Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M802077200 on May 8, 2008

J. Biol. Chem., Vol. 283, Issue 27, 18538-18544, July 4, 2008
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/27/18538    most recent
M802077200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Saliba, R. S.
Right arrow Articles by Moss, S. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Saliba, R. S.
Right arrow Articles by Moss, S. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The Ubiquitin-like Protein Plic-1 Enhances the Membrane Insertion of GABAA Receptors by Increasing Their Stability within the Endoplasmic Reticulum*

Richard S. Saliba{ddagger}§1, Menelas Pangalos, and Stephen J. Moss{ddagger}§2

From the {ddagger}Department of Neuroscience, University of Pennsylvania, Philadelphia, Pennsylvania 19104, §Department of Neuroscience, Pharmacology, and Physiology, University College London, London WC1E 6BT, United Kingdom, and Wyeth Research, Neuroscience Discovery, Princeton, New Jersey 08852

Received for publication, March 14, 2008 , and in revised form, April 30, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{gamma}-Aminobutyric acid receptors (GABAAR) are the major sites of fast inhibitory neurotransmission in the brain, and a critical determinant for the efficacy of neuronal inhibition is the number of these receptors that are expressed on the neuronal cell surface. GABAARs are heteropentamers that can be constructed from seven subunit classes with multiple members; {alpha}, β, {gamma}(1–3), {delta}, {epsilon}(1–3), {theta}, and {pi}. Receptor assembly occurs within the endoplasmic reticulum, and it is evident that transport-competent combinations exiting this organelle can access the cell surface, whereas unassembled subunits are ubiquitinated and subject to proteasomal degradation. In a previous report the ubiquitin-like protein Plic-1 was shown to directly interact with GABAARs and promote their accumulation at the cell surface. In this study we explore the mechanisms by which Plic-1 regulates the membrane trafficking of GABAARs. Using both recombinant and neuronal preparations it was apparent that Plic-1 increased the stability of endoplasmic reticulum resident GABAARs together with an increase in the abundance of poly-ubiquitinated receptor subunits. Furthermore, Plic-1 elevated cell surface expression levels by selectively increasing their rates of membrane insertion. Thus, Plic-1 may play a significant role in regulating the strength of synaptic inhibition by increasing the stability of GABAARs within the secretory pathway and thereby promoting their insertion into the neuronal plasma membrane.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
GABAARs3 are Cl–-selective ligand-gated ion channels and are the major mediators of fast synaptic inhibition in the brain. GABAARs are also the major sites of action for anxiolytics, sedatives, and anti-convulsants, including both barbiturates and benzodiazepines (1, 2). These receptors are heteropentamers that can be assembled from seven subunit classes, {alpha}, β, {gamma}(1–3), {delta}, {epsilon}(1–3), {theta}, and {pi}, providing the structural basis for receptor structure (1, 2). A combination of molecular, biochemical, and genetic approaches suggests that, in the brain, the majority of benzodiazepine-sensitive synaptic receptor subtypes are composed of {alpha}, β, and {gamma}2 subunits (3). In contrast, receptors that incorporate {alpha}4/5 and {delta} subunits are extrasynaptic and mediate tonic inhibition (4). The number of GABAA receptors on the neuronal cell surface is a critical determinant for the efficacy of synaptic inhibition and, at steady state, is determined by the rates of receptor insertion and removal from the plasma membrane (5).

It is evident that GABAARs are assembled within the endoplasmic reticulum (ER) and then transported to the plasma membrane for insertion while misfolded or unassembled receptor subunits are rapidly targeted for ER-associated degradation (6–9). Proteins targeted for ER-associated degradation are modified by ubiquitination, resulting in their recognition and degradation by the proteasome (10). In addition, neuronal activity can regulate the abundance of cell surface GABAARs by altering subunit ubiquitination, a process that in turn controls receptor insertion into the plasma membrane and subsequent accumulation at postsynaptic inhibitory specializations (11).

The ubiquitination and proteasomal degradation of proteins is subject to modulation by regulatory proteins that contain an N-terminal ubiquitin-like domain and one or two ubiquitin-associated domains (12). One member of this class of proteins termed Plic-1 (protein linking integrin-associated protein to cytoskeleton-1) (13) binds to GABAARs. These interactions are dependent upon the ubiquitin-associated domain of Plic-1 that binds to conserved amino acids within the intracellular domains of GABAAR {alpha} and β subunit isoforms. Moreover, this interaction has been demonstrated to be of significance in mediating the cell surface accumulation of GABAARs as revealed using dominant negative peptides (14).

