is ADAM22, a member of a large family of catalytically inactive metalloproteases (25, 26), which, via its binding to the secreted protein LGI1, governs transsynaptic nanoalignment. Deleting either ADAM22 (27) or LGI1 (28) reduces AMPAR synaptic transmission. Critical for ADAM22’s function is the presence of a PDZ binding motif (PBM) at the cytoplasmic C terminus. Thus, expressing a mutated form of ADAM22, which lacks the PBM (ADAM22ΔC5) fails to rescue the defect, resulting from the deletion of ADAM22 (27). Furthermore, AMPAR responses are depressed in ADAM22^ΔC5/ΔC5 knockin (KI) mice (29). Previous results found that the typical enhancement in AMPAR responses seen with the overexpression of PSD-95 or the depression observed with the knockdown (KD) of PSD-95 is absent in LGI1 knockout (KO) mice (27). Interestingly, the depression observed with the KD of SAP102 remained intact (27). Complimentary results are seen with ADAM22^ΔC5/ΔC5 KI mice (29) where overexpression of PSD-95 failed to enhance synaptic transmission. Surprisingly, LTP was intact in these mice. What could account for the dissociation of the enhancing action of PSD-95 and LTP?Here we show an essential role for MAGUKs in both basal synaptic transmission and in LTP. Any one of the MAGUKs can substitute for each other. However, coexpression of the MAGUKs suggests differences in their action. The synaptic enhancement seen with the coexpression of PSD-95 and PSD-93 is no greater than the enhancement observed when singly expressed. In contrast the enhancement seen with the coexpression of PSD-95 and SAP102 is additive. In addition, when MAGUK binding to ADAM22 is eliminated in ADAM22^ΔC5/ΔC5 KI mice, PSD-95 is no longer functional, but the action of SAP102 remains intact. Finally, KD of SAP102 in ADAM22^ΔC5/ΔC5 KI mice abolishes LTP. Based on these results we propose a model in which the AMPAR/TARP/PSD-95 complex binds to ADAM22, a transsynaptic adhesion protein essential for nanocolumn stability, which tethers AMPAR receptors in the nanocolumn. An additional pathway involving SAP102 would hold the AMPAR/TARP/SAP102 complex in the nanocolumn by an ADAM22-independent mechanism.     

膜相关性鸟苷酸激酶与脑缺血

膜相关性鸟苷酸激酶（membrane associated guanylate kinase,MAGUK）是一类突触后致密蛋白（postsynaptic density,PSD）,定位于突触后膜的突触后致密区,包括PSD-95、SAP-102、PSD-93和SAP-97.MAGUK家族成员含有多个蛋白结合位点,这些特殊的结合位点负责介导多种信号的转导.MAGUK参与中枢神经系统疾病发生和发展机制,已成为神经科学研究的热点.文章简要综述了MAGUK在缺血性脑损伤中的作用.     

Direct interactions between PSD-95 and stargazin control synaptic AMPA receptor number

Excitatory synapses in the brain exhibit a remarkable degree of functional plasticity, which largely reflects changes in the number of synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). However, mechanisms involved in recruiting AMPARs to synapses are unknown. Here we use hippocampal slice cultures and biolistic gene transfections to study the targeting of AMPARs to synapses. We show that AMPARs are localized to synapses through direct binding of the first two PDZ domains of synaptic PSD-95 (postsynaptic density protein of 95 kDa) to the AMPAR-associated protein, stargazin. Increasing the level of synaptic PSD-95 recruits new AMPARs to synapses without changing the number of surface AMPARs. At the same time, we show that stargazin overexpression drastically increases the number of extra-synaptic AMPARs, but fails to alter synaptic currents if synaptic PSD-95 levels are kept constant. Finally, we make compensatory mutations to both PSD-95 and stargazin to demonstrate the central role of direct interactions between them in determining the number of synaptic AMPARs.     

