(NAS Colloquium) Molecular Kinesis in Cellular Function and Plasticity (2002)

Morgan Sheng^*

Department of Neurobiology, and Howard Hughes Medical Institute, Massachusetts General Hospital and Harvard Medical School, 50 Blossom Street (Wel 423), Boston, MA 02114

A specific set of molecules including glutamate receptors is targeted to the postsynaptic specialization of excitatory synapses in the brain, gathering in a structure known as the postsynaptic density (PSD). Synaptic targeting of glutamate receptors depends on interactions between the C-terminal tails of receptor subunits and specific PDZ domain-containing scaffold proteins in the PSD. These scaffold proteins assemble a specialized protein complex around each class of glutamate receptor that functions in signal transduction, cytoskeletal anchoring, and trafficking of the receptors. Among the glutamate receptor subtypes, the N-methyl-D-aspartate receptor is relatively stably integrated in the PSD, whereas the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor moves in and out of the postsynaptic membrane in highly dynamic fashion. The distinctive cell biological behaviors of N-methyl-D-aspartate and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors can be explained by their differential interactions with cytoplasmic proteins.

Excitatory synapses predominantly use glutamate as the neurotransmitter. When viewed by electron microscopy, excitatory synapses are characterized by an electron-dense thickening of the postsynaptic membrane, termed the postsynaptic density (PSD). Containing specific receptors for the neurotransmitter glutamate, as well as numerous receptor-associated proteins, the PSD can be regarded as a proteinaceous “organelle” specialized for postsynaptic signal transduction. A disk-like structure ˜30–40 nm thick and up to a few hundred nm wide, the PSD is relatively insoluble in nonionic detergents and can be purified to a considerable degree by differential centrifugation (1). Because it is a prime example of a subcellular molecular microdomain and contains the critical proteins involved in synaptic plasticity, the PSD has been intensively studied in recent years (reviewed in refs. 2 and 3).

In neuronal excitatory synapses, glutamate receptors are cardinal components of the postsynaptic specialization and are highly concentrated in the PSD. Recent advances in understanding the molecular organization of the PSD has stemmed largely from studies of glutamate receptors and their interacting proteins. The major postsynaptic glutamate receptors include N-methyl-D-aspartate (NMDA) receptors, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and the group I metabotropic glutamate receptors (mGluRs), which are linked to phospholipase C and phosphoinositide turnover. These glutamate receptors are specifically targeted to the postsynaptic membrane, indeed, even to specific subdomains within the postsynaptic specialization (4).

NMDA receptors are a consistent feature of excitatory synapses of the forebrain, whereas AMPA receptor content is highly variable; indeed, a significant fraction of excitatory synapses lack AMPA receptors altogether (5–7). This differential regulation implies that NMDA and AMPA receptors use distinct mechanisms for synaptic targeting.

NMDA receptors are heteromeric (probably tetrameric) complexes composed of NR1 and NR2 subunits (8). The four different NR2 subunits (NR2A to NR2D) possess long cytoplasmic tails, the C termini of which end in the conserved sequence -ESDV or -ESEV. This C-terminal peptide motif binds to the PDZ domains of PSD-95/SAP90, an abundant constituent of the PSD (9–15). PSD-95/SAP90 belongs to the MAGUK superfamily of proteins, which are characterized by the presence of PDZ domains, a Src homology 3 domain, and a guanylate kinase-like (GK) domain. PDZ domains are modular protein domains of ˜90 aa that are specialized for binding to C-terminal peptides in a sequence-specific fashion (16–18). The interaction between NR2 subunits of the NMDA receptor and PSD-95 is important for specific localization of NMDA receptors in the PSD (19, 20) and in the coupling of NMDA receptors to cytoplasmic signaling pathways (21, 22). For instance, by binding to neuronal nitric oxide synthase (nNOS), PSD-95 facilitates the activation of nNOS by NMDA receptor-mediated calcium influx (23, 24). In addition, PSD-95 is likely to aid in the anchoring of NMDA receptors to the postsynaptic cytoskeleton (25). The general concept of a postsynaptic scaffolding function for MAGUK proteins is supported by genetic studies of Discs large in Drosophila. Discs large (the fly homolog of PSD-95) is important for development of the neuromuscular junction in Drosophila larvae and required for synaptic localization of its binding partners: the Shaker potassium channel and the Fasciclin II adhesion molecule (26–28).