Here we have begun to explore the mechanisms by which Plic-1 regulates GABAAR cell surface stability. We demonstrate that Plic-1 increases the stability of ER resident GABAAR β3 subunits and also elevates the levels of a ubiquitinated β3 species. As a result, Plic-1 increases the insertion of GABAARs into the neuronal cell surface without affecting their endocytic sorting. Therefore, Plic-1 regulation of the ubiquitin-dependent, proteasomal degradation of GABAARs may provide a dynamic mechanism regulating the efficacy of inhibitory synaptic transmission.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—Rabbit anti-Myc IgGs were obtained from Santa Cruz Biotechnology. Monoclonal anti-HA IgGs were obtained from Roche Applied Science. Rabbit polyclonal anti-β3 IgGs have been described previously (15, 16). Secondary horseradish peroxidase-conjugated antibodies were from Jackson Immunological Laboratories and protein A horseradish peroxidase was from Pierce.

Cell Culture and Transfections—HEK-293 cells were maintained in Dulbecco's modified Eagle's medium/F12 (1:1) nutrient mix with 10% fetal bovine serum. HEK-293 cells were transfected with pRK5{alpha}1, pRK5β3, and pRK5{gamma}2s (17, 18) with or without pRK5Plic-1 (vector ratio 1:1:1, respectively) using the amaxa biosystems nucleofection kit. Cortical neurons were obtained from embryonic day 18 (E18) rats (16, 19). Dissociated E18 rat cortical neurons were transfected with 3 µg of plasmid DNA/5 x 106 neurons using the Rat NucleofectorTM kit (amaxa) and then used in experiments after 3–4 days in vitro (20–22).

Biotinylation—HEK-293 cells or cortical neurons were chilled on ice for 5 min and then washed twice in PBS plus 1 mM CaCl2 and 0.5 mM MgCl2 (PBS-CM) at 4 °C. Cells were incubated for 15 min at 4 °C in 1.5 mg/ml sulfosuccinimidyl 2-(biotinamido)-ethyl-1, 3-dithiopropionate (NHS-SS-Biotin) (Pierce) dissolved in PBS-CM. To quench unreacted biotin, neurons were washed three times (10 min each wash, at 4 °C) in PBS-CM plus 75–100 mM glycine and then washed twice in PBS and either lysed in RIPA buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 2 mM EDTA, and a mammalian protease inhibitor mixture; Sigma) or returned to a 37 °C incubator. Protein concentrations were determined using the micro BCA protein assay kit (Pierce), and equal amounts of protein were added to immobilized avidin (neutravidin; Pierce) for 2 h at 4 °C. Avidin beads were washed twice, for 15 min at 4 °C, in high salt (500 mM NaCl) RIPA buffer, followed by a 15-min wash at 4 °C, in (150 mM NaCl) RIPA buffer. Precipitated biotinylated proteins were resolved by SDS-PAGE, and β3 was detected using immunoblotting with rabbit anti-β3, followed by peroxidase-conjugated anti-rabbit IgGs and detection with ECL. Blots were imaged using the Fujifilm LAS-3000 imaging system and bands quantified with Fujifilm Multi Gauge software.

Deglycosylation—Endoglycosidase H (Endo H) and peptide N-glycosidase-F (PNGaseF) were purchased from New England BioLabs. Transfected HEK-293 cells expressing {alpha}1Mβ3 with or without Plic-1 were lysed in RIPA buffer plus protease inhibitor mixture (Sigma). Total protein (10 µg) from lysates was denatured in 0.5% SDS and 40 mM dithiothreitol for 10 min at room temperature and then incubated for 1 h with Endo H at 37 °C in 1x reaction buffer (0.05 M sodium citrate, pH 5.5). N-Glycosidase-F digestion of 10 µg of denatured total protein was performed in 0.05 M sodium phosphate, pH 7.5, and 1% Nonidet P-40 for 1 h at 37 °C. Digestion was terminated by the addition of Laemmli sample buffer. Protein products were resolved by SDS-PAGE, and the Mβ3 subunit was detected by immunoblotting.