The immunoglobulin family member dendrite arborization and synapse maturation 1 (Dasm1) controls excitatory synapse maturation

In the developing mammalian brain, a large fraction of excitatory synapses initially contain only N-methyl-D-aspartate receptor and thus are "silent" at the resting membrane potential. As development progresses, synapses acquire alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs). Although this maturation of excitatory synapses has been well characterized, the molecular basis for this developmental change is not known. Here, we report that dendrite arborization and synapse maturation 1 (Dasm1), an Ig superfamily member, controls excitatory synapse maturation. Dasm1 is localized at the excitatory synapses. Suppression of Dasm1 expression by using RNA interference or expression of dominant negative deletion mutants of Dasm1 in hippocampal neurons at late developmental stage specifically impairs AMPA-R-mediated, but not N-methyl-D-aspartate receptor-mediated, synaptic transmission. The ability of Dasm1 to regulate synaptic AMPA-Rs requires its intracellular C-terminal PDZ domain-binding motif, which interacts with two synaptic PDZ domain-containing proteins involved in spine/synapse maturation, Shank and S-SCAM. Moreover, expression of dominant negative deletion mutants of Dasm1 leads to more immature silent synapses. These results suggest that Dasm1, as a transmembrane molecule, likely provides a link to bridge extracellular signals and intracellular signaling complexes in controlling excitatory synapse maturation.     

Dynamic and specific interaction between synaptic NR2-NMDA receptor and PDZ proteins

The relative content of NR2 subunits in the NMDA receptor confers specific signaling properties and plasticity to synapses. However, the mechanisms that dynamically govern the retention of synaptic NMDARs, in particular 2A-NMDARs, remain poorly understood. Here, we investigate the dynamic interaction between NR2 C termini and proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) scaffold proteins at the single molecule level by using high-resolution imaging. We report that a biomimetic divalent competing ligand, mimicking the last 15 amino acids of NR2A C terminus, specifically and efficiently disrupts the interaction between 2A-NMDARs, but not 2B-NMDARs, and PDZ proteins on the time scale of minutes. Furthermore, displacing 2A-NMDARs out of synapses lead to a compensatory increase in synaptic NR2B-NMDARs, providing functional evidence that the anchoring mechanism of 2A- or 2B-NMDARs is different. These data reveal an unexpected role of the NR2 subunit divalent arrangement in providing specific anchoring within synapses, highlighting the need to study such dynamic interactions in native conditions.     

Synapse-specific regulation of AMPA receptor function by PSD-95

PSD-95 is a major protein found in virtually all mature excitatory glutamatergic synapses in the brain. Here, we have addressed the role of PSD-95 in controlling glutamatergic synapse function by generating and characterizing a PSD-95 KO mouse. We found that the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)subtype of glutamate receptor (AMPAR)-mediated synaptic transmission was reduced in these mice. Two-photon (2P) uncaging of MNI-glutamate onto individual spines suggested that the decrease in AMPAR function in the PSD-95 KO mouse stems from an increase in the proportion of "silent" synapses i.e., synapses containing N-methyl-d-aspartate (NMDA) receptors (NMDARs) but no AMPARs. Unexpectedly, the silent synapses in the KO mouse were located onto morphologically mature spines. We also observed that a significant population of synapses appeared unaffected by PSD-95 gene deletion, suggesting that the functional role of PSD-95 displays synapse-specificity. In addition, we report that the decay of NMDAR-mediated current was slower in KO mice: The contribution of NR2B subunit containing receptors to the NMDAR-mediated synaptic current was greater in KO mice. The greater occurrence of silent synapses might be related to the greater magnitude of potentiation after long-term potentiation induction observed in these mice. Together, these results suggest a synapse-specific role for PSD-95 in controlling synaptic function that is independent of spine morphology.     

Interaction of the AMPA receptor subunit GluR2/3 with PDZ domains regulates hippocampal long-term depression

The interaction of PDZ domain-containing proteins with the C termini of alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) receptors has been suggested to be important in the regulation of receptor targeting to excitatory synapses. Recent studies have shown that the rapid internalization of AMPA receptors at synapses may mediate, at least in part, the expression of long-term depression (LTD). We have previously shown that phosphorylation of Ser-880 on the AMPA receptor GluR2 subunit differentially regulated the interaction of GluR2 with the PDZ domain-containing proteins GRIP1 and PICK1. Here, we show that induction of LTD in hippocampal slices increases phosphorylation of Ser-880 within the GluR2 C-terminal PDZ ligand, suggesting that the modulation of GluR2 interaction with GRIP1 and PICK1 may regulate AMPA receptor internalization during LTD. Moreover, postsynaptic intracellular perfusion of GluR2 C-terminal peptides that disrupt GluR2 interaction with PICK1 inhibit the expression of hippocampal LTD. These results suggest that the interaction of GluR2 with PICK1 may play a regulatory role in the expression of LTD in the hippocampus.     

