Molecular Properties and Cell Biology of the NMDA Receptor

NMDA receptors (NMDARs) play a distinct role at excitatory glutamatergic synapses, where they are usually localized with other ionotropic glutamate receptors, including tors. Two features are essential to their specialized roles in synaptic plasticity and the excitodependent magnesium block, the removal of which requires depolarization of the membrane potential. Second, upon activation, the NMDAR channel passes sodium and, importantly, calcium into the neuron. Calcium is the universal second messenger in numerous intracellular signaling cascades and is critical in synaptic plasticity and mechanisms of neurotoxicity (288). 1 NMDAR Structure and Subunits The functional NMDAR is a tetrameric protein complex with subunits comprised of three gene families: NR1, NR2, and NR3. All NMDAR subunits have similar membrane topology: an extracellular amino-terminal domain (ATD), 3 transmembrane domains (TM1, 3, 4), a re-entrant P loop at TM2 that lines the pore of the channel, and an intracellular C-terminal tail. The ATD is implicated in subunit assembly (210, 213) and in receptor sensitivity to modulators such as protons, polyamines, Zn, and ifenprodil (76, 198, 256) (for review, see (122)). The extracellular S1 (N-terminal) and S2 (between TM3 and TM4) regions comprise the glycine-binding domains of NR1 (124, 162, 350) and NR3 (10, 40), and the glutamate-binding domains of the NR2 subunits (172, 318). The intracellular C-terminal tail, the sequence and length of which varies considerably among the different subunits, interacts with numerous binding partners (252, 364) and contains multiple phosphorylation sites (145, 283, 364). NR1 subunits contain three alternatively spliced exonsone near the N-terminus (N1; exon 5) and two in the C-terminal tail (C1 and C2/C2’; exons 21 and 22). Thus, there are eight different splice forms, seven of which have been identified in vivo (174), that exhibit distinct regional distributions over development (175, 269, 392). Disorders, National Institutes of Health, Bethesda, MD, USA, [email protected] AMPA receptors (AMPARs) and kainate receptors, as well as metabotropic glutamate receptoxic cell death that results from their deregulation (66). First, NMDARs exhibit a voltageJ.W. Hell, M.D. Ehlers (eds.), Structural and Functional Organization of the Synapse, Science+Business Media, LLC 2008 DO I : 10.1007/978-0-387-77232-5_1 © Springer 2, 318 R.J. Wenthold et al. The most common isoforms either contain the C1 and C2 cassettes (i.e., NR1-1a/b) or lack C1 but contain C2 (i.e. NR1-2a/b) with “a” and “b” denoting the absence or presence of the N1 cassette, respectively (174, 175). Importantly, NR1 isoforms differ in properties such as deactivation kinetics (280) and sensitivity to H, polyamines, and other modulators (77, 337). Knockout of NR1 leads to death shortly after birth (88), indicating that the NMDAR plays an essential role in normal nervous system function after birth. Four genes constitute the NR2 subunit family: NR2A-D. Expression of these subunits is regulated developmentally and regionally (135, 173, 226). Briefly, NR2A and NR2B are the most widely expressed NR2 subunits in the forebrain. NR2B is expressed early in development, and decreases with age as NR2A expression increases. NR2C is abundant in adult cerebellar granule cells (324), and NR2D expression peaks at postnatal day (P)7 in the diencephalon and brainstem (75, 226). Physiological properties, including probability of opening, single channel conductance, and sensitivity to block by magnesium, differ significantly among the subunits (66). Additionally, subunit composition specifies binding partners and sensitivity to regulation by phosphorylation (145, 283, 364). Transgenic mice have revealed important roles of the NR2 subunits. Mice lacking NR2A exhibit impaired synaptic maturation (91, 336) and synaptic plasticity (264). The NR2B knockout is fatal, indicating that NR2B is essential for normal neuronal function after birth. Mice overexpressing NR2B show enhanced learning and memory (326) and altered behavioral responses to inflammatory pain (361). Mice lacking both NR2A and NR2C exhibit a dramatic loss of spontaneous and evoked EPSCs in cerebellar granule cells and deficits in motor coordination (139). Mice lacking NR2D are less sensitive to stress and have altered monoaminergic neuronal function (222); overexpression of NR2D impairs NMDAR-dependent long-term potentiation (LTP) (243). NR3A and NR3B are the most recently identified NMDAR subunits. NR3A expression is highest in early postnatal development (4, 50, 321). In contrast, NR3B remains high in adult brains and is expressed predominantly in motor neurons of the brainstem and spinal cord (19, 206). Numerous studies have reported incorporation of NR3 subunits into NR1and NR2-containing NMDAR complexes (4, 254), resulting in decreased channel conductance and calcium permeability. Additionally, NR3 subunits associate with NR1 to form an excitatory glycinergic receptor (40). NR3A knockout mice exhibit enhanced NMDA responses and an increased density of dendritic spines in the cerebral cortex (58), consistent with a role for NR3A in depressing NMDAR activity during postnatal development. Additional diversity of the NMDAR response arises from the complexity of subunit composition and localization. For example, different NMDARs may be expressed at different synapses within the same neuron (for review, see (154)). Furthermore, evidence exists for the incorporation of more than one type of NR2 subunit in each complex (i.e., di-heteromeric NR1/NR2X or tri-heteromeric NR1/NR2X/NR2Y), such as NR1/NR2A/NR2B receptors in hippocampal neuron synapses, NR1/NR2A/NR2C in cerebellar granule cell synapses, and NR1/NR2B/NR2D in substantia nigra dopaminergic neurons (for review, see (55)). Additionally, subcellular localization differs as some receptors are targeted to synapMolecular Properties and Cell Biology of the NMDA Receptor 319 tic sites, both preand postsynaptic, while others are localized extrasynaptically (183, 319, 333) (reviewed in (154)). Thus, NMDAR subunits assemble in complexes that exhibit a range of physiological properties, binding partners, and subcellular localization. 