Immunoprecipitation—Transfected HEK-293 cells were lysed in 100 µl of 1% SDS, 25 mM Tris, pH 7.4. Lysates were then diluted 10-fold, with 900 µl of RIPA buffer (lacking SDS) plus 10 mM N-ethylmaleimide (Sigma) and mammalian protease inhibitor mixture (Sigma). Lysates were then sonicated in 1.5 ml of microcentrifuge tubes for 10 s at 10 amplitude microns (Sanyo Soniprep 150). Following centrifugation at 14,000 rpm to pellet insoluble material, lysates were precleared with nonspecific rabbit IgGs and protein A-Sepharose for 1 h at 4 °C. Lysates were then incubated for 1 h at 4 °C with 3 µg of rabbit anti-Myc (Santa Cruz Biotechnology) and then a further 1 h with protein A-Sepharose. Precipitated immunocomplexes were washed, alternatively, in low salt (150 NaCl) and high salt (350 mM NaCl) RIPA buffer for 20 min at 4 °C. Immunoprecipitated antigen was resolved on a 10% SDS-PAGE gel and immunoblotted with mouse anti-HA, followed by horseradish peroxidase-conjugated donkey anti-mouse IgGs and detection with ECL (Super-Signal®; Pierce). Blots were stripped in 62.5 mM Tris-HCl, pH 6.7, 2% SDS, 100 mM β-mercaptoethanol at 60 °C for 30 min and reprobed with rabbit anti-Myc, followed by protein A-horseradish peroxidase (Pierce) and detection with ECL.

BBSβ3 Cell Surface Labeling/Receptor Insertion Assay—Hippocampal neurons (9 DIV) were transfected with equimolar amounts of cDNAs encoding BBSβ3 (23) and Plic-1 (14) or empty vector (pRK5) using Effectene (Qiagen) according to the manufacturer's instructions. Following transfection, neurons were grown for a further 24 h. To measure steady state cell surface levels of GABAAR incorporating BBSβ3 subunits, neurons were labeled with 1 µg/ml rhodamine-conjugated {alpha}-bungarotoxin (Invitrogen) for 15 min at 15 °C. Neurons were washed three times in PBS at 15 °C and then fixed. To measure the membrane insertion of neuronal GABAARs incorporating BBSβ3 subunits in the presence or absence of Plic-1, neurons were labeled with 10 µg/ml unlabeled {alpha}-bungarotoxin for 15 min at 15 °C to block cell surface receptors. The cells were washed three times in PBS at 15 °C followed by a 4-min incubation at 37 °C with 1 µg/ml rhodamine-conjugated {alpha}-bungarotoxin (Invitrogen). All incubations were performed in the presence of 200 µM tubocurarine (Sigma) to block {alpha}-bungarotoxin (Bgt) binding to endogenous acetylcholine receptors (23, 24). Cells were fixed in 4% paraformaldehyde, and confocal images were collected using a x60 objective lens acquired with Olympus FluoView Version 1.5 software; the same image acquisition settings for BBSβ3 with or without Plic-1 were used. These images were analyzed using MetaMorph (Universal Imaging Corp.) imaging software. First, a three-dimensional reconstruction of an imaged neuron was made from a series of Z sections, and then the average fluorescence intensity of rhodamine-Bgt staining was measured along 30 µmoftwo proximal dendrites/neuron, after subtraction of background fluorescence.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Plic-1 Increases the Cell Surface Expression Levels of Recombinant GABAARs—To analyze the role that Plic-1 plays in regulating GABAAR functional expression, we expressed {alpha}1, β3, and {gamma}2 subunits in HEK-293 cells with a cDNA encoding pRK5-Plic-1 or with control empty vector (pRK5). We focused on GABAARs composed of these subunits because they reproduce many of the physiological and pharmacological properties of their neuronal counterparts (1, 2). Following transfection, HEK cells were lysed and equal amounts of detergent-soluble extracts were resolved by SDS-PAGE and subjected to immunoblotting with anti-Plic-1 and anti-β3 IgGs. Although abundant Plic-1 immunoreactivity was seen in cells transfected with the Plic-1 construct, significant levels were absent in cells transfected with empty vector (Fig. 1A). Consistent with this we have previously demonstrated that HEK-293 only express low endogenous levels of Plic-1 (14). In cells expressing Plic-1, total expression levels of the β3 subunit were increased by 67.2 + 4.5% compared with control (Fig. 1A). Using biotinylation to label and isolate cell surface proteins, it was also apparent that Plic-1 increased the cell surface accumulation of GABAARs relative to control cells (Fig. 1A), which is consistent with previous observations (14).


Figure 1
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 1.
Plic-1 modulates the cell surface accumulation of GABAARs expressed in HEK-293 cells. A, Plic-1 increases steady state levels of GABAAR β3 subunits. HEK cells were transfected with {alpha}1β3{gamma}2s plus Plic-1 or control empty vector (pRK5). Cells were biotinylated and lysed, and cell surface receptors were isolated with immobilized avidin. Immunoblots show total and cell surface levels of GABAAR β3 subunits and total levels of Plic-1 as indicated. Graph represents quantification of total and cell surface β3 subunits from blots in upper panel. Data represent mean ± S.E. percentage of control values. *, significantly different from control p < 0.01; t test, n = 4. B, cell surface degradation of GABAARs. HEK cells were transfected with {alpha}1β3{gamma}2s plus Plic-1 or control empty vector (pRK5). Cells were biotinylated and incubated at 37 °C for varying time points as indicated. Upper panel, immunoblot of cell surface degradation of β3 in the absence of Plic-1. Lower panel, immunoblot showing cell surface degradation of β3 in the presence of Plic-1. Graph shows percent β3 remaining over time. Open squares represent control, and filled squares represent +Plic-1 Error bars are ± S.E., n = 3.