An RBCC protein implicated in maintenance of steady-state neuregulin receptor levels

Despite numerous recent advances in our understanding of the molecular mechanisms underlying receptor tyrosine kinase down-regulation and degradation in response to growth factor binding, relatively little is known about ligand-independent receptor tyrosine kinase degradation mechanisms. In a screen for proteins that might regulate the trafficking or localization of the ErbB3 receptor, we have identified a tripartite or RBCC (RING, B-box, coiled-coil) protein that interacts with the cytoplasmic tail of the receptor in an activation-independent manner. We have named this protein Nrdp1 for neuregulin receptor degradation protein-1. Northern blotting reveals ubiquitous distribution of Nrdp1 in human adult tissues, but message is particularly prominent in heart, brain, and skeletal muscle. Nrdp1 interacts specifically with the neuregulin receptors ErbB3 and ErbB4 and not with epidermal growth factor receptor or ErbB2. When coexpressed in COS7 cells, Nrdp1 mediates the redistribution of ErbB3 from the cell surface to intracellular compartments and induces the suppression of ErbB3 and ErbB4 receptor levels but not epidermal growth factor receptor or ErbB2 levels. A putative dominant-negative form of Nrdp1 potentiates neuregulin-stimulated Erk1/2 activity in transfected MCF7 breast tumor cells. Our observations suggest that Nrdp1 may act to regulate steady-state cell surface neuregulin receptor levels, thereby influencing the efficiency of neuregulin signaling.     

Emerging therapies in gastrointestinal cancers

EPIDEMIOLOGY Gastrointestinal cancers account for 21% of all cancers incidences and 25% of the cancer mortality in the United States[1]. Despite recent advances in diagnosis and treatment, gastric carcinoma, ductal carcinoma of the pancreas and colorectal…     

Postsynaptic assembly induced by neurexin-neuroligin interaction and neurotransmitter

Presynaptic and postsynaptic differentiation occurs at axodendritic contacts between CNS neurons. Synaptic adhesion mediated by synaptic cell adhesion molecule (SynCAM) and beta-neurexins/neuroligins triggers presynaptic differentiation. The signals that trigger postsynaptic differentiation are, however, unknown. Here we report that beta-neurexin expressed in nonneuronal cells induced postsynaptic density (PSD)-95 clustering in contacting dendrites of hippocampal neurons. The effect is specific to beta-neurexin and was not observed with other synaptic cell adhesion molecules such as N-cadherin or SynCAM. NMDA receptors, but not alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptors (AMPARs), were recruited to this beta-neurexin-induced PSD-95 scaffold. Remarkably, AMPARs were inserted into this scaffold upon glutamate application or expression of a constitutively active form of calmodulin kinase II in neurons. Expression of a dominant-negative neuroligin-1 in cultured neurons markedly reduced the sizes and densities of PSD-95 puncta and AMPAR clusters. In addition, excitatory, but not inhibitory, synaptic functions were impaired in these neurons, confirming that PSD-95/neuroligin-1 interaction is involved in postsynaptic assembly at glutamatergic synapses. These results demonstrate that postsynaptic assembly of the glutamatergic synapse may be initiated by presynaptic beta-neurexin and that glutamate release also is required for maturation of synapses.     

ErbB receptors, their ligands, and the consequences of their activation and inhibition in the myocardium

The epidermal growth factor (EGF) receptor (or ErbB1) and the related ErbB4 are transmembrane receptor protein tyrosine kinases which bind extracellular ligands of the EGF family. ErbB2 and ErbB3 are “co-receptors” structurally related to ErbB1/ErbB4, but ErbB2 is an “orphan” receptor and ErbB3 lacks tyrosine kinase activity. However, both are important in transmembrane signalling. All ErbB receptors/ligands are intimately involved in the regulation of cell growth, differentiation and survival, and their dysregulation contributes to some human malignancies. After extracellular ligand binding, receptor dimerisation and transautophosphorylation of intracellular C-terminal tyrosine residues, they bind signalling proteins which recognise specific tyrosine-phosphorylated motifs. This leads to activation of multiple signalling pathways, notably the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade and the phosphoinositide 3-kinase (PI3K)/protein kinase B [PKB/(Akt)] pathway. In heart, targeted deletion of ErbB2, ErbB3, ErbB4 and some ErbB receptor extracellular ligands leads to embryonic lethality resulting from cardiovascular defects. ErbB receptor ligands improve cardiac myocyte viability and are hypertrophic, partly because of activation of ERK1/2 and/or PI3K/PKB(Akt). Furthermore, ErbB transactivation by Gq protein-coupled receptor (GqPCR) signalling may mediate the hypertrophic effects of GqPCR agonists. The utility of anthracyclines in cancer chemotherapy can be limited by their cardiotoxic side effects and these may be counteracted by ErbB receptor ligands. ErbB2 is the target of anti-cancer monoclonal antibody trastuzumab (Herceptin), and its myocardial downregulation may account for the occasional cardiotoxicity of this therapy. Here, we review the basic biochemistry of ErbB receptors/ligands, and emphasise their particular roles in the myocardium.     