It should be emphasized that in addition to NMDA receptors, PSD-95 probably organizes other membrane proteins (such as adhesion molecules, receptor tyrosine kinases, and ion channels) in the postsynaptic specialization of mammalian neurons. Thus PSD-95-associated proteins may serve anchoring and signaling functions that are not exclusively related to NMDA receptors. For instance, PSD-95 has been reported to bind kainate receptors, a less well-characterized class of ionotropic glutamate receptor that also exists at postsynaptic sites (29). The NMDA receptor/PSD-95 protein complex in the PSD is growing rapidly in size and complexity as newer technologies such as mass spectrometry are used to study its components (30). The total number of proteins in the PSD may be as high as a few hundred, especially if one includes proteins that are only weakly enriched in, or transiently associated with, the PSD. In general, our

understanding of the functional significance of proteins in the PSD lags behind the pace of their identification.

Many of the proteins in the NMDA receptor/PSD-95 complex are specifically and highly enriched in the postsynaptic specialization. An example is SynGAP, a GTPase-activating protein for Ras, which has a C terminus that interacts with all three PDZ domains of PSD-95 (31, 32). The function of SynGAP remains unclear, but it may be involved in regulation of Ras activation in response to NMDA receptor stimulation. A protein termed SPAR, a GTPase protein for Rap, which binds to the GK domain of PSD-95, has been identified (D.Pak and M.S., unpublished observations). SPAR contains two domains that associate with actin and dramatically reorganize the actin cytoskeleton in heterologous cells. SPAR appears to regulate the size and shape of dendritic spines via its GAP activity, thus implicating Rap signaling in the control of postsynaptic structure.

In addition to SPAR, the GK domain of PSD-95 family proteins binds to an abundant family of proteins in the PSD, termed GKAP (also named SAPAP or DAP) (33–36). The C terminus of GKAP in turn binds to the PDZ domain of Shank, a family of scaffold proteins containing multiple additional protein interaction domains including ankyrin repeats, Src homology 3 domain, and proline-rich motifs (37, 38). Via one of these proline-rich motifs, Shank interacts with Homer (37, 38), a cytoplasmic adaptor protein originally discovered by Worley and coworkers (39) as a binding partner of group I mGluRs. The NMDA receptor/PSD-95 complex therefore is potentially linked to mGluRs via Shank and Homer.

The EVH1 domain of Homer binds to an internal sequence motif (consensus sequence PPXXF) in the proline-rich region of Shank and in the cytoplasmic tail of mGluR1/5 (40, 41). Homer proteins typically contain a coiled-coil domain that mediates self-association (41, 42). These “CC-Homers” multimerize to form multivalent complexes that can crosslink multiple proteins that contain the PPXXF motif (41). Several other proteins have been noted to contain the PPXXF Homer-binding consensus, including the IP3 receptor (IP3R), a downstream effector in the mGluR signaling pathway. Multimeric Homer has the potential therefore to link together mGluRs with IP3Rs, mGluRs with

Shank, and IP3Rs with Shank. IP3Rs are concentrated in the smooth endoplasmic reticulum, an intracellular calcium store that extends into dendritic spines and often approaches the postsynaptic specialization (43). Thus the morphological basis exists in dendritic spines for a close interaction between postsynaptic mGluRs, the NMDA receptor complex, and intracellular calcium stores. It is believed that Homer brings IP3Rs into close proximity of the group 1 mGluRs, thereby allowing for more efficient coupling between surface mGluRs and intracellular calcium stores (40). Because Shank is a component of the NMDA receptor complex via binding to GKAP (37), the Homer-Shank interaction potentially links the group 1 mGluRs to the NMDA receptor and its associated proteins (38). Shank and Homer also may contribute to a functional coupling between NMDA receptors and intracellular calcium stores. Shank and Homer are highly and specifically enriched in the PSD and are located at the cytoplasmic face of the PSD (in contrast to PSD-95, which is located close to the postsynaptic membrane). This “deep” location within the PSD is well-suited for potential interactions of Shank and Homer with cytoplasmic proteins and the smooth endoplasmic reticulum. In addition, Shank and Homer could interact with the postsynaptic cytoskeleton that impinges on the cytoplasmic face of the PSD. Indeed, an interaction between Shank and the actin-binding protein cortactin has been discovered (37). Consistent with a role in cytoskeletal regulation, overexpression of Shank in cultured neurons induces enlargement of dendritic spines (C.Sala and M.S., unpublished work). The spine promoting effect depends on synaptic targeting of Shank and the ability of Shank to bind Homer. Thus Shank and Homer cooperate to induce enlargement of dendritic spines. In addition, Shank and Homer act synergistically to recruit IP3R to dendritic spines, presumably by direct binding of IP3R to Homer (C.Sala and M.S., unpublished work). Because they are indirectly associated with NMDA receptors and mGluRs, Shank and Homer may be able to couple morphological responses of dendritic spines to changes in synaptic activity.

