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Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2013 November 29; 288(48): 34897–34905.
Published online 2013 October 10. doi:  10.1074/jbc.M113.514745
PMCID: PMC3843101

Endoplasmic Reticulum-associated Degradation Controls Cell Surface Expression of γ-Aminobutyric Acid, Type B Receptors*

Abstract

Metabotropic GABAB receptors are crucial for controlling the excitability of neurons by mediating slow inhibition in the CNS. The strength of receptor signaling depends on the number of cell surface receptors, which is thought to be regulated by trafficking and degradation mechanisms. Although the mechanisms of GABAB receptor trafficking are studied to some extent, it is currently unclear whether receptor degradation actively controls the number of GABAB receptors available for signaling. Here we tested the hypothesis that proteasomal degradation contributes to the regulation of GABAB receptor expression levels. Blocking proteasomal activity in cultured cortical neurons considerably enhanced total and cell surface expression of GABAB receptors, indicating the constitutive degradation of the receptors by proteasomes. Proteasomal degradation required Lys48-linked polyubiquitination of lysines 767/771 in the C-terminal domain of the GABAB2 subunit. Inactivation of these ubiquitination sites increased receptor levels and GABAB receptor signaling in neurons. Proteasomal degradation was mediated by endoplasmic reticulum-associated degradation (ERAD) as shown by the accumulation of receptors in the endoplasmic reticulum upon inhibition of proteasomes, by the increase of receptor levels, as well as receptor signaling upon blocking ERAD function, and by the interaction of GABAB receptors with the essential ERAD components Hrd1 and p97. In conclusion, the data support a model in which the fraction of GABAB receptors available for plasma membrane trafficking is regulated by degradation via the ERAD machinery. Thus, modulation of ERAD activity by changes in physiological conditions may represent a mechanism to adjust receptor numbers and thereby signaling strength.

Keywords: Endoplasmic Reticulum (ER), ER-associated Degradation, GABA Receptors, Neurons, Proteasome, Ubiquitination

Introduction

The signaling strength of neurotransmitter receptors is significantly controlled by the number of receptors in the plasma membrane. Protein synthesis, cell surface trafficking, endocytotic removal from the plasma membrane, and degradation of the receptors need to be precisely balanced to maintain an appropriate level of cell surface receptors. These mechanisms thus provide means for adapting receptor numbers in response to plastic changes in neurons. There is accumulating evidence that regulated protein degradation via the ubiquitin-proteasome system plays an important integrative role in synaptic plasticity (13). Proteasomal degradation at the endoplasmic reticulum (ER)3 is crucial for the quality control of newly synthesized receptors. Incorrectly folded and misassembled receptor proteins are efficiently eliminated from the endoplasmic reticulum via the ER-associated degradation (ERAD) (4). Defective receptor proteins are polyubiquitinated, exported from the ER membrane and degraded by proteasomes in the cytoplasm. There is evidence that ERAD may also be involved in the regulation of the number of functional receptors in response to physiological stimuli. Prolonged activation of IP3 receptors, which release Ca2+ from the ER, down-regulates the expression of the receptors in ER membranes via ERAD-dependent proteasomal degradation (5). This is thought to be a homeostatic response to counterbalance excessive accumulation of Ca2+ in the cytoplasm. However, it is currently unclear whether the ERAD machinery contributes to the regulation of the cell surface density of neurotransmitter receptors.

GABAB receptors are G protein-coupled receptors assembled from the two subunits GABAB1 and GABAB2. They mediate slow inhibitory neurotransmission in the CNS and are thought to be involved in a variety of neurological disorders (6). It is meanwhile well established that GABAB receptors are endocytosed from the plasma membrane via the classical dynamin- and clathrin-dependent pathway and are eventually degraded in lysosomes (7). Lysosomal targeting appears to be mediated by the ESCRT (endosomal sorting complex required for transport) machinery (8) that sorts mono- and Lys63-linked polyubiquitinated proteins to lysosomes (9). It is currently unclear whether proteasomal degradation contributes to the regulation of GABAB receptors available for signal transduction. Therefore, we tested in this study the hypothesis that cell surface levels of GABAB receptors might be controlled by proteasomal degradation.

