α-Amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptors (AMPARs) are heterotetrameric glutamate-gated ion channels that mediate most of the synaptic transmission in the brain. Alteration in AMPAR synaptic expression has been considered the most important molecular mechanism in the formation of both Hebbian-type and homeostatic synaptic plasticity (Malinow and Malenka 2002; Collingridge et al. 2004; Turrigiano 2008). As AMPARs traffic rapidly between the plasma membrane and cytosolic compartments via vesicle-mediated membrane insertion, internalization and recycling (Malinow and Malenka 2002; Song and Huganir 2002; Newpher and Ehlers 2008), levels of surface AMPAR accumulation can be efficiently regulated by altering receptor dynamics. Ultimately, the total abundance of AMPARs is determined through a balance between receptor synthesis and degradation. However, exactly how neurons regulate AMPAR trafficking and turnover, a question critical to our understanding of synaptic plasticity and higher brain functions, remains less well understood.
Ubiquitin is a small 76 amino acid protein ubiquitously expressed in all eukaryotes. Ubiquitin can be covalently conjugated to other proteins (ubiquitination) through a series of reactions catalyzed by three enzymes: E1–E3. The ubiquitin-activating enzyme E1 activates ubiquitin in an ATP-dependent manner, while E3 is the ligase that links ubiquitin to its substrate at lysine residues and determines substrate specificity. Once a single ubiquitin is conjugated to the target protein (monoubiquitination), an internal lysine in ubiquitin itself can be linked to a second ubiquitin and so on to form a ubiquitin chain (polyubiquitination). Ubiquitination of membrane proteins functions as a tag that can be readily recognized by endocytotic machinery, leading to receptor internalization. Polyubiquitinated proteins are often sorted to the proteasome or lysosome for degradation (Nandi et al. 2006; Schmitt 2006). Of particular interest is the ubiquitin-proteasome system (UPS), which is present in synapses (Bingol and Schuman 2006; Bingol et al. 2010) and plays an important role in synaptic function, including synapse development and maturation (DiAntonio et al. 2001; Ding and Shen 2008), synaptic plasticity (Hegde 2004), pre-synaptic vesicle release (Willeumier et al. 2006) and post-synaptic reorganization through proteolysis of post-synaptic proteins including postsynaptic density protein 95 (PSD-95) and glutamate receptor interacting protein (GRIP) (Colledge et al. 2003; Ehlers 2003; Bingol and Schuman 2004; Guo and Wang 2007). Furthermore, ubiquitination has been implicated in glutamate receptor trafficking and turnover, including NMDA receptors (Kato et al. 2005) and AMPARs (Patrick et al. 2003; Bingol and Schuman 2004). In C. elegans, ubiquitination of AMPARs regulates glutamate receptor (GluR) synaptic accumulation (Burbea et al. 2002). Consistently, in Drosophila, inhibition of the proteasome by subunit mutation increases GluRIIB expression and enhances synaptic transmission at the neuromuscular junction (Haas et al. 2007). However, in the mammalian system, direct evidence for AMPAR ubiquitination and the identity of the participating E3 ligase(s) remain to be established.
Here, we have examined the existence and functions of AMPAR ubiquitination in a mammalian system. We find that AMPARs in rat neurons are subject to direct ubiquitination. Conjugation of multiple ubiquitin molecules to lysine residues at the intracellular C-terminals of GluA1 subunits facilitates AMPAR internalization and reduces receptor cell-surface expression. Importantly, we identify neural precursor cell expressed, developmentally down-regulated 4 (Nedd4) as the E3 ligase involved in AMPAR ubiquitination. Nedd4 is enriched in synapses, co-distributes and physically associates with AMPAR subunits. Nedd4 expression induces GluA1 ubiquitination, resulting in a reduction in AMPAR surface expression. Consistently, Nedd4 knockdown suppresses ubiquitin-induced GluA1 ubiquitination. These results strongly indicate an important role for Nedd4-mediated ubiquitination in AMPAR trafficking.