2 Assembly of Functional NMDARs 2.1 Processing in the Endoplasmic Reticulum The NMDAR is a typical integral membrane protein that is thought to follow a standard pathway in its biosynthesis. Most integral membrane proteins are synthesized by ribosomes that bind to the membrane of the endoplasmic reticulum (ER) shortly after the initiation of translation. This initial interaction with the ER occurs at sites known as translocons where a complex of proteins facilitates the translocation of the polypeptide across the ER membrane. As it is being translated, the growing polypeptide undergoes a complex process that will lead to its correct folding and orientation in the membrane. For multi-subunit complexes, assembly may occur cotranslationally (86) or after translation is complete (45). The NR1 subunit is made in large stoichiometric excess relative to NR2 (42, 127). Such a mechanism would ensure that sufficient amounts of NR1 partners are available for all newly synthesized NR2 subunits, and is consistent with the fact that the NR2 subunit is the main determinant of the function of the receptor complex (364). Unassembled NR1 is rapidly degraded with a half-life of one to two hours (127); this is a common fate for unneeded proteins in the ER. For the NMDAR, the first step is expected to be assembly of the NR1/NR2 dimer, followed by the formation of the tetrameric complex. Evidence for the initial formation of an NR1/NR2 dimer is based on the crystal structure of the mature protein (93), although the formation of such intermediates has not been detected biochemically. The mechanism underlying NR1/NR2 assembly remains largely unexplored. Interestingly, biochemical studies have shown that unassembled NR1 is present primarily as a homodimer (213) raising the question of whether this is simply a default route after failure to assemble with NR2 or if NR1 dimer formation is functionally important, perhaps as an intermediate in the interaction with NR2. NR1 can assemble with NR3 to form a glycine receptor (254), and the homodimerization of NR1 may play a role in the formation of NR1/NR3 complexes. 2.2 Retention Mechanisms for NMDARs To prevent use of unassembled, misfolded and otherwise malformed complexes, the ER uses a rigorous quality control mechanism for proteins exported from the ER (83). All NR2 subunits and some NR1 splice variants are retained in the ER unless assembled (209). The NR1 subunit has at least one identified ER retention motif in its C-terminus. Of the NR1 splice variants, only NR1-1, one of the most abundant splice variants, does not reach the cell surface when expressed alone in heterologous cells (244). Using chimeric proteins of the C-terminus and single transmembrane 320 R.J. Wenthold et al. proteins (tac = interleukin-2 receptor α subunit), a consensus ER retention motif, RXR, was identified in the C1 cassette (295, 314, 373). Retention by this site can be modulated by phosphorylation of nearby serine residues by PKA and PKC (293). The NR1-3 splice variant, which contains the C1 and C2’ cassettes, is also expressed on the cell surface due to an export signal in the distal part of the C2’ cassette (295, 314, 373). This site (-STVV) contains a PDZ binding domain as well as a binding site for COPII, which is involved in the export of proteins from the ER. In addition to regulating retention and export of the unassembled subunits, the same sites may affect the processing rate of the assembled complex, as discussed below. Assembly with NR2 negates the retention sites, and the assembled complex leaves the ER. Most of these studies have been done on chimeric proteins, so the role of these retention and export sites in full-length NR1 or the assembled NR1/NR2 complex, is not clear. However, the general principle of ER retention has been demonstrated in the intact animal. Mice in which the NR1 subunit is deleted in the hippocampus show an accumulation of NR2 subunits in the ER (92). While less is known about NR3, it may also use RXR motifs for ER retention and require association with NR1 for export from the ER (206). The ER retention of the NR2 subunits is less studied and likely more complex. None of the four NR2 subunits has been shown to exit the ER unless assembled with NR1. The isolated C-terminus of NR2B appended to tac is retained in the ER, suggesting an ER retention site (119). Efforts to find a specific motif responsible for the retention have been unsuccessful thus far, although truncation of the C-terminus appended to tac leads to increased surface expression in constructs containing the Cterminus up to residue 1070 of NR2B (119). The NR2 subunit, truncated a few amino acids after TM4, is retained in the ER suggesting that there are one or more ER retention signals in the remaining part of the molecule. While NR1 and NR2 subunits can assemble and form functional receptors without most of the C-terminus, a short segment of the C-terminus that includes the motif (HLFY), which begins with the second amino acid after TM4 in NR2A and NR2B, is required to form functional receptors (119). Constructs lacking this motif or containing mutations in this motif are not functional. This seems to be due to the fact that without this motif, the assembled complex does not leave the ER, since the complex appears to be properly folded and assembled based on its ability to bind MK801 (119). Whether the HLFY motif functions as an export signal or is simply an area of the molecule