AMPAR Subunits are subject to ubiquitination
To examine whether AMPARs are capable of being modified by ubiquitination, we co-expressed HA-tagged ubiquitin (Ub) together with GFP-tagged GluA1 subunits (GluA1) in HEK 293A cells. 2 days after transfection, GluA1 subunits were immunoprecipitated with anti-GFP antibodies and anti-HA antibodies were used to confirm the presence of ubiquitination. In support of AMPAR ubiquitination, a strong ubiquitin smear, which is a typical biochemical signature for protein ubiquitination resulting from conjugation with a varied number of ubiquitin moieties, was reliably observed in isolated GluA1 ( left and 1B). In contrast, despite similar levels of intense total ubiquitination species in lysates (, right), control cells expressing free GFP and HA-ubiquitin showed no ubiquitin signals in GFP immunoprecipitates (, left). All the cell lysates used for ubiquitination assays in this study were prepared under denaturing conditions to avoid conventional protein-protein association. Therefore, the ubiquitination signals represent a bona fide conjugation of ubiquitin to GluA1 subunits, rather than a conventional protein-protein association. The high molecular weight of the ubiquitin smear indicates the conjugation of multiple ubiquitin molecules with GluA1 subunits. To examine whether AMPAR ubiquitination occurs at the plasma membrane, we incubated GluA1-expressing HEK cells with anti-GFP antibodies to immunoprecipitate surface GluA1. Following a first round of immunoprecipitation to isolate surface GluA1, the remaining supernatants were incubated with anti-GluA1 antibodies to isolate intracellular GluA1. Western analysis revealed intense ubiquitination of surface GluA1 ( left and 1D). However, although more GluA1 was isolated from the cytosolic compartment, no significant ubiquitination signals were detected ( right and 1D). These results indicate that ubiquitination occurs mainly on surface GluA1 subunits. The minimal ubiquitination of intracellular receptors may indicate that the ubiquitinated intracellular receptors are degraded with high efficiency.
Figure 1 AMPAR GluA1 subunits are subject to ubiquitination. (A) HEK cells were co-transfected with GFP-GluA1 or GFP as a control, together with HA-ubiquitin (Ub). pcDNA vector was also transfected as a control. Cell lysates were prepared in a denaturing lysis (more ...)
Ubiquitination facilitates GluA1 degradation
A physiological consequence of protein ubiquitination, especially polyubiquitination, is to direct the protein to degradation pathways. To examine the effect of ubiquitination on AMPAR turnover, we co-expressed GFP-GluA1 in HEK 293A cells with varied amounts of ubiquitin. We found that the abundance of GluA1 showed linear downregulation along with higher levels of ubiquitin expression (). To directly investigate GluA1 degradation, we treated cells with 5 mM anisomycin to inhibit protein synthesis and time chased the remaining GluA1 abundance up to 4 hrs. GluA1 levels showed a time-dependent decrease, with about 40% decrease (0.63 ± 0.12, n=3) after 4 hrs of anisomycin incubation, indicating constitutive receptor degradation. In contrast, in cells transfected with HA-ubiquitin, GluA1 abundance showed a facilitated rate of reduction. At 4 hrs of anisomycin treatment, GluA1 was reduced by approximately 80% (0.23 ± 0.03, n=3) (), strongly indicating that ubiquitination enhances AMPAR turnover.
Figure 2 Ubiquitination causes a reduction in GluA1 protein abundance. (A) GluA1 was transfected in HEK 293A cells with varied amounts of HA-ubiquitin. GluA1 abundance (top) showed an inverse relationship to the level of ubiquitination (middle). Tubulin was probed (more ...)