The NR1 subunit of the NMDA receptor also participates in a variety of interactions with specific cytoskeletal and signaling proteins (25). Together, the NMDA receptor subunits interact with a multitude of intracellular proteins, either directly or indirectly via scaffold proteins like PSD-95. The immediate envelope of protein interactions that anchors and integrates NMDA receptors in the PSD (the PSD-95 protein complex) can be regarded as a key modular subdomain of the postsynaptic specialization.

Although AMPA receptors also are concentrated at postsynaptic sites of excitatory synapses, the synaptic levels of AMPA receptors are much more heterogeneous than those of NMDA receptors. Some excitatory synapses contain NMDA receptors but not AMPA receptors, especially early in development (5–7). It is also apparent that a large fraction of AMPA receptors lies within intracellular compartments. The synaptic distribution of AMPA receptors can be altered by activity (44, 45), and recent studies suggest rapid activity-regulated delivery of AMPA receptors to synapses (46–48). Thus the synaptic targeting of AMPA receptors appears to be regulated on a much shorter time scale than for NMDA receptors. The rapid movements of AMPA receptors into and out of the postsynaptic membrane has revealed a surprisingly dynamic regulation of the postsynaptic specialization.

AMPA receptors are typically composed of heteromeric combinations of GluR1–4 subunits (8, 49), whose membrane topology is similar to that of NMDA receptor subunits. The C-terminal cytoplasmic tails of AMPA receptor subunits interact with a distinct set of cytoplasmic proteins than do NMDA receptors. These differential protein interactions presumably underlie the differential regulation of synaptic targeting of NMDA and AMPA receptor-channels. The GluR2 and GluR3 subunits of AMPA receptors share a C-terminal sequence (-SVKI) that interacts with the fifth PDZ domain of GRIP/ABP, a family of proteins containing six or seven PDZ domains (50–52). GRIP is enriched in synapses in the brain, but to only a modest degree when compared with PSD-95. GRIP also differs from PSD-95 in being relatively abundant in intracellular compartments in dendrites and cell bodies of neurons, suggesting that GRIP may be involved in trafficking of AMPA receptors, rather than/in addition to synaptic anchoring (51, 53, 54). The fact that overexpression of the C-terminal tail of GluR2 in neurons inhibits synaptic clustering of AMPA receptors (50) is consistent with either an anchoring or trafficking role for GRIP. Blocking GluR2-GRIP interactions also prevents potentiation of synaptic responses, suggesting that binding to GRIP is involved in recruitment of functional AMPA receptors to the synapse (55). Similarly, mutation of the C terminus of GluR1 (which binds to the PDZ domain protein SAP97; ref. 56) also prevents its functional recruitment to synapses (47). Thus interactions between the C terminus of AMPA receptor subunits and PDZ domain scaffold proteins appear to be important for synaptic targeting and/or stabilization of AMPA receptors (57).

In addition to GRIP/ABP, the C-terminal sequence of GluR2/3 mediates binding to PICK-1 (58), another PDZ-containing protein previously shown to bind protein kinase C (59). Phosphorylation of the C terminus of GluR2 prevents its binding to GRIP but not to PICK-1 (60–62), suggesting the

possibility of a phosphorylation-dependent switch in AMPA receptor interaction with PDZ proteins. Phosphorylation-dependent changes in PDZ interactions could regulate the sorting of AMPA receptors during exocytosis and/or endocytosis (63).