critical to its proper folding remains to be determined. A later study (376) showed that the HLFY motif is not necessary, but can be replaced by alanines if the remainder of the Cterminus is absent. This may indicate the HLFY provides more of a structural role to ensure the proper orientation of the C-terminus, rather than an export motif that is involved in overriding ER retention. Interestingly, the NR2 subunit can form functional receptors when expressed in two distinct pieces (291). Two segments split shortly before TM4 can form functional receptors when expressed in heterologous cells with NR1. Presumably the C-terminus segment can assemble with the remainder of the molecule either in the ER or after exit from the ER. In either case, the retention mechanisms for both segments are overridden. Molecular Properties and Cell Biology of the NMDA Receptor 321 2.3 Preferential Assembly of Complexes While the ER is an essential station through which nearly all integral membrane proteins must pass and is required for their assembly and folding, very little is known about how these events occur. In neurons expressing the two most common NR2 subunits, NR2A and NR2B, three types of receptor are formed, NR1/NR2A, NR1/NR2B and NR1/NR2A/NR2B. Since the functional properties of these three receptors are dramatically different, it is interesting to speculate that their formation may not be due simply to the random association of the subunits, but may be influenced by other factors. In the young adult CA1/CA2 neurons, where both NR2 subunits are relatively abundantly expressed, about 60% of NR2A and 70% of NR2B are present as diheteromeric complexes (NR1/NR2B and NR1/NR2A) based on coimmunoprecipitation results using detergent solubilized extracts (3). Interestingly, at P7, when NR2A is just beginning to increase in abundance, the diheteromeric complexes are present at the same relative amounts, suggesting that although NR2B is present in much greater abundance, only a fraction of NR2A assembles with NR2B. This would argue that formation of complexes is not dependent solely on the relative abundance of the NR2 subunits. It is interesting to speculate that assembly is influenced by factors such as the developmental stage or neuronal activity and that the proportion of receptors containing 2A, 2B or both 2A and 2B can be modulated. Similar effects could be expected for the NR1 splice variants. Although they have a less important effect on receptor function than NR2, there is evidence that they can influence receptor trafficking. Variants containing the C2’ domain show an accelerated trafficking from the ER (228, 244) probably through the interaction with COPII, as noted above. Neuronal activity preferentially leads to increased expression of C2’containing variants (228), and the combined effect would be an increased production of mature NMDARs. It has also been reported that there may be a preference for pairing NR2 subunits with different NR1 splice variants (300). The high level of regulation involving the ER retention of both NR1 and NR2 as well as the exit of the assembled receptor complex reflects the critical role of this receptor in neurons. 3 Post Golgi Sorting and Dendritic Trafficking 3.1 Packaging into Organelles Following release from the ER, integral membrane proteins enter the Golgi apparatus and the trans-Golgi network (TGN) for additional processing and packaging into vesicular and tubulovesicular carriers for transport to the plasma membrane. In neurons many proteins are segregated into either axonal or dendritic compartments, which may involve the packaging of specific proteins into transport vesicles destined for either axons or dendrites either at the level of the TGN or at endosomal compartments soon after release from the TGN. The sorting may also occur later, and the mechanisms by which proteins are sorted into axonal and dendritic compartments can vary. For example, vesicle-associated membrane protein 2 (VAMP2) is delivered to both axons and dendrites but is preferentially removed from dendrites through 322 R.J. Wenthold et al. endocytosis, leading to an accumulation in axons (284). These findings would imply that trafficking in neurons cannot be accomplished with only two transport vesicles, one for axons and one for dendrites, but that multiple routes of delivery must exist for each domain. It is equally unlikely that each protein has its own transport vesicle, but how many there are and how proteins are sorted for packaging into specific vesicle populations remains largely unexplored. Another complication involves explaining sorting of proteins that are present in both axons and dendrites in subsets of neurons, as is the case for NMDARs (discussed below). This would require that different mechanisms be used in different neurons. While evidence suggests that most axonal and dendritic membrane proteins are delivered while associated with some sort of intracellular organelle, proteins can move by lateral diffusion within the plasma membrane. NMDARs and AMPARs can both move rapidly within the plasma memNMDARs can be shown in adult neurons with immunocytochemistry to be associated with intracellular tubulovesicular structures near the TGN and in dendrites (Fig. 1). In young (3 days in vitro (DIV)) cultures of cortical neurons, mobile vesicular structures containing NMDARs can be identified through live imaging (358). In these studies, the NR1 subunit was tagged with EGFP or DsRed and transfected into cultured neurons. Live imaging showed that organelles containing the tagged NR1 move along microtubules at rates averaging about 4 μm/min, somewhat slower than that seen with fast axonal transport (about 30 μm/min). Using co-transfection with tagged AMPARs, about 61% of the NR1 clusters also contained AMPARs. However, when only mobile clusters were analyzed, 28% of