As discussed previously [1.2-4], PSD-95 and SAP97 are PSD-MAGUKs and therefore, are involved in the functional localisation and trafficking of receptors in the PSD, via PDZ domain-mediated interactions. PSD-95 has been shown to predominantly interact with the N-methyl-ᴅ-aspartate receptors (NMDARs) (El- Hussein et al., 2000, Cui et al., 2007), whereas the most prominent PDZ domain- mediated receptor interaction of SAP97 is with the α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptors (AMPARs) (Waites et al., 2009a, Howard et al., 2010); research has shown the presence of PSD-95 – AMPAR and SAP97 – NMDAR interactions also (Xu et al., 2008, Li et al., 2011, Marcello et al., 2012, Opazo et al., 2012).
NMDARs/AMPARs belong to the glutamate subset of the ionotropic neurotransmitter receptor family and so, share some structural similarities. NMDARs are glutamate- and glycine-gated cation channels that are formed by the heterotetrameric assembly of subunits belonging to three classes: NR1, NR2A-D and
Introduction
18 NR3A/B (Figure 1.8) (Gottschalk et al., 2009). Two NR1 subunits are a compulsory component of NMDARs, along with two of the other subunits; NR2A and NR2B are the primary other subunits in most central neurons. The NR1 subunit has been found to bind glycine, whereas the NR2 subunit binds glutamate; the NR2 subunit also contains a C-terminal class I PDZ domain binding motif and so, it is this subunit that participates in the PDZ domain-mediated interactions with PSD-95/SAP97 (Sheng and Sala, 2001).
Figure 1.8: A simplistic overview of the structure of the N-methyl-ᴅ-aspartate receptors (NMDARs). a) A front-on perspective showing an individual NRX subunit, along with the different binding sites of various ions/chemicals and the different ions the NMDAR is responsible for transporting. b) A plan view illustrating the tetrameric structure forming a transmembrane pore; the NR1 (red) glycine and NR2 (blue) glutamate binding sites are shown. Image a) taken from http://sunburst.usd.edu/.
AMPARs comprise of four closely related subunits GluR1-4, each of which consists of four transmembrane domains (Figure 1.9a) and these subunits combine in different stoichiometries to form a tetramer ion channel (Figure 1.9b); these tetrameric channels usually consist of a pair of subunit dimers (Shepherd and Huganirl, 2007). The GluR1 subunit contains a C-terminal class I PDZ domain binding motif and so, it is this subunit that participates in the PDZ domain-mediated interactions with PSD-95/SAP97 (Sheng and Sala, 2001).
Glutamate binding site Glycine binding site Transmembrane pore
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Introduction
19
Figure 1.9: A simplistic overview of the structure of the α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptors (AMPARs). a) A front-on perspective showing an individual GluRX subunit. b) A front-on perspective illustrating how the tetrameric structure of individual subunits forms a transmembrane pore. Image taken from (Shepherd and Huganirl, 2007).
Research has shown that the PDZ domain-mediated association of PSD-95 and SAP97 with the C-termini of the NMDARs/AMPARS occurs via the PDZ1 and PDZ2 domains of the PDZ proteins (Wang et al., 2005, Marcello et al., 2012); the solution state NMR structures of SAP97 PDZ2 bound independently to NR2B (Wang et al., 2005) and GluR1 (von Ossowski et al., 2006) have been previously determined. The PDZ domain mediated-interaction between PSD-95/SAP97 and the NMDARs/AMPARs are vital in facilitating the role of PSD-95/SAP97 in the localisation of the NMDARs/AMPARs at the postsynaptic membrane and their transport to/from the membrane; therefore, PSD-95 and SAP97 are believed to have an important role in synaptic plasticity (Craven and Bredt, 1998).
Synaptic plasticity is defined as the ability of a synapse to change in strength and it is the most important foundation in learning and memory storage within the brain (Montgomery et al., 2004). Two of the key processes underlying synaptic plasticity are long-term potentiation (LTP) and long-term depression (LTD) (Shepherd and Huganirl, 2007); these processes act by causing changes in the efficacy of synaptic connections via the addition or removal of receptors to/from the postsynaptic membrane (Montgomery et al., 2004).
Introduction
20 LTP is an activity-dependent enhancement in synaptic transmission, this is usually induced by brief high frequency stimulation and is long lasting (more than 1 hour) (Shepherd and Huganirl, 2007); LTP is widely believed to be a key cellular mechanism in learning and long-term memory (Waites et al., 2009b). NMDARs are postulated to function as a cellular trigger for the phosphorylation of the AMPAR GluR1 subunit by CAMKII (Lau and Zukin, 2007); this facilitates AMPAR insertion into the PSD or directly at the synapse, this process is known as exocytosis (Figure 1.10a).
Figure 1.10: Two of the key processes underlying synaptic plasticity: long-term potentiation (LTP) and long-term depression (LTD). a) An overview of the exocytosis on-going during LTP; AMPAR insertion due to phosphorylation by NMDAR-mediated CaMKII activity occurs either directly at the synapse or at extrasynaptic sites and subsequent diffusion into the synapse (red arrow). b) An overview of the endocytosis occurring during LTD; AMPARs are either recycled or degraded. Image taken from (Shepherd and Huganirl, 2007).
LTD is a long-lasting suppression of synaptic strength and is typically dependent on NMDAR-activated dephosphorylation of the GluR1 AMPAR subunit (Xu et al., 2008); this allows retrieval of the AMPARs from synaptic sites and causes the AMPARs to diffuse out of the PSD, via endocytosis (Figure 1.10b) (Shepherd and Huganirl, 2007, Lau and Zukin, 2007). LTD is widely believed to be an important means of information storage in the brain and is implicated in the process of removing old memories.
The research to-date on the interaction between the PSD-MAGUKs PSD-95/SAP97 and the glutamate receptors NMDAR/AMPAR suggests that this is an important and
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Introduction
21 beneficial set of PDZ domain-mediated interactions. However, there is evidence to suggest that the PDZ domain-mediated interaction between PSD-95 and NMDAR is a potential therapeutic target in the treatment of stroke (Aarts et al., 2002, Cook et al., 2012) and chronic pain (Florio et al., 2009, LeBlanc et al., 2010); this is discussed further in [1.6].