EXPERIMENTAL PROCEDURES

Antibodies

The following primary antibodies were used: rabbit GABAB1a,b (10, 11) directed against the C terminus of GABAB1 (affinity-purified, 1:500 for in-cell Western assay and immunofluorescence), rabbit GABAB2N (10, 11) directed against the N terminus of GABAB2 (affinity-purified, 1:250 for in-cell Western assay and immunofluorescence, 1:50 for in situ PLA), guinea pig GABAB2 (1:1,000 for immunofluorescence in neurons and 1:4,000 in HEK 293 cells, 1:1,000 for Western blotting; Chemicon International), mouse PDI (1:1,000 for immunofluorescence; Santa Cruz Biotechnology), mouse ubiquitin (P4D1, 1:50 for Western blotting; Santa Cruz Biotechnology), mouse ubiquitin Lys48-specific (clone Apu2, 1:50 for in situ PLA; Millipore), mouse VCP (p97) (1:50 for in situ PLA, 3E8DC11; Abcam), mouse actin (1:1,000 for in-cell Western assay; Chemicon International), mouse HA (1:500 for immunofluorescence; Santa Cruz Biotechnology), and rabbit SYVN1/Hrd1 (1:50 for in situ PLA; Bioss). Secondary antibodies were coupled either to horseradish peroxidase (1:5,000; Jackson ImmunoResearch), Alexa Fluor 488 (1:1,000; Invitrogen), Cy-3 (1:500; Jackson ImmunoResearch), IRDye680 (1:400; LI-COR Biosciences), or IRDye800CW (1:400; LI-COR Biosciences).

Drugs

The following drugs were used: baclofen (50 μm; Tocris Bioscience), betulinic acid (20 μg/ml; Sigma-Aldrich), bicucullin (4 μm; Tocris Bioscience), Eeyarestatin I (5 μm; Chembridge), 7-nitro-2,3-dioxo-1,4-dihydroquinoxaline-6-carbonitrile (CNQX 2 μm; Tocris Bioscience), lactacystin (50 μm; Sigma-Aldrich), MG132 (10 μm; Sigma-Aldrich), pyrenebutyric acid (50 μm; Sigma-Aldrich), SMI-UPS14 (5 μm; BostonBiochem), and tetrodotoxin (0.5 μm; Tocris Bioscience).

Plasmids

The following cDNAs in the appropriate expression vectors were used: GABAB(1a) (12) (pcDNA1), GABAB2 (13) (pcI) (pcDNA1, GABAB plasmids were kindly provided by Dr. B. Bettler (University of Basle) and Dr. K. Kaupmann (Novartis, Basle)), ubiquitin and ubiquitin (K48R) (14) (pRK5-HA; Addgene plasmids 17604 and 17608), VCP/p97-EGFP and VCP/p97(DKO)-EGFP (15) (pEGFP-N1; Addgene plasmids 23971 and 23974).

Mutation of GABAB2

Lysines 767 and 771 in GABAB2 were mutated to arginines using the QuikChange II XL site-directed mutagenesis kit from Stratagene according to the manufacturer's instructions.

Culture and Transfection of Cortical Neurons

Primary neuronal cultures of cerebral cortex were prepared from day 18 embryos of time-pregnant Wistar rats as described previously (10, 11). Neurons were kept in culture for 12–17 days before being used. Neurons were transfected with plasmid DNA using magnetofection as detailed by Buerli et al. (16).

Culture and Transfection of HEK 293 Cells

HEK 293 cells were cultured in minimum essential medium (Invitrogen) containing 10% fetal calf serum (Invitrogen), 2 mm glutamine (Invitrogen), and 4% gentamicin (Invitrogen). HEK 293 cells were transfected with plasmids using the calcium phosphate precipitation method.

Proteasome Activity Assay

Neurons cultured in 96-well plates were incubated for 12 h with either 10 μm MG132, 50 μm lactacystin, or 20 μm betulinic acid followed by determination of proteasome activity using the Proteasome Glo Chymotrypsin-like cell-based assay (Promega) according to the manufacturer's instructions.

Immunoprecipitation and Western Blotting

Immunoprecipitation of GABAB receptors from deoxycholate extracts of rat brain membranes and Western blotting for the detection of GABAB2 and ubiquitin was done as described previously (10, 17).