the NR1 was associated with AMPARs; this latter figure gives a more accurate estimate of actual transport vesicles that contain both receptors since the total contains stationary clusters that may be nascent synapses, which contain both receptors. Nevertheless, this modest overlap (28%) indicates neither a specific exclusion of the two populations nor an active mechanism for co-localization. Rather, it suggests that the packaging is poorly controlled or entirely random. An important caveat, however, is that these studies were done using over-expressed proteins, and aberrant packaging due to artificially high levels of proteins may have occurred. In a subsequent study (359), the same authors found that transport of NMDARs involved a continuous recycling of receptors from the cell surface to intracellular organelles. Interestingly, these receptors were associated with SAP102, which appeared to remain associated with the receptor complex. In their earlier study (358), these authors had found that PSD-95 was not associated with NMDARs during their transport. Some studies have addressed the molecular nature of transport vesicles and motors that may be involved in the movement of NMDARs in dendrites. These studies show that the NMDAR is part of a large molecular complex, although the different studies identify different associated proteins; such results would fit with the idea that the brane ((338); described more in Section 5), and one study showed that the first appearance of new AMPARs following irreversible inactivation of existing receptors is at the cell body (2). 3.2 Association with Proteins During Transport Molecular Properties and Cell Biology of the NMDA Receptor 323 Fig. 1. NMDARs can exit the Golgi/TGN via clathrin-coated vesicles. Double-labeling using rabbit polyclonal antibodies to AMPARs (combination of 3 antibodies: GluR1, GluR2, GluR2/3) and 10 nm immunogold, and mouse monoclonal antibodies to NR1 (3 antibodies) and 5 nm immunogold, in a section of a neuron soma in the CA1 stratum pyramidale of the hippocampus from a P10 rat. In these studies, identification of 5 nm gold particles, where unclear, was confirmed by detailed examination and photography at higher magnifications. On the right, the micrograph is repeated and major structures are highlighted in color (green for membranous structures and clathrin coats; red for AMPAR-10 nm gold; blue for NR1-5 nm gold). Note that AMPA and NMDA receptor labeling is localized mainly separately in both the Golgi apparatus and trans-Golgi network (TGN), and NR1 is found alone in several clathrin-coated vesicles (CCVs) budding off of the TGN (R.S. Petralia, Y.-X. Wang, and R.J. Wenthold, unpublished data; (259, 260)). receptor is associated with more than one type of protein complex and transport vesicle during its transport to and from synapses and extrasynaptic locations. KIF17, a dendrite-specific microtubule-dependent molecular motor binds directly to the first PDZ domain of mLin-10 and transports a large protein complex containing the NMDAR (297). The NMDAR is linked to the complex through a PDZ interaction of NR2B and mLin-7, which then binds to mLin-2, which forms a link with mLin-10. KIF17 complexes move in dendrites at an average speed of 46 μm/min, which is similar to fast axonal transport but significantly faster than the movement of NR1 (4 μm/min) as described above. The expression of KIF17 and NR2B are closely linked since decreasing KIF17 with antisense oligonucleotides leads to a corresponding decrease in NR2B (110). Interestingly, this decrease in NR2B is offset by a corresponding increase in NR2A, which suggests that the KIF17 model applies only to NR2B-containing receptors. Mice overexpressing KIF17 also up-regulate NR2B expression and showed enhanced learning and memory (368). Further evidence for a microtubule-based movement of NMDARs is that microtubule-destabilizing drugs decrease the number of NMDARs on the surface of dendrites (383). Transport of NMDARs may also involve association with membrane-associated guanylate kinases (MAGUKs), particularly SAP102. With its multiple domains, 324 R.J. Wenthold et al. SAP102 is capable of interacting simultaneously with a number of proteins in addition to the NMDARs. SAP102 (and other MAGUKs) interacts directly through its PDZ domain with Sec8 (285), a member of the mammalian exocyst complex. The exocyst is a complex of eight proteins that has been studied in both yeast and mammalian cells and has been shown to play a role in the intracellular trafficking and delivery to the plasma membrane of a subset of membrane proteins (126, 378). It is not clear why only some proteins are delivered via the exocyst route, although some studies have suggested that the exocyst may provide a mechanism to more specifically target delivery to a particular location on the cell membrane (215). Expression of a Sec8 construct with a mutated PDZ binding domain blocks the surface delivery of the NMDAR in both transfected heterologous cells and in neurons (285). This effect can be negated in heterologous cells by expressing an NR2 subunit with a mutated PDZ binding domain, leading to normal cell surface expression of the receptor, indicating that a separate pathway can be used for delivery of non-PDZ interacting proteins. These results suggest that NMDARs are routinely associated with SAP102 (or another MAGUK) and the exocyst complex at some point in their biosynthetic pathway. Expression of Sec8 with a mutated PDZ binding domain leads to a buildup of NMDAR at several intracellular sites, including the TGN, but the exact point where the exocyst interaction begins and ends is not known. Another protein that interacts with SAP102 (and other MAGUKs) and influences NMDAR trafficking is mPins (the mammalian homologue of the Drosophila melanogaster partner of inscuteable), also referred to as