N-ethylmaleimide-sensitive factor is required for the synaptic incorporation and removal of AMPA receptors during cerebellar long-term depression

Cerebellar long-term depression (LTD) is a persistent attenuation of synaptic transmission at the parallel fiber-Purkinje cell synapse mediated by the removal of GluR2 subunit-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The removal of AMPA receptors requires protein kinase C phosphorylation of the GluR2 subunit within its carboxyl-terminal PSD-95/Discs Large/Zona Occludens-1 (PDZ) ligand and binding of the PDZ domain-containing protein, PICK1. The sequence of the GluR2 subunit is similar to that of the GluR3 and GluR4c subunits, which also contain PDZ ligands and protein kinase C consensus sites. Although GluR3 and GluR4c are also expressed in Purkinje cells, we have previously shown that cerebellar LTD is absent in GluR2(-/-) mice, suggesting that these subunits are unable to substitute functionally for GluR2. Here, we examine the apparent difference in the regulation of these AMPA receptor subunits by attempting to rescue LTD in GluR2(-/-) Purkinje cells with WT and mutant GluR2 and GluR3 subunits. Our results show that the selective interaction of the GluR2 subunit with the N-ethylmaleimide-sensitive factor protein is required for synaptic, but not extrasynaptic, incorporation of AMPA receptors as well as for their competence to undergo LTD. In addition, perfusion of a synthetic peptide that acutely disrupts the interaction of GluR2 with N-ethylmaleimide-sensitive factor selectively depletes GluR2-containing receptors from synapses and occludes LTD. These findings demonstrate that interaction of AMPA receptors with N-ethylmaleimide-sensitive factor plays a critical role in incorporation of AMPA receptors into synapses and for their subsequent removal during cerebellar LTD.     

A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage

Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-D-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are lacking. Here we report the design and synthesis of a novel dimeric inhibitor, Tat-NPEG4(IETDV)(2) (Tat-N-dimer), which binds the tandem PDZ1-2 domain of PSD-95 with an unprecedented high affinity of 4.6 nM, and displays extensive protease-resistance as evaluated in vitro by stability-measurements in human blood plasma. X-ray crystallography, NMR, and small-angle X-ray scattering (SAXS) deduced a true bivalent interaction between dimeric inhibitor and PDZ1-2, and also provided a dynamic model of the conformational changes of PDZ1-2 induced by the dimeric inhibitor. A single intravenous injection of Tat-N-dimer (3 nmol/g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-N-dimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke.     

Supertertiary structure of the synaptic MAGuK scaffold proteins is conserved

Scaffold proteins form a framework to organize signal transduction by binding multiple partners within a signaling pathway. This shapes the output of signal responses as well as providing specificity and localization. The Membrane Associated Guanylate Kinases (MAGuKs) are scaffold proteins at cellular junctions that localize cell surface receptors and link them to downstream signaling enzymes. Scaffold proteins often contain protein-binding domains that are connected in series by disordered linkers. The tertiary structure of the folded domains is well understood, but describing the dynamic inter-domain interactions (the superteritary structure) of such multidomain proteins remains a challenge to structural biology. We used 65 distance restraints from single-molecule fluorescence resonance energy transfer (smFRET) to describe the superteritary structure of the canonical MAGuK scaffold protein PSD-95. By combining multiple fluorescence techniques, the conformational dynamics of PSD-95 could be characterized across the biologically relevant timescales for protein domain motions. Relying only on a qualitative interpretation of FRET data, we were able to distinguish stable interdomain interactions from freely orienting domains. This revealed that the five domains in PSD-95 partitioned into two independent supramodules: PDZ1-PDZ2 and PDZ3-SH3-GuK. We used our smFRET data for hybrid structural refinement to model the PDZ3-SH3-GuK supramodule and include explicit dye simulations to provide complete characterization of potential uncertainties inherent to quantitative interpretation of FRET as distance. Comparative structural analysis of synaptic MAGuK homologues showed a conservation of this supertertiary structure. Our approach represents a general solution to describing the supertertiary structure of multidomain proteins.