|คาสิโนไทย||Structural Insights into
the Regulation of NMDA Receptors by CRMP2
In the dentate gyrus of the hippocampus, more than 90% of granule cells are silent, that is, they do not respond to environmental stimuli, and no longer engage in acquiring new memory (Alme et al., 2010). Silent cells are also quite prevalent in the neocortex (Barth and Poulet, 2012), and other subregions of the hippocampus (Thompson and Best, 1989). What causes these cells to become silent?
Neuronal silence is closely related to memory extinction - inhibition of memory retrieval. Mounting evidence revealed that protein kinase A (PKA) could influence memory extinction (Koh et al., 2002; Isiegas et al., 2006; Mueller et al., 2008; Nijholt et al., 2008; Menezes et al., 2015), but the underlying mechanism remains elusive. The NMDA receptor (NMDAR) has been known to play pivotal roles in all aspects of learning and memory. In particular, its GluN2B (formerly NR2B) subunit is involved in memory extinction (Corcoran et al., 2013; Jacobs et al., 2014; Sun et al., 2018). Furthermore, GluN2B could act as gateways for addictive behavior (Hopf, 2017), which may arise from impairment in memory extinction (Hyman, 2005). What makes GluN2B so special in memory extinction?
TAT is a small peptide (YGRKKRRQRRR) rich in arginine (R). Intriguingly, TAT-containing peptides are also implicated in memory extinction (Marek et al., 2011; Corcoran et al., 2015). CBD3 is a small peptide derived from CRMP2, which binds preferentially to GluN2B over GluN2A (Al-Hallaq et al., 2007). The conjugated TAT-CBD3 peptide has been demonstrated to attenuate NMDAR activity (Brustovetsky et al., 2014). Even poly-arginine peptides (e.g., RRRRRRRRR) can alleviate NMDAR-mediated excitotoxicity (Meloni et al., 2015). How can arginine residues be involved in NMDAR activity? This paper will address these questions on the basis of structural data.
The PKA-Dependent NMDAR Closure
An NMDAR consists of two GluN1 (formerly NR1) subunits and two additional subunits which are predominately either GluN2A (NR2A) or GluN2B (NR2B). Other subunits, GluN3, GluN2C and GluN2D, are relatively rare. Inhibition of PKA has been shown to lengthen the closed duration of GluN2B- but not GluN2A-containing NMDARs (Aman et al., 2014), suggesting that PKA targets GluN2B, not GluN2A. Aman et al. refer to the PKA-dependent closed state as "desensitized state". Since this state could give rise to memory extinction, it will be called the "extinction state".
PKA is an enzyme that catalyzes protein phosphorylation - addition of a phosphate group PO43-. The crucial PKA phosphorylation site has been identified as Serine 1166 (S1166), located in the C-terminal domain (CTD) of GluN2B. Loss of this single phosphorylation site abolishes PKA-dependent potentiation of NMDAR Ca2+ permeation, synaptic currents, and Ca2+ rises in dendritic spines (Murphy et al., 2014).
The following will present evidence to support the "CABT Hypothesis", namely, the extinction state of NMDAR is caused by the binding between the CTD of GluN2B and the CABT complex, which consists of a CRMP2 monomer, alpha (α) and beta (β) tubulin.
The Functions of CRMP2
The collapsin response mediator protein-2 (CRMP2) was first discovered as a principal regulator of axonal extension (Goshima et al., 1995). Since then, its functions have expanded to include dendritic branching (Niisato et al., 2013), axonal transport, protein endocytosis, calcium channel regulation and neurotransmitter release (Khanna et al., 2012; Hensley and Kursula, 2016). More recently, a number of studies demonstrated that CRMP2 also played a key role in dendritic spine development and synaptic integrity (Zhang et al., 2018; Mita et al., 2016; Jin et al., 2016; Zhang et al., 2016; Xing et al., 2016). Hyperphosphorylation of CRMP2 may causes spine loss as observed in Alzheimer's disease (Cole et al., 2007). Strikingly, the gene encoding CRMP2, DPYSL2, is linked to schizophrenia (Fallin et al., 2005; Pham et al., 2016), which may account for the decreased spine density (Glantz and Lewis, 2000) and impaired memory extinction (Holt et al., 2009; Holt et al., 2012) in the disorder.
The Structure of CRMP2
A human CRMP2 consists of 572 amino acids. It may interact with the GluN2B subunit of NMDAR via the region around 480 - 495 (Brustovetsky et al., 2014). The residues between 505 and 525 are involved in the binding to tubulin, as hyperphosphorylation in this region disrupts their interaction (Yoshimura et al., 2005).
Binding Between CRMP2 and Tubulin
Tubulin is the canonical binding partner of CRMP2. It has two isoforms, α and β, that usually form a heterodimer. The dendritic spine contains abundant tubulin heterodimers. In the postsynaptic density (PSD, a structure just beneath the postsynaptic membrane), the amount of α-tubulin molecules accounts for 8% of the PSD protein mass, far exceeding the amount of PSD-95 molecules, which account for only ~ 0.8% (Yun-Hong et al., 2011).