A surprising finding was that GluR2 binds to NSF, an ATPase involved in membrane fusion and vesicle trafficking (64–66). NSF binding is mediated by a membrane proximal segment of GluR2’s cytoplasmic tail, distinct from the C terminus that binds to GRIP or PICK-1. Surface expression of AMPA receptors is inhibited by peptides that block the GluR-NSF interaction, suggesting that NSF is involved in the insertion or stabilization of AMPA receptors in the postsynaptic membrane (67). The binding of NSF to AMPA receptor GluR2 subunits in particular seems to allude to the dynamic nature of the trafficking and regulation of AMPA receptors. It is possible that the NSFGluR2 interaction is relevant to synaptic plasticity by regulating the vesicle trafficking or protein unfolding of AMPA receptors (reviewed in ref. 68).

Endocytosis of postsynaptic AMPA receptors is likely to be an important means of depressing excitatory transmission (69–71). The underlying dynamics and molecular mechanisms are being uncovered. Using immunofluorescence and surface biotinylation assays, a rapid rate of basal AMPA receptor endocytosis in cultured hippocampal neurons, which is further accelerated in response to synaptic activity, ligand binding, and insulin, has been measured (63). AMPA-induced AMPA receptor internalization is mediated in part by depolarization and calcium influx through voltage-dependent calcium channels and in part by a novel ligand-binding mechanism that is independent of receptor activation. The endocytosis of AMPA receptors depends on dynamin, but multiple signaling pathways converge on this final mechanism (63, 72). For instance, insulin- and AMPA-induced AMPA receptor internalization differentially depend on protein phosphatases; furthermore, they require distinct sequence determinants within the cytoplasmic tails of GluR1 and GluR2 subunits. Once internalized AMPA receptors can be sorted to different destinations. AMPA receptors internalized in response to AMPA stimulation enter a recycling endosome system, whereas those internalized in response to insulin diverge into a distinct (possibly degradative) compartment. Thus the molecular mechanisms and intracellular sorting of AMPA receptors are diverse and depend on the internalizing stimulus (63, 72). In contrast to AMPA receptors, NMDA receptors show negligible internalization over the time course of minutes to an hour (63).

From the many recent studies reviewed above, a daunting picture is emerging of the molecular complexity of the postsynaptic specialization of glutamatergic synapses. Glutamate receptors (which are fundamental components of the postsynaptic membrane) use their cytoplasmic domains to interact with a variety of intracellular proteins. The receptor is thus anchored by, and integrated into, a sophisticated protein network that supports the receptor’s postsynaptic actions and that modulates the receptor’s activity. Within individual synapses, different subclasses of neurotransmitter receptors (e.g., AMPA, NMDA, and mGluRs) are segregated by differential protein interactions into distinct molecular environments that correspond to localized signaling microdomains. Examples include the PSD-95-based protein complex, which brings (among other things) calcium-regulated molecules into the sphere of influence of the NMDA receptor-calcium channel. These distinct microdomains are linked together in the overall PSD architecture by scaffold proteins such as Shank lying deep in the PSD. The NMDA receptor/PSD-95 complex can be regarded as a relatively stable core substructure of the PSD, whereas AMPA receptors and their associated proteins are much more dynamically regulated. The differential protein interactions of NMDA receptors and AMPA receptors ultimately will explain the contrasting cell biological behaviors of these two different glutamate receptors.

Obviously, we have only reached a qualitative descriptive phase in the analysis of the molecular organization of the PSD. It will be critical to determine the stoichiometry and geometry of interactions involving glutamate receptors and other proteins, if we are to appreciate the true functional architecture of this postsynaptic organelle. It should be also clear that we have a rather static view of postsynaptic structure. An important future challenge is to uncover the developmental and activity-dependent regulation of the protein interactions that underlie the dynamics of the postsynaptic specialization.

I am an Assistant Investigator of the Howard Hughes Medical Institute. This work is supported by the National Institutes of Health (Grant NS35050).

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