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- Materials and methods
- Supporting Information
We show that mammalian AMPARs are subject to direct modulation by ubiquitination. Covalent conjugation with ubiquitin molecules shifts the molecular weight of GluA1 from 100 kDa to as high as approximately 300 kDa, indicating the addition of a large number (more than 20) of ubiquitin moieties. Given the spectrum of the ubiquitin smear in isolated GluA1, AMPARs are likely modified mainly by polyubiquitination, but could be multi- or monoubiquitinated as well. This is consistent with recent studies showing high molecular weight ubiquitinated receptors including glutamate receptors (Rezvani et al. 2007) and GABAA receptors (Saliba et al. 2007). In C. elegans, only five ubiquitin moieties or less are conjugated with GluA1 (Burbea et al. 2002), suggesting a more intense ubiquitination process in mammalian AMPARs. At basal conditions, only a limited amount of ubiquitin conjugation can be detected on AMPARs, similar to previous work on C. elegans AMPARs (Burbea et al. 2002) and mammalian GABAA receptors (Saliba et al. 2007) This might result from an inactive ubiquitination process at basal neuronal activity levels, or short-lived ubiquitinated receptors. In neurons, AMPARs are normally assembled into heterotetrameric complexes with the most typical combinations being either GluA1 and GluA2 or GluA2 and GluA3 subunits (Lu et al. 2009). In heterologous cells, transfected GluA1 subunits are known to form homotetrameric channels which, like endogenous receptors, can be targeted onto the plasma membrane and respond to agonist activation. Nevertheless, transfected GluA1 might be processed differently; for instance, proteins may be misfolded, leading to an endoplasmic reticulum-associated degradation response, where defective proteins are selectively ubiquitinated prior to proteasome-mediated degradation (Plemper and Wolf 1999). If this is the case, ubiquitination should occur on GluA1 localized within intracellular compartments, but not at the plasma membrane. Contrary to this possibility, our results from surface pulldown assays reveal that ubiquitin conjugation occurs mainly on cell-surface, rather than intracellular AMPARs, which argues against the involvement of endoplasmic reticulum-associated degradation. The preference of ubiquitination on surface AMPARs may be because of plasma membrane-limited localization of participating ubiquitin ligases such as Nedd4 (Dunn et al. 2004; Ingham et al. 2004). There are four lysine residues residing at the intracellular C-terminus of GluA1. Mutation of K868 or all four lysines to arginines dramatically suppressed the intensity of GluA1 ubiquitination, indicating that the last lysine residue at GluA1 C-terminus K868 is the key site for ubiquitin conjugation, consistent with previous work showing the involvement of C-terminal lysines in C. elegans AMPAR ubiquitination. Reasons for the incomplete knockdown of ubiquitination in the lysine mutant remain unclear, but it might be attributable to ubiquitination at non-lysine residues (Ikeda et al. 2002).
Our results demonstrate that over-expression of ubiquitin increases receptor internalization and reduces GluA1 surface expression, indicating the role of the UPS in AMPAR internalization. Similar results have been observed in a recent study published during the revision of this paper (Schwarz et al. 2010). Interestingly, Schwarz et al. show that AMPAR ubiquitination is facilitated by AMPA treatment, indicating the existence of activity-dependent receptor autoregulation. The observed effects might potentially be a result of ubiquitination of other synaptic proteins, such as PSD-95, which subsequently destabilizes surface AMPARs (Colledge et al. 2003; Bingol and Schuman 2004). However, our data show that mutation of the lysine residues at the GluA1 intracellular terminus suppresses both constitutive and glutamate-induced receptor internalization, supporting a role for the direct involvement of GluA1 ubiquitination in AMPAR internalization. The molecular basis underlying ubiquitination-mediated endocytosis remains unclear. It has been established that AMPARs internalize via the clathrin-coated pit pathway following the binding of the clathrin adaptor protein AP2 to the GluR C-terminus (Man et al. 2000; Lee et al. 2002). Given that AP2 binding is not directly regulated by ubiquitination, it is intriguing to postulate that a distinct, ubiquitination-sensitive clathrin adaptor, such as EPS15 (d’Azzo et al. 2005; Piper and Luzio 2007), might be implicated in ubiquitination-dependent AMPAR trafficking.