Immunocytochemistry and Confocal Laser Scanning Microscopy

Double labeling immunocytochemistry was performed with cortical neurons cultured on coverslips as described previously (10, 11, 17). Neurons were analyzed by confocal laser scanning microscopy (LSM510 Meta; Zeiss, 100× plan apochromat oil differential interference contrast objective, 1.4 NA) at a resolution of 1,024 × 1,024 pixels in the sequential mode. Quantification of fluorescence signals and image processing was done as detailed in Ref. 11. Images shown represent a single optical layer.

In-cell Western Assay

The in-cell Western assay was exactly done as in Ref. 11. Neurons cultured in 96-well plates were treated with the drug to be tested for the indicated time at 37 °C and 5% CO2. After fixation and permeabilization, the neurons were incubated simultaneously with GABAB receptor and actin antibodies. Nonspecific GABAB receptor antibody binding was determined in parallel cultures by competition using the respective peptide-antigen (10 μg/ml). After incubation with the appropriate secondary antibodies, the fluorescence was measured with the Odyssey infrared imaging system (LI-COR Biosciences). Specific GABAB signals were normalized to the actin signal determined in parallel.

In Situ Proximity Ligation Assay (PLA)

The in situ PLA technology is a highly sensitive antibody-based method for the microscopic detection of protein-protein interactions and post-translational protein modifications in cultured cells and tissue section (18, 19). For in situ PLA, we used Duolink PLA probes and detection reagents according to the manufacturer's instructions (Olink Bioscience). The specificity of the PLA signal was validated for each pair of antibodies in HEK 293 cell expressing or not expressing GABAB receptors. In addition, in neurons, omitting one of the primary antibodies did not generate PLA signals.

For signal quantification, cells were imaged for GABAB receptor expression and PLA signals by confocal microscopy (LSM510 Meta; Zeiss, 100× plan apochromat oil differential interference contrast objective, 1.4 NA, resolution 1,024 × 1,024 pixels, sequential mode). GABAB receptor fluorescence intensities, PLA spots, and the cell area were quantified using ImageJ. PLA signals were normalized to the GABAB receptor signal and the cell area.

Electrophysiology

Cortical neurons at 13–15 days in vitro were recorded in the whole cell voltage clamp configuration at room temperature. Total spontaneous postsynaptic currents (sPSCs) were recorded at a holding potential of −60 mV. Baclofen-evoked potassium currents were elicited using a 10-s pulse of 50 μm baclofen at −90 mV. Patch electrodes were filled with 120 mm CsCl/KCl, 10 mm EGTA, 10 mm HEPES (pH 7.4), 4 mm MgCl2, 0.5 mm GTP, and 2 mm ATP. Spontaneous PSCs recordings were performed using intracellular CsCl, whereas the potassium currents were recorded using an intracellular solution containing KCl. The external solution contained 140 mm NaCl, 10 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 10 mm HEPES (pH 7.4), and 10 mm glucose. Potassium currents were recorded in the presence of tetrodotoxin (0.5 μm), 7-nitro-2,3-dioxo-1,4-dihydroquinoxaline-6-carbonitrile (CNQX, 2 μm), and bicuculline (4 μm). To enhance the amplitude of the baclofen-evoked currents, the potassium concentration of the extracellular solution was increased to 30 mm, and the sodium concentration was reduced to 120 mm (to keep osmolarity constant) before the application of the GABAB agonist. All the synaptic events displaying amplitudes above the background noise (5–12 pA) were identified and analyzed off-line using MiniAnalysis 6.0.7 software (Synaptosoft). Mean amplitudes and frequency values were obtained from 1 min of epoch recordings on each experimental condition and normalized to the control condition of the individual neuron.

Statistical Analysis

The data are presented as means ± S.E. The statistical analysis of data were performed with the GraphPad Prism 5 software. Unpaired t test was used for comparing two conditions and one-way ANOVA followed by Dunnett's post hoc test for analysis of multiple conditions. The level of significance and the n values are indicated in the figure legends. Differences were considered statistically significant when p < 0.05.