Lysine residues at the GluA1 C-terminal are targets of ubiquitination and responsible for degradation
During ubiquitination, a ubiquitin molecule is covalently conjugated to a lysine residue of the target protein. When the intracellular domain of the GluA1 subunit was examined, we found four lysine residues all localized on the C-terminus. To identify the site(s) of ubiquitination, we replaced each lysine with arginine at each individual site (K813R, K819R, K822R and K868R) or all four lysines together (4KR) () in an N-terminal GFP-tagged GluA1 construct. Mutation sites were confirmed by sequencing. In HEK 293A cells co-transfected with the KR mutants and HA-ubiquitin, GFP-tagged GluA1 mutants were immunoprecipitated with anti-GFP antibodies and ubiquitinated species in the immunoprecipitates were probed with anti-HA antibodies. We found that typical ubiquitination smears were still detected in K813R, K819R and, to a lesser extent, K822R. In contrast, the intensity of the ubiquitin conjugates in K868R was markedly reduced. Consistently, GluA1 ubiquitination intensity was also significantly reduced when all four lysines were mutated (4KR) (). Since all the KR mutants showed higher protein levels compared to the wildtype control, ubiquitination signals were normalized to GluA1 protein abundance. A clear reduction in ubiquitination intensity on K868R and 4KR was revealed following normalization (). Results from these experiments indicate lysine 868 as the principal residue for GluA1 ubiquitination.
Figure 3 Lysine residues at GluA1 C-terminus are sites of ubiquitination. (A) Illustration of the lysine residues at the C-terminus of GluA1 and various forms of KR mutants. (B) Ubiquitination assays on HEK 293A cells overexpressing GFP-GluA1 wildtype or KR mutants, (more ...)
We found that elevated protein ubiquitination was accompanied with enhanced GluA1 degradation (). However, this degradation could be a consequence of ubiquitination of other proteins, which in turn modulates GluA1 proteolysis. We reasoned that if this ubiquitin-dependent receptor degradation is due to direct ubiquitination on GluA1 subunits, the KR mutants should become resistant to the degradation process. To test this, we expressed GluA1WT and the GluA1 KR mutants in HEK cells to compare their degradation rates. Following incubation with 5 mM anisomycin to inhibit protein synthesis for 0, 3 and 6 hrs, transfected cells were lysed and probed for total GluA1 abundance. We found that protein levels of K813R, K819R and K822R decreased at a rate comparable to that of the wild type GluA1. In contrast, the amount of K868R and 4KR showed little change over a course of 6 hrs (). The reduced degradation rate in K868R and 4KR mutants is consistent with the important role of K868 in ubiquitination.
Ubiquitination and proteasomal degradation of AMPARs in neurons
We then explored the occurrence of AMPAR ubiquitination in neurons. Because AMPARs are enriched at the postsynaptic domain on dendritic spines, we first examined whether ubiquitin is localized at the vicinity of synapses. In cultured cortical neurons, ubiquitin and a synaptic marker protein PSD-95 were immunostained. Ubiquitin signals were detected throughout the neuron. In dendrites, ubiquitin formed intense clusters, most of which co-distributed with the postsynaptic scaffolding molecule PSD-95 (), indicating a concentrated localization of ubiquitin in spines. Consistently, western blotting demonstrated strong ubiquitin immunosignals in brain synaptosomal preparations compared to the lysate (), indicating the occurrence of ubiquitination modification on synaptic proteins.
Figure 4 Ubiquitination and proteasome-mediated degradation of AMPARs in neurons. (A) Double staining of ubiquitin (green) and the postsynaptic marker protein PSD-95 (red) in 2 wk old cultured cortical neurons. The boxed area was zoomed in for clarity (bottom). (more ...)
Polyubiquitinated proteins are normally directed to the proteasome for degradation. Many membrane receptors can be degraded by the proteasome, including the IL-2 receptor, growth hormone receptor, opioid receptor and the inhibitory GABAA
receptors (Hegde 2004
). Degradation of synaptic scaffolding proteins such as PSD-95 is also proteasome dependent (Colledge et al. 2003
). In cultured cortical neurons at basal conditions, we failed to observe obvious AMPAR ubiquitination. This might be due to either a minimal level of ubiquitination or rapid degradation and removal of ubiquitinated receptors by the proteasome. To accumulate ubiquitinated receptors for better detection, we pre-incubated neurons with the proteasome inhibitor MG-132 (5 μM) for 24 hours. Under this condition, a strong ubiquitination smear was detected in immunoprecipitated GluA1 subunits (), indicating the presence of receptor ubiquitination in neurons. In agreement with proteasome-mediated degradation, we found a rapid elevation of AMPAR amount when proteasomal activity was inhibited. Treatment of cultured cortical neurons with MG-132 (5 μM) for 3 hrs significantly increased GluA1 levels (). To confirm that the effect was indeed proteasome dependent, we found a similar increase in GluA1 by other proteasome specific inhibitors including lactacystin (10 μM) and epoxomicin (10 μM), as well as PR11 (0.5 μM) (), indicating proteasome-mediated AMPAR degradation at basal conditions.