In line with the role of ubiquitination in directing proteins to the degradation pathway, we observed that over-expressing ubiquitin reduces total receptor abundance in neurons. Similarly, in C. elegans GluR ubiquitination also leads to a reduction in GluR synaptic accumulation (Burbea et al. 2002). In Drosophila, inhibition of the proteasome by subunit mutation increases GluRIIB expression and enhances synaptic transmission at the neuromuscular junction (Haas et al. 2007). These findings strongly indicate the involvement of the UPS in AMPAR turnover. Because AMPARs can also be degraded by the lysosome (Ehlers 2000), it would be interesting to know whether the distinct machineries involved in AMPAR degradation are exclusively utilized under different conditions, or are employed in a sequentially coordinated manner so as to accomplish complete proteolysis (Geetha and Wooten 2008).
The E3 ligase is a key component in the molecular machinery of AMPAR ubiquitination. Although activity of several E3 ligases such as the anaphase-promoting complex (Juo and Kaplan 2004) as well as the Skp1/Cullin/F Box component including LIN-23 (Dreier et al. 2005) and KEL-8 (Schaefer and Rongo 2006) have been implicated in AMPAR turnover, they do not seem to directly target AMPARs for ubiquitination. We find that Nedd4 is preferentially localized in synapses and associates with AMPARs in neurons. Over-expression of Nedd4 causes GluA1 ubiquitination, which is accompanied by suppression of AMPAR cell-surface expression and excitatory synaptic currents. Consistently, knockdown of endogenous Nedd4 reduces AMPAR ubiquitination. All of these results strongly indicate a role for Nedd4 as an AMPAR E3 ligase. For protein association, Nedd4 typically binds to a proline proline X tyrosine, where X is any amino acid (PPXY) domain in its substrates such as epithelial sodium channels via its tryptophan tryptophan (WW) domain (Staub et al. 1996; Snyder 2005). However, no such domain exists at the GluA1 C-terminus, suggesting the involvement of an unconventional interacting motif or an unidentified intermediate protein. Indeed, many substrates of Nedd4-like E3 ligases such as EPS15, Notch and transforming growth factor beta (TGF-β) receptor 1 do not contain the proline proline X tyrosine, where X is any amino acid (PPXY) domain (Chen and Matesic 2007). In addition, the tryptophan tryptophan (WW) domain has been shown to be able to interact with a motif containing a phosphorylated serine or threnonine (Lu et al. 1999). Interestingly, unlike the effect of ubiquitin, Nedd4 over-expression in neurons only decreases surface AMPAR expression without changing the total receptor amount. This could be because of a mild effect of Nedd4 on AMPAR ubiquitination compared to a stronger ubiquitin-induced effect. Alternatively, Nedd4 might participate in other cellular functions such as the facilitation of AMPAR gene transcription or translation, which in turn would counterbalance ubiquitination-dependent AMPAR degradation. Indeed, Nedd4 has been shown to be able to translocate into the nucleus and regulate nuclear targets (Hamilton et al. 2001). An important feature of Nedd4 is that it contains a C2 domain, through which Nedd4 associates with plasma membrane phospholipids in a calcium-dependent manner (Ingham et al. 2004; Wang et al. 2010). It is intriguing to postulate that calcium-induced membrane translocation of Nedd4 may be important in linking neuronal activity to surface receptor ubiquitination, endocytosis and degradation. In addition, whether Nedd4 is the sole E3 ligase for AMPAR ubiquitination in vivo remains to be addressed with more thorough investigation.
- Top of page
- Materials and methods
- Supporting Information
Appendix S1. Supplementary Materials and Methods.
Figure S1. Nedd4 localizes at the synapse and associates with AMPARs in neurons. Double staining in cortical neurons indicates co-localization of Nedd4 with AMPAR GluA2 (a) or the synaptic protein Shank (b). The boxed area was enlarged (bottom panel) for clarity. Arrows indicate puncta of co-distribution. (c) Western blots of synaptosome fractions prepared from cortical rat brain tissue. Protein assays were performed in lysates prior to westerns to ensure equal loading. AMPAR subunits and Nedd4 were enriched in synaptosomes. (d) Using lysates from rat primary culture, Nedd4 was detected in immunoprecipitates of anti-GluA1 antibodies, but not IgG control (left panel), indicating association of Nedd4 and AMPARs. Reprobing of the membrane confirmed specific pull-down of GluA1 (right panel). Scale bar, 10 µm.
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