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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termed SPAR, a GTPase protein for Rap, which binds to the GK domain of PSD-95, has been identified (D.Pak and M.S., unpublished observations).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) (3336). 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.

Regulated Synaptic Targeting of AMPA Receptors

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 (57). 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 (4648). 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 (5052). 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 (6062), suggesting the



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