Ubiquitination regulates AMPAR internalization and cell-surface expression
A major function of ubiquitination is to sort membrane proteins to the endocytotic pathway (d’Azzo et al. 2005
). To directly assess effects of ubiquitination on AMPAR trafficking, we performed internalization assays (Man et al. 2007
; Hou et al. 2008b
). Cortical neurons were transfected with GFP alone or together with HA-ubiquitin. 2 days after transfection, neurons were incubated with anti-GluA1 N-terminal antibodies at 4°C to label receptors on the cell surface. Cells were then treated with glutamate (50 μM) at 37°C for 15 minutes to induce receptor internalization. Following blockade of the remaining surface receptors with non-conjugated secondary antibodies, the internalized receptors were then specifically labeled under permeant conditions by fluorescent secondary antibodies. We found that GluA1 internalization was markedly enhanced in cells overexpressing ubiquitin compared to those transfected with GluA1 alone (internalized GluA1 intensity: 3301 ± 111, n=15 cells in GFP alone control; 3997 ± 140, n=17 cells in GFP+ubiquitin) ().
Figure 5 Ubiquitin regulates AMPAR internalization and surface expression. (A and B) Cultured cortical neurons were transfected with GFP or GFP plus HA-ubiquitin. Surface AMPARs were labeled by anti-GluA1Nt antibodies, followed by glutamate (50 μM, 15 (more ...)
A direct consequence of receptor endocytosis is to down regulate surface receptor expression. In addition, the internalized AMPARs can be recycled back to the plasma membrane, while a fraction of those can be sorted for degradation (Ehlers 2000
), resulting in a loss of total receptor amount. To examine the effect of ubiquitination on AMPAR surface localization and stability, we transfected cortical neurons with HA-ubiquitin plus GFP, or GFP alone as a control. We then immunolabeled the surface and total endogenous GluA1 subunits in transfected neurons under nonpermeant and permeant conditions with antibodies against GluA1Nt and GluA1Ct, respectively. We found that in neurons overexpressing ubiquitin, GluA1 surface expression was reduced by 45% (control, 1272 ± 11, n=19 cells; ubiquitin, 691 ± 13, n=16 cells) (). Meanwhile, ubiquitin expression also reduced the total amount of GluA1 by 25% (control, 399 ± 8, n=19 cells; ubiquitin, 302 ± 10, n=16 cells) (), consistent with ubiquitination-dependent AMPAR internalization and degradation in neurons. To ensure that the GFP signals could reliably indicate ubiquitin over-expression, we immunostained HA in neurons co-transfected with HA-ubiquitin and GFP. We found that all of the green cells contained red (50 green cells were all red), and most of the red cells contained green (in 50 red cells, 45 showed green) (, bottom), indicating reliable co-expression of double transfected constructs.
To further confirm that ubiquitination on GluA1 is necessary for receptor internalization, we evaluated the effects of the KR mutations on AMPAR trafficking. Cortical neurons were transfected with GFP-GluA1 or GFP-4KR with or without HA-ubiquitin. 24 hours later, cells were incubated with anti-GFP antibodies at 4°C to label surface GluA1 and then treated with glutamate (50 μM) at 37°C for 15 minutes to induce receptor endocytosis. Immunofluorescence intensity acquired following a blockade of the remaining surface receptors was used to indicate receptor internalization. Internalized GluA1 immunofluorescence of 4KR was significantly reduced compared with wildtype GFP-GluA1 (), indicating a role for GluA1 ubiquitination in basal receptor trafficking. More importantly, we found that whereas overexpression of HA-ubiquitin markedly enhanced internalization of wildtype GFP-GluA1, co-transfection with ubiquitin failed to facilitate 4KR internalization (1669 ± 125, n=23 cells in GluA1 alone; 2032 ± 119, n=23 cells in GluA1+ubiquitin; 1385 ± 138, n=20 cells in 4KR; 1411 ± 119, n=20 cells in 4KR+ubiquitin) (). These results strongly indicate that ubiquitin-induced receptor internalization results from direct ubiquitination of lysine residues at the GluA1 C-terminus.