RESULTS

The Expression Level of GABAB Receptors Is Controlled by Proteasomal Degradation

It is currently unknown whether the ubiquitin-proteasome system contributes to the regulation of GABAB receptor expression levels in neurons. To gain evidence for a potential degradation of GABAB receptors by proteasomes, we treated cultured cortical neurons for 12 h with the proteasome inhibitors MG132 or lactacystin and determined the GABAB1 and GABAB2 protein expression levels. Under these conditions, MG132 and lactacystin decreased proteasomal activity to 31 ± 2 and 17 ± 1% of untreated controls, respectively (Fig. 1A). Both drug treatments increased total GABAB receptor expression levels (MG132: GABAB1, 131 ± 2%; GABAB2, 143 ± 5%; lactacystin: GABAB1, 142 ± 4%; GABAB2, 147 ± 2% of control; Fig. 1B), suggesting that under basal conditions GABAB receptors were constitutively degraded to a certain extent by proteasomes.

FIGURE 1.
The expression level of GABAB receptors is controlled by proteasomes. A, verification of drug effects on proteasome activity. Neurons were incubated for 12 h with the indicated drugs and tested for proteasome activity (n = 12 cultures) **, p < ...

Prolonged inhibition of proteasomes depletes the pool of free ubiquitin (20, 21), which might also affect ubiquitin-dependent processes unrelated to proteasomal degradation. There is some evidence that GABAB receptors are sorted to lysosomes via the ubiquitin-dependent ESCRT (endosomal sorting complex required for transport) machinery (8). Hence, prolonged inhibition of proteasomes might indirectly compromise lysosomal degradation of the receptors. However, an indirect contribution of lysosomal degradation could be ruled out. Pharmacologically increasing proteasome activity by treating cortical neurons for 12 h with the proteasome activator betulinic acid (22), which enhanced proteasomal activity to 143 ± 17% of control (Fig. 1A), significantly decreased GABAB receptor levels (GABAB1, 69 ± 2%; GABAB2, 64 ± 3% of control; Fig. 1C).

It has recently been shown that inhibition of the proteasome-associated deubiquitinating enzyme USP14 enhanced the degradation of proteasome substrates (23). Inhibition of USP14 by incubation of cortical neurons with SMI-USP14 (small molecule inhibitor of USP14) strongly reduced GABAB receptor levels (GABAB1, 49 ± 4%; GABAB2, 29 ± 2% of control; Fig. 1D), further supporting the view that GABAB receptors are degraded by proteasomes.

Finally, we assessed the functional consequences of decreased GABAB receptor levels after enhancing proteasomal activity with betulinic acid by measuring spontaneous synaptic activity in electrophysiological experiments. Activation of GABAB receptors with the selective agonist baclofen considerably decreased the amplitude as well as the frequency of sPSCs to 43 ± 4 and 56 ± 7%, respectively (Fig. 1E). Treatment of cultures for 12 h with betulinic acid diminished baclofen-induced inhibition of sPSCs (amplitude, from 43 ± 4 to 90 ± 12% of control; frequency, from 56 ± 7 to 94 ± 15% of control; Fig. 1E), supporting the hypothesis that enhanced proteasomal activity leads to reduced levels of functional GABAB receptors available for neuronal inhibition.

GABAB Receptors Undergo Lys48-linked Polyubiquitination

Lys48-linked polyubiquitination of proteins serves as a signal for proteasomal degradation. Consistent with polyubiquitination, GABAB receptors immunoprecipitated from deoxycholate extracts of crude rat brain membranes exhibited on Western blots ubiquitin immunoreactivity in the high molecular range (Fig. 2A). This suggests that GABAB receptors are ubiquitinated under basal conditions to a certain extent. Likewise, using the in situ PLA, we found that GABAB receptors in cultured cortical neurons display Lys48-linked polyubiquitination, which was considerably increased upon inhibition of proteasomal activity with MG 132 (172 ± 11% of control; Fig. 2B). This indicates the accumulation of Lys48-linked polyubiquitinated GABAB receptors destined for proteasomal degradation.

FIGURE 2.
Lys48-linked ubiquitination controls the expression level of GABAB receptors. A, demonstration of ubiquitination of native GABAB receptors. GABAB receptors were immunoprecipitated from deoxycholate extracts of rat brain membranes using either GABAB1a,b ...