LGN (286). mPins, a multiple domain protein (26, 69), interacts with SAP102 through its domain known as its linker region and the SH3/GK domain of SAP102. Unlike the exocyst complex, which interacts with all NMDARs, mPins plays a more modulatory role in regulating NMDAR trafficking; while NMDAR surface expression can be nearly eliminated by dominantnegative Sec8, similar constructs of mPins have less dramatic effects. Over-expression of mPins or expression of individual domains of mPins, which may act as dominantnegative constructs, influences the number of NMDARs at synapses, as well as spine size and number. One binding partner of mPins is the G-protein subunit, Gαi (367), raising the interesting possibility that NMDAR trafficking could be influenced through mPins by G-protein signaling. A complex consisting of Gαi, SAP102 and the NMDAR can be identified in brain by co-immunoprecipitation, and over-expression of Gαi enhances NMDARs on dendrites of cultured hippocampal neurons (286). A particular motor that could move a SAP102 based complex has not been identified, although KIF1Bα interacts directly with several MAGUKs and could fill this purpose (224). While microtubule-based motors are expected to be involved throughout transport in the dendrite, myosin motors are likely involved in the delivery to and from the plasma membrane. NMDARs are regulated by myosin light chain kinase and interact directly with myosin regulatory light chain (6, 179). The studies discussed above point to two different mechanisms for the delivery of NMDARs, one SAP102-based and the other mLin-7/mLin-2/mLin-10-based. They have very different complexes of proteins that associate directly or indirectly with the NMDAR, but an important common feature is that both rely on PDZ interactions for the formation of the complex, pointing out the importance of PDZ proteins not only to NMDAR organization at the synapse, but also in NMDAR trafficking. With Molecular Properties and Cell Biology of the NMDA Receptor 325 the identification of at least two distinct mechanisms for dendritic transport of NMDARs, a challenge is to determine at which points in the trafficking of NMDARs the two mechanisms are functioning. Given the complex nature of the trafficking of membrane proteins through multiple organelles on their way to and from the cell surface, it is likely that there are additional mechanisms with other associated proteins involved. Like most other membrane proteins, NMDARs are endocytosed and recycled to the plasma membrane. As discussed above, this may be a normal process for their delivery in young neurons. Are new and recycled receptors simply mixed together or are their deliveries separated? Perhaps this is one role for the exocyst, which may associate only with newly-synthesized proteins. 4 NMDARs at the Synapse NMDARs are present at most glutamatergic synapses in the brain (Fig. 2). Although the NMDAR is relatively stable at the synapse, several studies have shown that a number of conditions can cause rapid changes in synaptic NMDARs. For example, activation of type 1 metabotropic receptors causes a rapid internalization of synaptic NMDARs (307), and some forms of LTP and long-term depression (LTD) involve changes in synaptic NMDARs (14, 22, 106, 117, 225, 238). Synaptic NMDARs recycle and exchange with cytoplasmic and extrasynaptic receptors (267, 279, 334). NMDARs may also be altered during homeostatic plasticity (255) as neurons maintain a normal firing rate by scaling their synaptic strength (340). As described in the section on structure, most NMDARs form tetramers containing two obligatory NR1 subunits and two NR2 subunits. Subunit composition of synaptic NMDARs shapes the kinetics of NMDAR-mediated currents. For instance, the presence of the NR2A subunit confers a faster decay time for NMDAR-mediated currents than the presence of the NR2B subunit (347). Therefore, mature synapses exhibit shorter periods of NMDAR activity than immature synapses, which may lead to greater precision of coincidence detection from converging synaptic inputs. Both the number and composition of synaptic NMDARs are closely regulated by factors including (1) their addition to, and removal from, the synapse, (2) their posttranslational modifications such as phosphorylation, and (3) their interaction with associated proteins; all of which can be modified in response to neuronal activity. Each of these factors will be discussed in further detail. 4.1 Regulation of Synaptic NMDARs 4.1.1 Addition and Removal of NMDARs The insertion of NR2Aand NR2B-containing NMDARs into synapses occurs under different conditions. In hippocampal slices cultured from rats at P7, NR2Bcontaining receptors are added constitutively, whereas NR2A-containing receptors are delivered after activation of pre-existing synaptic NMDARs (12). Similar results were found in the visual cortex, where visual experience leads to NMDAR activation that is responsible for the rapid insertion of NR2A-containing receptors (272) and is reversed after visual deprivation (263, 271). Likewise, an initial phase of learning in 326 R.J. Wenthold et al. Fig. 2. Summary of NMDAR trafficking at the synapse in mature forebrain. NMDARs are clustered at the postsynaptic membrane. Their presence in this region is regulated by interactions with PDZ proteins and adhesion molecules that can serve as scaffolds to bring them into contact with signaling and regulatory proteins (see sections on interacting proteins). The number of NMDARs at the synapse can also be modulated by their addition or removal from the synaptic membrane (see Section 4.1.1). To be internalized, NMDARs move to an endocytic zone located in the perisynaptic membrane to undergo clathrin-dependent endocytosis. NMDARs may also be internalized by a clathrin-independent pathway, although this mechanism is not well