The E3 ligase Nedd4 induces AMPAR ubiquitination in heterologous cells
During ubiquitination, an E3 ligase interacts with a particular substrate protein and conjugates ubiquitin molecules to a lysine residue, thus achieving specificity in protein ubiquitination. In searching for the E3 ligase responsible for AMPAR modification, our recent study demonstrated that intracellular accumulation of sodium triggers proteasome-mediated AMPAR degradation, suggesting the presence of sodium-related machinery in AMPAR ubiquitination. We therefore chose to examine the role of Nedd4 (neural precursor cell expressed, developmentally down-regulated 4) in AMPAR ubiquitination. Indeed, in our earlier work we have identified Nedd4 as an AMPAR E3 ligase (Zhang et al., 2009
). Nedd4 is a HECT domain-containing single molecule E3 ligase (Ingham et al., 2004
), which is highly expressed in neurons and has been shown to be regulated by sodium (Harvey et al., 1999
; Dinudom et al., 2001
; Kabra et al., 2008
). Nedd4 ligases have been implicated in regulating the trafficking and turnover of many membrane proteins including sodium channels (Staub et al., 2000
; Snyder, 2005
; Kabra et al., 2008
). To examine the interaction of Nedd4 with its substrate AMPARs, we transfected HEK cells with GFP-GluA1, and performed co-immunoprecipitation assays with antibodies against endogenous Nedd4. GluA1 was positively detected in immunoprecipitated Nedd4 complexes, but not in the IgG immunoprecipitation control (). In order to evaluate the role of Nedd4 in AMPAR ubiquitination, we transfected HEK cells with GFP-GluA1 alone, or together with Nedd4, or Nedd4 plus HA-ubiquitin. GluA1 was immunoprecipitated with anti-GFP and probed with anti-ubiquitin antibodies. As shown previously (), little ubiquitination of GluA1 from HEK lysate was detected under basal conditions (), indicating inefficient ubiquitination in heterologous cells. However, co-transfection of GFP-GluA1 with Nedd4 produced a significant amount of ubiquitination species in GFP-GluA1 immunoprecipitates, which was further enhanced by co-expressing ubiquitin (), indicating GluA1 as a substrate of the Nedd4 ligase.
Figure 6 Nedd4 mediates GluA1 ubiquitination. (A) Lysate of HEK cells expressing GFP-GluA1 was incubated with anti-Nedd4 antibodies to isolate endogenous Nedd4. GluA1 was positively detected in Nedd4 complexes, indicating an association. IgG was used as a control. (more ...)
To further confirm the involvement of endogenous Nedd4, we introduced an siRNA specifically targeting Nedd4. The effect of the siRNA was confirmed by a dramatic reduction in Nedd4 levels in HEK cells transfected with Nedd4 siRNA (). We then expressed GFP-GluA1 and HA-ubiquitin, together with Nedd4 siRNA or a scrambled siRNA control. As shown earlier in this study ), expression of ubiquitin induces strong ubiquitination of GluA1 (). However, in the presence of Nedd4 siRNA, which reduced total Nedd4 in the lysate, ubiquitin overexpression failed to induce GluA1 ubiquitination as compared to cells expressing scrambled siRNA control (). These results strongly indicate that endogenous Nedd4 mediates the ubiquitination effect and functions as the ubiquitin ligase for AMPAR ubiquitination.
Nedd4 co-localizes and associates with AMPAR in neurons
If Nedd4 functions as a ligase for AMPAR ubiquitination, it should be localized in close proximity to its target so as to promote efficient ubiquitin ligation. Towards this end, we double stained Nedd4 with AMPAR GluA2 subunits, and the synaptic protein Shank in cultured cortical neurons. The Nedd4 immunofluorescence signal showed a punctate pattern in dendrites, and co-distributed with both AMPARs (Supplemental Figure, SFig. A
) and Shank (SFig. B
), indicating an enrichment of Nedd4 at synaptic sites. Furthermore, rat brain lysates and synaptosome preparations of equal total protein amounts were analyzed by western blotting. The synaptosome sample showed markedly higher amounts of AMPAR subunits and Nedd4 compared to brain lysate, confirming synaptic distribution of the Nedd4 ligase (SFig. C
). Similar to the results observed from transfected HEK cells (), we found that GluA1 co-immunoprecipitated with Nedd4 using lysates from cultured cortical neurons (SFig. D
), suggesting a possible role for Nedd4 as an AMPAR ubiquitin ligase in neurons.
