Chapter 7
Molecular biology of postsynaptic structures
Flaminio Cattabeni, Fabrizio Gardoni, and Monica Di Luca
Synapses are specialized sites of communication between neurons in the brain that are vital for interneuronal signaling and necessary for the processing and integration of information. Efficient and plastic signal transduction at synapses is critical for the cor-rect functioning of the synapse and information processing in the nervous system. The strength of individual central nervous system (CNS) synapses is thought to be con-trolled by signaling machinery that regulates the number and activity of postsynaptic receptor ion channels.
The neurotransmitter glutamate mediates the majority of excitatory synaptic trans-mission in the brain. Excitatory glutamatergic synapses feature a prominent thickening at the cytoplasmic surface of the postsynaptic membrane at sites of close opposition to the presynaptic terminal for which the term Post Synaptic Density (PSD) was coined.
Electron-microscopy studies have identified in the 1950s the PSD as an electron-dense structure beneath the postsynaptic membrane in register with the active zone of the presynaptic compartment (Palay 1956). The thickness and density of PSD is variable and falls into two categories: type I, where PSD is electron dense and its size exceeds that of nerve terminals (excitatory glutamatergic synapse); it can be described as a kind of web adhering to the postsynaptic membrane; type II, where PSD is less electron-dense and its size is similar to the presynaptic thickening (GABAergic synapse).
PSDs isolated by Cotman et al. (1974) were purified from synaptosomal membranes washed with 3%-N-lauroyl sarcosinate, and contain approximately 10–15 protein bands visible on Coomassie blue stained polyacrylamide gels. The PSD fraction iso-lated by Siekevitz’s group was purified from synaptosomes washed with 0.5% Triton X-100, a milder detergent than sarcosinate, and has a more complex protein composi-tion, consisting of about 25–30 protein bands.
Biochemical fractionation showed that the PSD structure includes four major classes of components (see Table 7.1): i) plasma membrane proteins such as ionotropic and
Table 7.1
Protein Binding partners i) plasma membrane proteins
AMPA SAP-97, GRIP
mGluR Homer
Neuroligin SAP-97
NMDA CaM, α-CaMKII, PSD-95 ii) cytoskeletal proteins
Actin α-actinin
α-actinin NMDA, actin, CaMKII Spectrin
Tubulin
iii) signaling proteins
Calmodulin CaMKII, NMDA CaMKII NMDA
Fyn-Src PSD-95
IP3 Homer
nNOS PSD-95
Ras SynGap
SynGAP PSD-95, Ras
iv) linker proteins
GKAP PSD-95, Shank
GRIP AMPA
Homer mGluR, IP3
PSD-95 NMDA, nNOS, GKAP, SynGAP SAP-97 AMPA, Neuroligin
Shank GKAP, Homer
Yotiao NMDA, PP1, PKA
metabotropic glutamate receptor subunits (Kennedy 2000), and neuroligins (Kennedy 1997), ii) signalling proteins such as Ca2+/Calmodulin-dependent kinase II (CaMKII; Kennedy et al. 1983), tyrosine kinases, i.e. Fyn and Src (Elliss et al. 1988) and SynGap (Chen et al. 1998; Kim et al. 1998), iii) cytoskeletal proteins such as actin, spectrin, tubulin (Kelly and Cotman 1978; Carlin et al. 1980), and iv) linker proteins such as members of PSD-95/SAP family (Gomperts 1996; Sheng and Kim 1996).
Hence the PSD contains the receptors with associated signalling and scaffolding proteins that organize signal transduction pathways near the postsynaptic membrane (Fig. 7.1).
7.1.1 Plasma membrane proteins
The ionotropic glutamate N-Methyl-D-Aspartate (NMDA) and 1-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) type receptors are concentrated in the PSD of excitatory synapses (Kennedy 2000). More controversial is the presence in the PSD structure of mGluR1αand mGluR5 subunits of metabotropic glutamate receptors.