defined. The insertion of newly synthesized or recycled NMDARs may occur in the endocytic zone or the postsynaptic density. In addition, mobile pools of NMDARs may diffuse laterally between the extrasynaptic and synaptic membranes (see Section 5). Downstream signaling of extrasynaptic and synaptic NMDARs may differ, resulting in deactivation or activation, respectively, of CREB and BDNF. the olfactory system requires NMDAR activation and enhances the expression of NR2A-containing receptors relative to NR2B-containing receptors in the piriform cortex (270). However, there also may be regional differences in the mechanisms of synaptic insertion for NR2Aand NR2B-containing receptors. In hippocampal slices prepared from rats at P6-7 and cultured for approximately 2.5 days, the insertion of NR2B-containing receptors does not change the kinetics of synaptic NMDARs (12), whereas the insertion of NR2A-containing receptors induces a faster decay time (12, 272). This suggests that both are replacing pre-existing NR2B-containing receptors. In contrast, in cerebellar granule cells harvested at P6 and cultured for 6-8 DIV, overexpression of NR2B slows the decay of NMDAR-mediated currents and overMolecular Properties and Cell Biology of the NMDA Receptor 327 expression of NR2A quickens their kinetics (268), indicating that both types of NMDARs can replace the other form. The mechanism that determines the number of synaptic NMDARs is unknown, although it appears to be distinct from the mechanism regulating extrasynaptic receptors. In cultured cerebellar granule cells, overexpression of NR2A or NR2B subunits causes an increase in the total number of functional NMDARs, whereas overexpression of the NR1 subunit does not (268). However, overexpression of NR2 subunits does not change the number of synaptic receptors, indicating that synaptic receptors are not regulated solely by availability. On the other hand, extrasynaptic receptors appear to be determined, at least to some extent, by the number of receptors synthesized. It is important, however, to point out that this latter property may vary among different neuron types. The removal of synaptic receptors likely requires at least three stages. First, the receptor is released from its anchor that holds it at the synapse. Second, for clathrindependent internalization, the receptor moves in the plasma membrane to the edge of the synapse either by diffusion or some form of active transport (see Section 5). Finally, the receptor is removed from the plasma membrane. Most current evidence shows that NMDARs are endocytosed through a clathrin-dependent mechanism (176, 237, 258, 279, 348). For clathrin-dependent internalization from the plasma membrane, proteins often contain an endocytic motif that is recognized by the AP-2 adaptor complex. Interestingly, NR2A and NR2B subunits use different motifs for removal. The NR2B subunit has a tyrosine-based YXXФ consensus motif, YEKL, near its C-terminus that can bind to AP-2 and promote clathrin-dependent internalization (279). In contrast, a homologous tyrosine-based YKKM motif on the NR2A subunit is not involved with NMDAR endocytosis, but rather, a dileucine motif (LL), which also binds to the AP-2 complex, is involved in endocytosis of NR2A subunits (176). Another YXXФ consensus motif of the NR2A subunit near its last transmembrane domain binds to AP-2 and leads to use-dependent rundown of NMDAR current (348). In fact, tyrosine-containing motifs present near the last transmembrane domain of all NR2 subunits are involved in NMDAR internalization and may target NMDARs to late endosomes for degradation (294). Therefore, multiple sites on the C-termini of both NR1 and NR2 subunits are involved in endocytosis of NMDARs, and additional sites are likely to be identified. Recent evidence suggests that NMDARs may also be removed by nonclathrinmediated pathways. For instance, prolonged activation of NMDARs leads to their degradation by calpain in a process that is not dependent on clathrin-mediated endocytosis (371). In clathrin-independent pathways, endocytosis occurs via uncoated invaginations in lipid rafts (149, 234), microdomains of the membrane proposed to act as platforms for neurotransmitter signaling (5). Although little is known about clathrin-independent internalization of NMDARs, it may occur in concert with clathrin-mediated pathways. Similar to the paradigm for internalization of epidermal growth factor receptors (302), higher levels of glutamate in the synapse may trigger clathrin-independent removal of NMDARs. The site of clathrin-independent endocytosis is undefined. 328 R.J. Wenthold et al. Once NMDARs are removed from the synaptic membrane, the intracellular pathways of NR2Aand NR2B-containing receptors diverge. Both encounter early endosomes, but then most NR2B-containing receptors are sent to recycling endosomes, whereas most NR2A-containing receptors travel to late endosomes (176). This implies that although NR2B-containing receptors are endocytosed more readily than NR2A-containing receptors (176), they are more likely to recycle and return to the synapse. 