Nedd4 ubiquitinates and regulates AMPARs surface stability in neurons
In order to confirm the involvement of Nedd4 in AMPAR ubiquitination in neurons, we cloned Nedd4 into a lentiviral vector so that biochemical analysis could be performed. To test the Nedd4 lentiviral constructs, HEK cells were transfected with HA-ubiquitin and GFP-GluA1 followed by viral Nedd4 infection. A marked increase in Nedd4 amount was observed in viral Nedd4-infected cells ( right). Correspondingly, Nedd4 infection increased GluA1 ubiquitination levels, which was further enhanced by co-expression of HA-ubiquitin ( left). Consistently, incubation of viral Nedd4 with cultured neurons also increased Nedd4 expression and levels of GluA1 ubiquitination (Nedd4, 1.40 ± 0.22, n=3) (). To investigate the role of endogenous Nedd4 in AMPAR ubiquitination in neurons, cultured cortical neurons were transfected with Nedd4-specific siRNA followed by infection with an ubiquitin adenovirus to elevate basal ubiquitination levels. We found that siRNA knockdown reduced Nedd4 abundance in neurons, and significantly reduced GluA1 ubiquitination as compared to the scrambled siRNA control (siRNA, 0.73 ± 0.08, n=3) (), strongly indicating that Nedd4 functions as an endogenous AMPAR E3 ubiquitin ligase in neurons. If so, Nedd4 activity should have a similar effect on AMPAR surface localization as that from ubiquitin overexpression. We thus examined surface and total GluA1 levels under nonpermeant and permeant conditions, respectively, in cortical neurons. 2 days after Nedd4 transfection, a marked reduction in surface-localized GluA1 was observed compared to the GFP transfection control (control, 1246 ± 15, n=19 cells; Nedd4, 736 ± 7, n=17 cells) ( and ). Interestingly, unlike the effect of ubiquitin overexpression, Nedd4 showed no significant effect on total AMPAR protein amount (control, 412 ± 9, n=19 cells; Nedd4, 417 ± 12, n=17 cells) ( and ). These data strongly indicate that endogenous Nedd4 in neurons catalyzes AMPAR ubiquitination and regulates receptor cell-surface localization.
Figure 7 Nedd4 ubiquitinates AMPARs and suppresses receptor surface expression in neurons. (A) HEK cells were first transfected with GluA1 and HA-ubiquitin cDNA plasmids and then incubated with lentiviral Nedd4 and a viral vector control. GluA1 immunoprecipitates (more ...)
Figure 8 Nedd4 suppresses AMPAR-mediated excitatory synaptic transmission. (A) Whole-cell recording of mEPSCs in neurons expression EGFP or EGFP and Nedd4. Typical mEPSCs (left) and pooled data (right) show reduced mEPSC amplitude in Nedd4-expressing neurons (n=5 (more ...)
If Nedd4-dependent ubiquitination regulates AMPAR surface localization, we wanted to know whether Nedd4 also altered AMPAR-mediated synaptic transmission. We therefore examined miniature excitatory postsynaptic currents (mEPSCs) by whole cell patch clamp recording on transfected hippocampal neurons (). Compared to control neurons that expressed EGFP alone, neurons co-expressing EGFP and Nedd4 showed a significant reduction in mEPSCs amplitude (control 26.4 ± 1.1 pA, n=5 cells; Nedd4 20.3 ± 0.8 pA, n=4 cells), whereas the miniature frequency showed no change (control 0.94 ± 0.04 Hz, n=5 cells; Nedd4 1.02 ± 0.08 Hz, n=4 cells). This result is consistent with the Nedd4-induced reduction in AMPAR surface expression and indicates a removal of receptors from synaptic sites by locally localized Nedd4.