7.1.1.1 NMDA receptors
NMDA receptors are oligomeric complexes formed by the coassembly of members of 3 receptor subunit families: NR1, NR2 subfamily (NR2A–D; Hollman and Heinemann 1994), and NR3A (Das et al. 1998). One of these, the NR1 subunit, is a ubiquitous and necessary component of functional NMDA receptor channels. Diverse molecular forms of the NR1 subunit are present and generated by alternative RNA splicing;
PDZ
AMPA NMDA
actin nNOS
PSD-95
mGluR
Ras PKA
PP1 Yotiao
Syn Gap Ca2⫹
Na⫹
GKAP
Shank
CaM
Homer
IP3 Neuroligin
GRIP
-Neurexin
␣-actinin
SH3 GK
NR1 NR2
CaMKII
Fig. 7.1
differential splicing of three exons generate at least eight NR1 splice variants; the spliced exons encode a 21 amino acid sequence in the N-terminus domain (N1) and adjacent sequences of 37 and 38 amino acids in the C-terminus (C1 and C2 respectively).
Splicing out the exon segment that encodes for the C2 cassette removes the first stop codon resulting in a new open reading frame that encodes an unrelated sequence of 22 amino acids (C2′) before a second stop codon is reached. These alternative splice processes cause alteration of the structural, physiological, and pharmacological prop-erties of NR1. Additional diversity is given by the multiplicity of NR2 subunits com-posing the receptor. There are four species of the second subunit type, NR2A–D, each encoded by a different gene. The different NR2 subunits are differentially expressed during different stages of development and in different tissues (Monyer et al. 1994).
Each subunit possesses a large extracellular N-terminal domain and four membrane (M) regions — the M-2 region contains a membrane-reentrant hairpin structure that contributes to the receptor channel pore. The N-terminal domain, which is large, glycosylated, and extracellular, contributes to the agonist-binding site. NMDA receptors bind two agonist ligands: glutamate and the coagonist, glycine. The NR1 subunit contains the binding site for glycine and the NR2 subunit, the binding site for glutamate.
NMDA receptors respond to agonists more slowly than AMPA receptors, and require greater than 2 ms to open. However, they have a higher affinity for glutamate, and their currents persist longer than AMPA receptor currents. The NMDA receptor admits both Na+and Ca2+ions. At the resting potential of the cell, a Mg2+ion blocks the NMDA receptor pore, but the Mg2+ion is released from the pore upon cell depolari-zation. Therefore, opening of the channel requires binding of ligand and simultane-ous depolarization of the cell. The channel thus operates as a coincidence detector that admits current only when agonist binding and cell depolarization take place simultaneously.
NMDA receptors display activity-dependent current decreases of several types, two of which are ligand dependent. One of these, glycine-dependent desensitization, occurs following receptor stimulation by glutamate when glycine concentrations are subsatu-rating, in the nanomolar range. The second, glycine-independent desensitization takes place in the presence of saturating glycine, at concentrations in the micromolar range.
When glutamate binds to the receptor in the presence of low concentrations of glycine, the receptor rapidly enters a desensitized (low-conductance) state. This desensitizing transition is blocked by glycine. NMDA receptor desensitization may limit receptor currents during persistent stimulation by glutamate. Domains of NR2 that influence receptor desensitization characteristics have been defined. A different activity-dependent NMDA receptor current decrease is Ca2+-dependent inactivation. Ca2+-dependent inactivation may be induced by increases in intracellular [Ca2+] that follow activity-dependent fluxes of Ca2+through the receptor. The increase in intracellular [Ca2+] triggers biochemical modifications of the receptor that decrease receptor mean open-ing time. It has recently been shown that Ca2+-dependent inactivation results from
binding of Ca2+-calmodulin to the membrane proximal region of the C-terminal domain of the NR1 subunit.
7.1.1.2 AMPA receptors
AMPA receptors are complexes of four subunit types, GluR1–4, which may be homo-meric or heterohomo-meric. AMPA receptors account for the great majority of fast excitatory CNS synaptic transmission. AMPA receptors have a lower affinity for glutamate than NMDA receptors, and their currents are typically rapid, rising within less than 1 ms.