Next we tested whether preventing Lys48-linked polyubiquitination affects GABAB receptor levels. Overexpression in neurons of a Lys48 chain elongation-defective ubiquitin mutant, in which lysine 48 had been exchanged for an arginine (Ub(K48R)), considerably increased the level of GABAB receptors (GABAB1, 166 ± 7%; GABAB2, 140 ± 7% of control; Fig. 2C). This finding corroborates a Lys48-linked polyubiquitin-mediated proteasomal degradation of GABAB receptors.

The C-terminal Domain of GABAB2 Contains a Major Lys48-linked Polyubiquitination Site

An in silico analysis predicted two lysines in the C-terminal domain of GABAB2 at positions 767 and 771 as likely candidates for ubiquitination. We inactivated these potential ubiquitination sites by exchanging both lysines for arginines (GABAB2(RR)) (Fig. 3A). Upon transfection into HEK 293 cells, GABAB2(RR) displayed reduced Lys48-linked polyubiquitination (61 ± 6% of wild type; Fig. 3B), indicating that Lys767/771 is a main site for Lys48-linked polyubiquitination in GABAB2.

FIGURE 3.
The C-terminal domain of GABAB2 contains a major Lys48-linked ubiquitination site. A, scheme depicting the location of mutated lysines in GABAB2. B, decreased Lys48-linked polyubiquitination of a GABAB2 mutant in which lysines 767 and 771 had been changed ...

We then tested whether GABAB1 is also a target for Lys48-linked polyubiquitination. However, HEK 293 cells transfected with GABAB1 showed only marginal GABAB1/Lys48-linked ubiquitination PLA signals as compared with HEK 293 cells expressing GABAB1 and GABAB2 (12 ± 6%; Fig. 3C). In line with this finding, co-expression of GABAB1 with GABAB2(RR) yielded a similar reduction in GABAB receptor/Lys48-linked polyubiquitination signals (56 ± 8%; Fig. 3C) as observed for GABAB2(RR) alone (61 ± 6%; Fig. 3B). Thus, GABAB2 appears to be the main target for Lys48-linked polyubiquitination of GABAB receptors.

Overexpressing GABAB2(RR) in cultured neurons increased GABAB receptor levels to a similar level as observed after chronic proteasome inhibition (GABAB1, 152 ± 15%; GABAB2, 146 ± 9% of control; Fig. 3D). This suggests that Lys767/771 in GABAB2 is the major Lys48-linked polyubiquitination site required for proteasomal degradation of GABAB receptors.

The functional consequence of the increased GABAB2 cell surface density after transfecting neurons with GABAB2(RR) was analyzed by measuring baclofen-induced K+ currents using whole cell patch clamp recordings. Transfection of GABAB2(RR) in neurons resulted in 2.8 ± 0.6-fold increased K+ channel current amplitudes after activation of GABAB receptors with baclofen as compared with neurons transfected with wild type GABAB2 (Fig. 3E). Thus, preventing proteasomal degradation of GABAB2 by overexpression of GABAB2(RR) increased the number of functional cell surface GABAB receptors available for signaling.

Cell Surface Expression of GABAB Receptors Is Regulated by ERAD

The most likely mechanism for proteasomal degradation of GABAB receptors is the ERAD. If GABAB receptors are degraded by ERAD, inhibition of proteasomal activity should result in an accumulation of GABAB receptors in the ER. Indeed, blocking proteasomal activity in neurons for 12 h with MG132 increased the number of GABAB2 clusters (136 ± 6% of control) as well as the clusters co-localizing with a marker protein for the ER (protein-disulfide isomerase (PDI), 133 ± 7% of control; Fig. 4A).

FIGURE 4.
GABAB receptors are degraded via the ERAD pathway. A, blocking proteasomal activity increased the number of GABAB2 clusters co-localized with the ER marker protein PDI. Neurons were incubated for 12 h with MG132 and stained for GABAB2 (red) and PDI ( ...

To further establish the role of ERAD in regulating cellular GABAB receptor levels, we tested the effect of directly inhibiting ERAD. Treatment of neurons for 12 h with the ERAD inhibitor Eeyarestatin I (EerI) (24, 25) increased both total GABAB2 (183 ± 15% of control; Fig. 4B) and cell surface levels of GABAB2 (204 ± 32% of control; Fig. 4C). Overexpression of GABAB2(RR), which lack the main Lys48-linked polyubiquitination sites, did not further increase total (112 ± 7% of control; Fig. 4D) or cell surface GABAB receptor levels (93 ± 15% of control; Fig. 4E). These observations indicate that Lys48-linked polyubiquitinated GABAB receptors are degraded by ERAD.