4.1.2 Modification of NMDARs by Phosphorylation Members of the protein tyrosine kinase (PTK) family include Src, Fyn, Lyn, Lck, and Yes. Phosphorylation of tyrosine residues on NR2A and NR2B subunits potentiates NMDAR-mediated currents (357). Tyrosine residues exist on NR2A and NR2B subunits (283). The YEKL internalization motif on the NR2B subunit discussed above contains the tyrosine residue Tyr1472 that is a major substrate for Fyn kinase (231). In cerebellar granule cells, phosphorylation of this residue by Fyn increases the synaptic localization of NMDARs by preventing their internalization (267). Also, in striatal tissue of Fyn knockout mice, basal tyrosine phosphorylation of NR2A and NR2B is reduced and NMDARs fail to redistribute to the synapse upon stimulation of dopamine receptors (72). Calcium influx through NMDARs generates the production of cyclic AMP, which activates cyclic AMP-dependent protein kinase (PKA). PKA can phosphorylate NR1, NR2A, and NR2B subunits. This phosphorylation can increase NMDAR activity by promoting synaptic targeting (54) or enhancing calcium permeability of NMDARs (305). PKA also can associate indirectly with NMDARs via Yotiao, allowing it to overcome the constitutive activity of protein phosphatase 1 (366). In addition, PKA can have indirect effects on NMDARs, such as in its phosphorylation of the immediate early gene cAMP-responsive element-binding protein (CREB), which can then activate other signaling cascades such as the MAP kinase pathway or induce new protein synthesis (352). Due to its calcium-dependent role, PKA is important for the induction of LTP and spatial long-term memory (1). Protein kinase C (PKC) also phosphorylates NR1, NR2A, and NR2B subunits. PKC activation increases the amplitude of NMDAR-mediated currents and channel open probability (43, 101, 374), in part by increasing the number of NMDARs inserted into the synaptic membrane (167). However, these enhancements are probably due to indirect signaling through second messenger cascades (390). Direct phosphorylation of NMDARs by PKC induces rapid dispersal of NMDARs from the synaptic to extrasynaptic membrane (87, 329) and increases their sensitivity to inactivation by intracellular Ca (202). Activation of casein kinase II (CKII) potentiates NMDAR activity by prolonging channel open time (187) and may contribute to the induction of LTP (39). CKII may also facilitate the replacement of NR2B-containing receptors with NR2A-containing receptors because it phosphorylates serine residue Ser1480 in the PDZ binding domain of the NR2B subunit, thereby blocking the interaction of NR2B-containing receptors with PDZ proteins and decreasing their surface expression in an activitydependent manner (49). Molecular Properties and Cell Biology of the NMDA Receptor 329 Cyclin-dependent kinase 5 (Cdk5) phosphorylates NR2A subunits at serine residue Ser1232 (184). This modification is important for mediating NMDA-evoked synaptic currents during the induction of LTP (184), as well as ischemic degeneration of hippocampal CA1 pyramidal neurons (355). 4.1.3 Other Modifications Ubiquitin conjugation involves the sequential actions of ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) (362). The E3 ubiquitin ligase confers protein specificity, in part by forming a complex with F-box proteins. Many postsynaptic proteins undergo ubiquitination and subsequent degradation by proteasomes, and this process may be dependent on neuronal activity (78). It is unclear whether NMDARs undergo ubiquitination. In synaptosomal membranes from cortical neurons, ubiquitination of the NR1, NR2A, and NR2B subunits was not detectable (78). However, in a separate study, expression cloning identified F-box protein 2 (Fbx2) as an interactor of the NR1 subunit (143). Fbx2 was shown to induce the ubiquitination of the NR1 subunit in HEK293T cells through binding to high-mannose glycans on the extracellular domain of the NR1 subunit. Interestingly, effects of Fbx2 inhibition on NMDAR ubiquitination were only observed under conditions of enhanced neuronal activity (143). NMDARs are also modified by S-nitrosylation of cysteines of the NR2A subunit by nitric oxide, which can down regulate ion channel activity (47). 4.1.4 Interactions with PDZ Proteins NMDARs can bind to the PDZ domains of all MAGUKs (156, 171, 235). MAGUKs are abundant at all excitatory synapses, although their expression varies during development. In the hippocampus, the expression of SAP102 peaks during early development whereas the expressions of PSD-95 and PSD-93 are highest during adulthood (82, 287). This parallels the developmental shift from NR2B-containing receptors to NR2A-containing receptors at synapses, suggesting that SAP102 may preferentially bind to NR2B-containing receptors and PSD-95 and PSD-93 may selectively bind to NR2A-containing receptors (287, 336). MAGUKs may function as anchors that retain and cluster proteins such as NMDARs at a particular cellular location. Studies on animal models have shown that the NR2 C-terminus is required for synaptic localization of NMDARs (312, 317). However, these results, in which nearly the entire C-terminus was deleted, could be due to the loss of other components of the C-terminus rather than only the PDZ binding domain. Studies using transfection of wild type and mutant NMDAR subunits Activity-dependent calcium influx induces autophosphorylation of Ca/ calmodulin-dependent protein kinase II (CaMKII), leading to its increased localization at the postsynaptic density (67, 299). CaMKII can bind NR1, NR2A, or NR2B subunits (100, 180), although its interaction with NR2B-containing receptors is much stronger than with NR2A-containing receptors (320). The interaction of CaMKII and NR2B-containing receptors locks CaMKII in an active conformation (16), enabling it to phosphorylate AMPARs and mediate LTP (7, 13, 63). 