AMPA receptor channels that contain GluR1, GluR3, or GluR4 subunits, or subunit GluR2 that is encoded by an unmodified GluR2 mRNA, can admit both Ca2+ions and Na+ions. RNA editing of GluR2 mRNA changes the structure of the GluR2 subunit by replacing glutamine with arginine at the “Q/R site” in the pore filter region, at the apex of the M2 hairpin. AMPA receptors containing GluR2 subunits encoded by edited mRNA are impermeable to Ca2+ions. The effect of an edited subunit is dominant, such that inclusion of a single edited GluR2 subunit in an AMPA channel prevents Ca2+entry through a receptor otherwise composed of GluR1, -3, -4, or unedited GluR2 subunits.
7.1.1.3 Metabotropic glutamate receptors
A third class of glutamate receptors present at many excitatory synapses is the metabotropic or heterotrimeric GTP-binding protein-linked glutamate receptors (mGluRs). Subtypes mGluR1 and mGluR5 are concentrated around the outer rim of glutamatergic PSDs as well as in decreasing concentration in the spine membrane as a function of distance from the PSD. A lattice of scaffold proteins may link the cytoplas-mic face of mGluRs to IP3 receptors in the spine apparatus. The lattice may also be connected to PSD-95 and thus to the NMDA receptor complex.
In this aspect, the Shank protein family consists of proteins — core components of PSD structure — sharing a domain organization consisting of ankyrin repeats near the N-terminus followed by SH3 domain, PDZ domain, and a SAM domain at the C-terminus (Naisbitt et al. 1999). Consistent with a scaffolding function, Shank medi-ates multiple protein interactions: the PDZ domain binds the GKAP family of PSD-95 binding proteins, thereby linking Shank to NMDA receptor complexes, while two other distinct PDZ domains of Shank form the binding sites for Homer, which in turn binds mGluR1 and IP3 receptors. From these interactions Shank is acting as a glue between ionotropic and metabotropic receptors in the postsynaptic compartment.
7.1.2 Signaling proteins
PSDs contain different classes of enzymatic systems, most of which are responsible for regulating the phosphorylation state of several PSD substrates: CaMKII represents the most abundant signaling protein in the PSD fraction. There, the enzyme is ideally positioned to play a major role in synaptic plasticity events (Kennedy et al. 1983).
CaMKII is a target for transient Ca2+entry through the NMDA channel and is neces-sary for normal synaptic plasticity in pyramidal neurons (Silva et al. 1992). CaMKII is a multisubunit protein having 8–12 subunits assembled in stochastic combinations from two closely related catalytic subunits, alpha and beta (Hanson and Schulman 1992). In the forebrain, the alpha subunit is about three times as abundant as the beta subunit.
A large body of evidence suggests that αCaMKII is a critical player in Long Term Potentiation (LTP), and it has special properties that make it an attractive candidate for exhibiting persistent changes and serving as a memory molecule (Lisman 1994).
A simple and direct role for CaMKII in triggering and perhaps maintaining LTP is sup-ported by studies in which CaMKII activity was acutely increased either with viral transfection (Pettit et al. 1994), or injection of calcium and calmodulin. In these cases synaptic transmission is enhanced and LTP is occluded. An important property of CaMKII is that when autophosphorylated on Thr286, its activity is no longer depend-ent on Ca2+-calmodulin (CaM). This allows its activity to continue long after the Ca2+
signal has returned to baseline (see also Chapter 12). Biochemical studies have demon-strated that this autophosphorylation does in fact occur after triggering LTP (Liu et al.
1999). That CaMKII autophosphorylation is required for LTP was convincingly demonstrated by an elegant use of molecular genetic techniques in which replacement of endogenous CaMKII with a form of CaMKII containing a Thr286 point mutation was capable of shifting LTP towards LTD (Mayford et al. 1995). A final important piece of evidence implicating CaMKII in LTP is that it can directly phosphorylate the AMPA receptor subunit, GluR1, in situ, and this has been shown to occur following the gener-ation of LTP.
The cytosolic tails of the NR2A/B subunits of the NMDA receptor bind to CaMKII and thus can serve as docking sites for it in the PSD (Gardoni et al. 1998; Strack and Colbran 1998). In addition, both the NR2A and NR2B subunits are phosphorylated by CaMKII (Omkumar et al. 1996; Gardoni et al. 2001). Docking of CaMKII to the tail of the NMDA receptor would position its catalytic domains near the receptor mouth, ideally located for activation by Ca2+flowing through the channel.