GABAB Receptors Interact with the ERAD E3 Ubiquitin Ligase Hrd1

Hrd1 is one prototypical ERAD E3 ubiquitin ligase responsible for Lys48-linked polyubiquitination of ERAD substrates (26). Using in situ PLA, we further confirmed the potential degradation of GABAB receptors via ERAD by showing that GABAB receptors interact with Hrd1 (Fig. 5A). Inhibition of ERAD for 12 h with EerI increased the number of interactions (GABAB2/Hrd1, 490 ± 45%; GABAB1/Hrd1, 305 ± 18% of control; Fig. 5A), indicating the accumulation of GABAB receptors at this central ERAD multiprotein complex. In line with this observation, blocking ERAD function for 12 h with EerI considerably increased the level of Lys48-linked polyubiquitinated GABAB receptors (242 ± 21% of control; Fig. 5B).

FIGURE 5.
GABAB receptors interact with the ERAD E3 ligase Hrd1. A, demonstration of the interaction of GABAB receptors with the ERAD E3 ligase Hrd1 using in situ PLA with Hrd1 and GABAB2 antibodies (left, upper panels) or GABAB1 antibodies in cortical neurons ...

GABAB Receptors Interact with the Essential ERAD Component p97

The AAA-ATPase p97 is a central constituent of the ERAD machinery involved in the retrotranslocation of proteins to the cytoplasm for proteasomal degradation (27). Using in situ PLA, we found that GABAB receptors interact with p97 (Fig. 6A). This finding further demonstrates the ERAD-mediated degradation of GABAB receptors. Inhibition of p97 by EerI decreased the interaction of GABAB2 with p97 (40 ± 8% of control; Fig. 6A), suggesting that the association is activity-dependent.

FIGURE 6.
GABAB receptors interact with the ERAD AAA-ATPase p97. A, demonstration of the interaction of GABAB receptors with the ERAD AAA-ATPase p97 in cortical neurons using in situ PLA. Treatment of neurons for 12 h with the p97 inhibitor EerI strongly reduced ...

Inhibition of p97 function in neurons by overexpression of a dominant-negative mutant of p97 (p97[DKO]) considerably increased total (176 ± 11% of control; Fig. 6B) as well as cell surface GABAB receptor levels (143 ± 11% of control; Fig. 6C) as compared with neurons overexpressing wild type p97. Overexpressing in addition GABAB2(RR) did not further increase total (wild type p97, 100 ± 6%; p97(DKO), 104 ± 6%; Fig. 6D) or cell surface GABAB receptor levels (wild type p97, 100 ± 12%; p97(DKO), 100 ± 11%; Fig. 6E), indicating that ubiquitination of GABAB2 is required for being recognized by the ERAD machinery.

Whole cell patch clamp recordings finally verified that inhibition of ERAD function by overexpression of p97(DKO) increased the level of functional cell surface GABAB receptors (Fig. 6F). Neurons overexpressing p97(DKO) displayed considerably increased amplitudes of baclofen-induced K+ currents (control, 72 ± 14 pA; p97(DKO), 139 ± 14 pA; Fig. 6F). These experiments show that GABAB receptors are degraded by ERAD, which affects the levels of total and cell surface GABAB receptors.

DISCUSSION

Mechanisms controlling the cell surface density of GABAB receptors are of pivotal importance for determining the level of GABAB receptor-mediated neuronal inhibition. Because GABAB receptors control glutamatergic neurotransmission (28), modulation of their cell surface density is presumed to significantly contribute to synaptic plasticity. However, the mechanisms that control cell surface expression of GABAB receptors are largely unknown. In the present study, we identified proteosomal degradation via the ER-resident ERAD machinery as a mechanism that determines cell surface expression of GABAB receptors.