330 R.J. Wenthold et al. have shown that NR2B-containing receptors depend on the PDZ binding domain for entry/retention at the synapse (267), while NR2A-containing receptors do not (12, 267, 327). Overexpression studies are conflicting, for in hippocampal neurons PSD95 overexpression does not affect the synaptic clustering or function of NMDARs (81, 290), although in cerebellar granule cells it does promote the synaptic insertion of NR2A-containing receptors (196). Knockout mice lacking PSD-95 (218) or PSD93 (208) have no significant loss of synaptic NMDARs. However, double knockout mice lacking both PSD-95 and PSD-93 show a reduction in NMDAR-mediated currents (82), indicating that compensatory mechanisms may stabilize NMDARs at MAGUKs also function as scaffolds that bring NMDARs into contact with regulatory and signaling molecules. For example, interactions with MAGUKs may inhibit internalization of NR2B-containing NMDARs by promoting the phosphorylation of Tyr1472 by Fyn kinase (72, 267), thus preventing the interaction of the NR2B subunit with AP-2 (see Section 4.1.1). Some proteins can be linked to NMDARs by binding to adjacent PDZ domains of MAGUKs. Examples include neuronal nitric oxide synthase (nNOS), which can mediate NMDAR-induced excitotoxicity (36), and synaptic Ras-GTPase activating protein (SynGAP), which couples NMDARs to the MAP kinase pathway (148). Other proteins may be linked to NMDARs by guanylate kinase-associated protein (GKAP), a central molecule that binds to the GK domain of MAGUKs (146). For instance, GKAP binds to Shank, which binds to Homer dimers that then bind to various other proteins including metabotropic glutamate receptors or IP3 receptors (230, 339). NMDARs are linked to the actin cytoskeleton through interactions (147) such as PSD-95-SPAR-actin, GKAP-Shank-cortactin-actin, or PSD-95-citron, which is a target of Rho (94). NMDARs can also associate with actin filaments via actinin (372) or α1-chimerin, an inhibitor of Rac1 (344). This suggests a mechanism for NMDAR activity to control overall spine structure (see Section 6.2). Other PDZ proteins that can also act as scaffolding molecules for NMDARs include synaptic scaffolding molecule (S-SCAM) (125) and channel interacting PDZ domain protein (CIPP) (161). CIPP is highly expressed in thalamus, colliculi, cerebellum, and brainstem, and can also bind to adhesion molecules (161). S-SCAM can link the MAP kinase pathway to NMDARs by binding to nRap GEP (neural GDP/GTP exchange protein for Rap1 small G-protein). S-SCAM (or PSD-95) also binds to membrane-associated guanylate kinase interacting protein (MAGUIN) (377), which may affect cell polarity (242). 4.1.5 Interactions with Adhesion Molecules NMDARs interact directly and indirectly with a variety of adhesion molecules at the synapse, including some that act transynaptically to link NMDARs to the presynaptic terminal. Adhesion molecules play a role in the formation, maturation, function, and plasticity of synapses (56). Similar to PDZ proteins, one of their functions may be to synapses. NMDARs are less affected than AMPARs by changes in MAGUK expression. Acute knockdown studies using shRNA for PSD-95 show either no change in NMDARs while AMPARs decrease (82) or a moderate reduction in NMDA EPSCs (79). Molecular Properties and Cell Biology of the NMDA Receptor 331 cluster NMDARs at synapses. Recent evidence suggests that PDZ proteins and adhesion molecules work together (95, 278), perhaps in a compensatory manner. The binding of presynaptic ephrin-B to postsynaptic EphB receptors induces a direct interaction of EphB receptors with the extracellular domain of NR1 subunits (57) that enhances NMDAR-mediated synaptic function (108, 120). Activated EphB receptors also have tyrosine kinase activity that can indirectly potentiate NMDARmediated Ca influx (325) and lead to synapse formation (57). EphB is critical for the formation of the postsynaptic density and dendritic spines (121). A family of synaptic adhesion-like molecules (SALMs) was recently discovered on the basis of its interactions with MAGUKs (153, 353). Of the five members, SALMS1-3 have PDZ binding domains which could allow SALMs to be linked indirectly with NMDARs at the synapse. However, SALM1 can also bind directly to the extracellular domain of the NR1 subunit when expressed in heterologous cells (353). Although a presynaptic ligand has not yet been found, SALM1 and SALM2 can induce synapse formation and neurite outgrowth (153, 353). Other families of transynaptic adhesion molecules may act indirectly to affect synaptic NMDARs ((56); see also the Section 6.1 below). Presynaptic neurexin binds to postsynaptic neuroligin, which interacts with the MAGUKs. Neuroligin localization at synapses may help recruit NMDARs to synapses and determine if developing synapses become excitatory or inhibitory (182). Similarly, homophilic interactions between presynaptic and postsynaptic cadherins induce interaction with postsynaptic β-catenin, which can bind directly to the PDZ scaffold S-SCAM and may control its synaptic targeting (236). NrCAM binds directly to SAP102 (59) and has been implicated in neuronal positioning and dendrite orientation (62). NCAM180 may interact directly with the NR2A subunit (96) to help mediate synaptic plasticity (31). Furthermore, integrins mediate the developmental switch from NR2B-containing receptors to NR2A-containing receptors at synapses (41) and potentiate NMDARmediated currents by activating src tyrosine kinases (21, 190). 4.2 Presynaptic NMDARs NMDARs are also found at the presynaptic terminal. The effect of presynaptic NMDARs on synaptic transmission varies among regions of the central nervous system. In some areas they negatively regulate neuronal excitability. They can inhibit glutamate release in spinal cord (11), enhance GABA release in cerebellum (71), or mediate LTD in the cerebellum (38), cortex (304), and retinotectal projections (188). However, in other brain regions they positively impact synaptic transmission. Activation of presynaptic NMDARs enhances glutamate release in hippocampal CA1 pyramidal neurons during early postnatal development (203) and is involved in associative LTP of the lateral amygdala (128). Interestingly, the function of presynaptic NMDARs in the entorhinal cortex decreases during development but can be elevated during epileptic states (375). Presynaptic NMDARs may preferentially contain NR2B (138, 375) or NR2D subunits (11, 203, 328) along with NR1 subunits with exon 5 inserts in the N-terminal domain (356). 332 R.J. Wenthold et al. 5 Extrasynaptic NMDARs