In the PSD fraction, phosphotyrosine-mediated pathways interact both physically and biochemically with NMDA receptor (Tezuka et al. 1999). The NMDA receptor NR2B subunit is the major synaptic phosphotyrosine peptide of the excitatory synapse, although the precise effect of tyrosine phosphorylation on NR2B function at the synapse is not yet known.
However, the receptor associates via PSD-95 with Syn-GAP, a PSD protein with a GTPase-activating domain that induces hydrolysis of GTP in complexes with the G protein Ras (Chen et al. 1998). Ras, in its active, GTP-bound state transduces signals from a large number of tyrosine kinases. One of these, Src, is a nonreceptor tyrosine kinase that is abundant in the brain. A related kinase, Fyn, is also found in the PSD.
SynGAP is specifically expressed in neurons and is highly concentrated at synaptic sites in hippocampal neurons, where it is tightly colocalized with PSD-95 (Kim et al.
1998). SynGAP is almost as abundant in the PSD fraction as PSD-95 itself, suggesting
that many synaptic PSD-95 molecules are bound to at least one copy of SynGAP.
The function of RasGAPs is to accelerate the intrinsic guanosine triphosphatase (GTPase) activity of Ras, thus accelerating the rate of inactivation of the GTP-bound form of Ras. Because the most common down-stream effect of GTP-Ras is activation of the MAP kinase (ERK 1 and ERK 2) cascade, RasGAPs can be thought of as brakes on the MAP kinase pathway. How might the function of SynGAP be linked to the NMDA receptor? The RasGAP activity is strongly inhibited by phosphorylation of SynGAP by CaMKII, an early target of calcium flowing through the NMDA receptor.
Hence, activation of the NMDA receptor may lead directly to inhibition of SynGAP and release of the brake on the MAP kinase pathway. An important missing link in this scheme is the nature of signaling pathways at glutamatergic synapses that can activate Ras. Possible candidates include Src or Fyn, which can activate Ras through the N-Shc adaptor protein, or the BDNF and Ephrin/EPH pathways (see Chapter 11). Postulated dendritic targets for regulation by MAP kinase include A-type K+channels that modu-late the sizes of EPSPs and of back-propagating action potentials and MAP2, which may mediate cellular remodeling.
Neuronal NOS, a Ca2+-activated form of NOS, can bind to PSD-95 through a class III PDZ domain interaction in which its own amino-terminal PDZ domain binds to a PDZ domain of PSD-95 (Sattler et al. 1999). Neuronal NOS is not abundant in the PSD fraction and is not expressed at high levels in pyramidal neurons. However, it is highly expressed in certain [gamma]-aminobutyric acid-containing neurons, which also express members of the PSD-95 family. Therefore, PSD-95 may concentrate nNOS near the NMDA receptor at postsynaptic sites in these neurons.
Biochemical and pharmacological evidence indicates that a number of other signal-ing complexes are located in spines within or near the PSD. For example, AMPA recep-tors may be bound to their own unique set of signaling complexes. In addition, the cyclic adenosine monophosphate (cAMP) signaling pathway is implicated in the regu-lation of glutamatergic transmission. cAMP potentiates induction of LTP in the Schaeffer collateral pathway. The favored mechanism involves a regulatory cycle first postulated in liver and muscle and now well documented in dopaminergic transmis-sion. Activated cAMP-dependent protein kinase phosphorylates a protein called Inhibitor-1 (DARPP-32 in the dopaminergic pathway). Upon phosphorylation, Inhibitor-1 becomes an inhibitor of protein phosphatase-1. This inhibition potentiates phosphorylation of proteins that can be dephosphorylated by protein phosphatase-1.
In Schaeffer collateral synapses, the cAMP pathway ‘gates’ autophosphorylation of CaMKII and subsequent induction of LTP.