Our data indicate that a fraction of GABAB receptors in the ER is constitutively Lys48-linked polyubiqutinated and degraded by the ERAD machinery. This conclusion is based on the observation that blocking proteasomal activity, inhibiting ERAD function, or interfering with GABAB receptor Lys48-linked polyubiquitination increased the expression levels of GABAB receptors in neurons. Lysines 767/771 in the C-terminal domain of GABAB2 appear to represent the main Lys48-linked polyubiquitination sites required for proteasomal degradation because their mutational inactivation rendered GABAB receptors largely immune to degradation. It is currently unclear whether Lys48-linked polyubiquitination of both lysines or only of Lys767 or Lys771 serves as a tag for proteasomal degradation. A recent proteomic study analyzing the ubiquitination state of rat brain synaptic proteins identified Lys771 in GABAB2 as being ubiquitinated (29). This observation favors Lys771 as the main Lys48-linked polyubiquitination site in GABAB2.

There are several lines of evidence that in particular GABAB receptors residing in the ER are degraded by proteasomes via ERAD. First, upon blocking proteasomal activity, the receptors accumulated in the ER. Second, blocking ERAD function pharmacologically or by overexpressing a dominant-negative mutant of the AAA-ATPase p97, which mediates the retrotranslocation of proteins to the cytoplasm for proteasomal degradation (27), increased GABAB receptor levels. Third, GABAB receptors interacted with the ERAD proteins p97 and Hrd1. Hrd1 is the prototypical ERAD E3 ligase (26) and most likely one of the ubiquitin ligases that mediate ubiquitination of GABAB receptors because stalling proteasomal degradation considerably increased its interaction with GABAB receptors and the level of Lys48-linked polyubiquitinated GABAB receptors.

In all cases tested, GABAB1 and GABAB2 were concomitantly up- or down-regulated to a similar extent, suggesting that assembled GABAB receptor complexes are degraded by ERAD. This notion is further strengthened by the finding that 1) inactivation of the ubiquitination sites in GABAB2 increased the expression levels of GABAB1 and GABAB2 as well as GABAB receptor-activated K+ current amplitudes, 2) that interfering with ERAD function increased GABAB receptor function (baclofen-induced K+ currents), and 3) that both GABAB1 and GABAB2 generated in situ PLA signals with the ERAD E3 ubiquitin ligase Hrd1, although only Lys48-linked polyubiquitination of Lys767/771 in GABAB2 appears to be required for proteasomal degradation of the receptors.

What might be the physiological implications of this mechanism? The most firmly established function of ERAD is the degradation of aberrant proteins in the ER (30). In addition, ERAD has been shown to rapidly degrade activated IP3 receptors in the ER to prevent excessive elevation of cytosolic Ca2+ concentrations (5), indicating that ERAD may also contribute to the regulation of functional receptors. Because blocking ERAD increased the level of functional GABAB receptors, and ERAD appears to degrade assembled heterodimeric receptors, it is rather unlikely that the role of ERAD is simply the degradation of un- or misfolded GABAB receptor subunits. The constitutive degradation of GABAB receptors suggests that ERAD controls the amount of receptors available for cell surface trafficking. This view is supported by recent studies on the regulation of cell surface GABAA receptors. Chronic suppression of neuronal activity or inhibition of L-type voltage-gated calcium channels decreased the level of functional GABAA receptors in the neuronal plasma membrane by a mechanism dependent on the ubiquitination of the GABAA receptor β3-subunit and proteasome activity, most likely via the ERAD pathway (31, 32). These findings imply that regulation of ERAD activity is a potential mechanism to adjust the level of functional GABAB receptors to changing physiological condition. Our finding that modulation of proteasomal activity up- or down-regulates the level of functional GABAB receptors supports this view. Interestingly, the level of proteasomal activity correlates with the activity state of neurons (33). We therefore hypothesize that the amount of functional GABAB receptors inserted into the plasma membrane is regulated by neuronal activity via ERAD.

Acknowledgments

We thank Dr. J.-M. Fritschy for support in confocal microscopy and for providing embryonic day 18 rat cortex, Corinne Sidler and Giovanna Bosshard for preparation of embryonic day 18 rat cortex, and Thomas Grampp for technical assistance.

*This work was supported by Swiss National Science Foundation Grants 31003A_121963 and 31003A_138382 (to D. B.).

3The abbreviations used are:

ER
endoplasmic reticulum
ERAD
ER-associated degradation
PLA
proximity ligation assay
sPSC
spontaneous postsynaptic current
ANOVA
analysis of variance
PDI
protein-disulfide isomerase.

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