The interaction between CRMP2 and tubulin is directly controlled by the phosphorylation state of CRMP2. Phosphorylation at Thr-514 by glycogen synthase kinase-3beta (GSK-3β) disrupts its binding to tubulin and inhibits axon outgrowth (Yoshimura et al., 2005). However, the phosphorylation at Thr-514 must first be primed by phosphorylation at Ser-522, which is catalyzed by cyclin-dependent kinase 5 (Cdk5) (Cole et al., 2006). Therefore, both GSK-3β and Cdk5 are critical for the CRMP2-tubulin interaction.
CRMP2 forms a tetramer in solution. Interaction with tubulin heterodimers breaks the tetramer of CRMP2 into monomers to form a hetero-trimeric complex, consisting of a CRMP2 monomer, α and β tubulin (Niwa et al., 2017). The hetero-trimeric complex will be referred to as the CABT complex (Figure 3), which could be essential for the integrity of dendritic spines, as hyperphosphorylation of CRMP2 by GSK-3β may causes spine loss (Cole et al., 2007).
The Extinction State of NMDAR
In humans, GluN2B contains a long CTD, including residues 867 - 1484. In rats and mice, their CTD is only slightly shorter, starting from residue 867 to 1482. Both CRMP2 and tubulin have been shown to interact with the GluN2B subunit of NMDARs. Tubulin may bind to the CTD of GluN2B at the region 1243 - 1376 (van Rossum et al., 1999). By utilizing a peptide array, Brittain et al. (2012) identified two peptides in GluN2B that may bind to CRMP2: peptide KPGMVFSISRGIYSC (residues 857–871 of the rat GluN2B sequence) and DWEDRSGGNFCRSCP (residues 1205–1219 of the rat GluN2B sequence). Notably, the second peptide contains the negatively charged sequence, DWED, which could interact with the positively charged H19 in CRMP2. Therefore, the CABT complex may bind to the CTD of GluN2B, with H19 near DWED (Figure 3). Such binding should close NMDARs (Figure 4), abolishing the NMDA plateau, consequently resulting in neuronal silence. With this function, CRMP2 is expected to play an important role in memory processing. Indeed, experiments have demonstrated that the antibody against CRMP2 causes amnesia (Mileusnic and Rose, 2011).
The NMDAR occluded by CABT will be called the "extinction state", because it may give rise to macroscopic memory extinction. Biophysically, the extinction state of an NMDAR resembles the inactivated state of a sodium channel, where the channel pore is occluded by a segment in the intracellular loop (Yu and Catterall, 2003).
Regulation of NMDAR Extinction by PKA
By analyzing the amino acid sequence of the GluN2B CTD, it was found that the CTD is intrinsically disordered, but may switch dynamically between folded and unfolded conformation (Ryan et al., 2008). Since PKA plays a crucial role in NMDAR extinction, it is reasonable to assume that the dynamic switch between folded and unfolded conformation could be controlled by PKA phosphorylation. The unfolded conformation, as shown in Figures 3 and 4, may associate with CABT, resulting in NMDAR extinction, whereas the folded conformation could prevent CABT-GluN2B binding.
The effect of PKA on the Ca2+ influx through NMDARs indicates that phosphorylation of S1166 may prevent CABT-GluN2B binding, thereby reducing NMDAR closure. This in turn suggests that S1166 phosphorylation could promote a folded conformation to block DWED-H19 interaction. The folded conformation is proposed in Figure 5, where DWED is buried within the GluN2B CTD, unable to interact with H19. It is important to note that, for PKA to regulate conformational switch, DWED should be in close proximity to S1166. This can be achieved by two turns: one at GGGP (residues 1169 - 1172) and another at GGVP (residues 1191 - 1194). Hence, this model is consistent with the fact that glycine (G) and proline (P) residues are frequently found in turn and loop structures of proteins (Krieger et al., 2005).
The Effects of Arginine Residues on the Folding of GluN2B CTD
The attractive interaction between a phosphate group and an arginine residue is very strong (Woods and Ferré, 2005). In Figure 5B, there is an arginine that may interact with S1166. The last section assumes that the phosphorylation state of S1166 is sufficient to control the switch between folded and unfolded conformation of the GluN2B CTD. Phosphorylation at S1166 could stabilize the folded structure, preventing CABT-GluN2B binding, consequently enhancing the Ca2+ influx through GluN2B-containing NMDARs. Conversely, dephosphorylation at S1166 should reduce Ca2+ influx as it may promote unfolded structure, permitting CABT to occlude the channel pore.
TAT is a peptide (YGRKKRRQRRR) enriched with arginine. The interaction between a phosphate group and two or more arginine residues is as strong as a covalent bond (see this figure). Therefore, TAT should be able to associate tightly with the phosphorylated S1166, interrupting the DFKRDS - DWEDRS binding, thereby altering the folded conformation and facilitating CABT-GluN2B binding. Consequently, TAT itself can have profound influence on the NMDAR-mediated Ca2+ influx and memory extinction. This notion is supported by the following observations:
Author: Frank Lee