Phosphatase-1 and the cAMP-dependent protein kinase can be complexed with the NR1 subunit of the NMDA receptor by the scaffold protein Yotiao, a splice variant of a family of AKAP (A-kinase-associated protein) proteins that target the cAMP-depend-ent protein kinase to subcellular compartmcAMP-depend-ents (Westphal et al. 1999). Yotiao and a sec-ond AKAP, AKAP75/150, which targets protein kinase A (PKA), protein kinase C (PKC), and the Ca2+-dependent protein phosphatase calcineurin to dendritic
microtubules, can be detected by immunoblot in the PSD fraction and in immunopre-cipitates of the NMDA receptor. Their relatively low abundance in the PSD fraction suggests that they may be present in a subset of PSDs in the brain. Differential distribution of AKAPs could alter the forms of synaptic plasticity displayed by different synapses.
Pharmacological evidence also indicates that PKC and the MAP kinase pathway participate in postsynaptic regulation of synaptic plasticity at glutamatergic synapses.
The structural basis for their localization in spines is not yet firmly established.
Just as for protein kinases, appropriate location and regulation of protein phosphatases are crucial for proper metabolic control. The calcium-dependent protein phosphatase calcineurin is localized in dendritic spines, perhaps by AKAP75/150. Both Yotiao and the neurabin/spinophilin family of proteins could target protein phosphatase-1 (PP1) to dendritic spines where it can dephosphorylate a variety of substrates. The ubiquitous protein phosphatase 2A (PP2A) is regulated and targeted by tissue-specific subunits; its brain-specific regulatory subunits have just begun to be studied.
7.1.3 Cytoskeletal proteins
The major cytoskeletal components in PSD are actin,α-actinin, spectrin, tubulin, and an homologue of the neurofilament NF-L subunit. A close link between cytoskeleton and glutamate receptors is present in PSD. In fact, the rundown of NMDA channel activity can be prevented by the presence of the microfilament-stabilizing drug, phalloidin. In addition, NMDA currents are increased in hippocampal neurons lacking gelsolin, an actin-severing protein. A direct molecular binding exists between α-actinin and C-terminal domains of NMDA receptor subunits (Wyszynski et al. 1999). Inside the PSD, also brain spectrin, or fodrin, are able to interact directly with NMDA recep-tor complex. Actin filaments bind directly to the PSD cytoplasmic face and associate with PSD components. The integrity of microfilament web secures the synaptic locali-zation of ionotropic glutamate receptors; dissolution of microfilaments within the dendritic spines shifts a portion of NMDA clusters from a synaptic to a non-synaptic localization over a 24 h period.
7.1.4 Linker proteins
Linker proteins belong to a family of synaptic proteins homologous to the product of the Drosophila gene disc large and comprises four closely related proteins called PSD-95 protein family (sometimes also MAGUK proteins), each of which contains five protein-binding domains (Cho et al. 1992). Three amino-terminal PDZ (PSD-95, Discs-large, ZO-1) domains are followed by an SH3 domain and a GK domain homologous to yeast guanylate kinase but lacking enzymatic activity. The first and second PDZ domains bind tightly to the tails of the NR2 subunits of the NMDA recep-tor. The three PDZ domains each have slightly different binding specificities and can interact with a variety of different neuronal membrane proteins. The tight colocaliza-tion of NMDA receptors and PSD-95 at synapses and the abundance of both proteins
in the PSD fraction suggest that in the forebrain, many synaptic NMDA receptors are attached to the PDZ domains of PSD-95 or one of its family members.
Neuroligin is an adhesion molecule that is present throughout the soma and dendrites of many neurons and has been localized to the synaptic cleft and the post-synaptic density of some neurons. It has not been detected in substantial amounts in the PSD fraction; thus, its association with PSD-95 may be transient, or more easily disrupted, than that of other proteins by extraction with detergent during purification of the PSD fraction (Irie et al. 1997). Alternatively, it may associate with PSD-95 in a relatively small proportion of synapses. The recent finding that expression of neuroligin in heterologous cells can induce clustering of presynaptic vesicles in contacting axons suggests that neuroligin may help to induce synapse formation at potential postsynaptic sites that contain NMDA receptor-associated signaling complexes.