 
The increase in GABAAR cell surface levels on expression with Plic-1 may be a consequence of decreased endocytosis, increased recycling of GABAAR from an endocytic pool, or an increase in insertion from the secretory pathway. To distinguish among these possibilities, we compared the cell surface degradation of GABAARs in the presence or absence of Plic-1. HEK cells expressing {alpha}1β3{gamma}2 with or without Plic-1 were labeled with cell-impermeant NHS-SS-Biotin and incubated for varying time periods at 37 °C. The levels of remaining biotinylated β3 subunits were then measured by immunoblotting of the avidin-purified fraction with rabbit anti-β3 IgGs. These experiments revealed similar levels of degradation of cell surface GABAARs in the presence or absence of Plic-1 over a 20-h time period (Fig. 1B).


Figure 2
View larger version (11K):
[in this window]
[in a new window]

  		FIGURE 2.
Plic-1 stabilizes an immature GABAAR β3 subunit glycoform. A, HEK-293 cells were transfected with {alpha}1 and β3 plus Plic-1 or control vector pRK5. After 24 h, expression cells were lysed, and 20 µg of cell lysate was incubated with (lanes 2 and 4) or without (lanes 1 and 3) Endo H or N-glycosidase-F (PNGase F) (lane 5). Endo H-sensitive and -resistant forms of β3 are indicated. B, quantification of Endo H-sensitive β3. Data represent mean ± S.E. percentage of control values. *, significantly different from control; p < 0.05, t test, n = 4.

 
The Abundance of ER-retained GABAARs Is Increased by Plic-1—The results in Fig. 1 suggest that Plic-1 primarily acts to increase GABAAR cell surface abundance by modifying receptor stability within the secretory pathway. Given that GABAARs are assembled from their constitutive subunits within the ER, we assessed the significance of Plic-1 in regulating receptor stability within this intracellular compartment. For these experiments, we examined the sensitivity of N-linked glycans within the GABAAR β3 subunit to digestion with Endo H. It is well known that transmembrane proteins residing within the ER carry high mannose N-linked glycans that are sensitive to cleavage by Endo H (26, 27). In contrast, transmembrane proteins that have exited the ER have mature N-linked glycans that are Endo H-insensitive. In our experiments, detergent-soluble extracts from HEK cells expressing {alpha}1β3 with or without Plic-1 were digested with Endo-H and then immunoblotted with rabbit anti-β3 IgGs. Endo H-resistant and -sensitive β3 was detected in control cells and those expressing Plic-1 (Fig. 2A). For comparison, samples were also treated with N-glycosidase-F, which cleaves all N-linked glycans, resulting in the detection of a single band of 52 kDa (Fig. 2A) (17, 28). We measured the levels of Endo H-sensitive β3 subunits in the absence or presence of Plic-1. Plic-1 increased the amount of Endo H-sensitive GABAARβ3 subunits by 32.5 + 5.4% compared with the β3 subunit co-expressed with empty vector (Fig. 2B). These results suggest that Plic-1 stabilizes ER resident β3 subunits.

Plic-1 Increases the Accumulation of Ubiquitinated GABAARs—GABAAR β3 subunits isolated from recombinant expression systems and neurons have been found to be ubiquitinated. Furthermore, proteasome inhibitors stabilize β3-ubiquitin conjugates. Given that Plic-1 is known to associate with proteasomal subunits (29–31), we speculated that Plic-1 might stabilize GABAAR β3 subunits by modulating their direct ubiquitination or the abundance of β3-ubiquitin conjugates and, thus, proteasomal degradation. To assess these possibilities, transfected HEK-293 cells expressing Myc-tagged β3 (Mβ3), Plic-1, and hemagglutinin (HA)-tagged ubiquitin (HA-Ub) were lysed in 1% SDS and diluted 10-fold in RIPA buffer. The Mβ3subunit was then immunoprecipitated with rabbit anti-Myc IgGs and immunoblotted with mouse anti-HA IgGs to detect ubiquitinated Mβ3 (Fig. 3A). Co-expression of Plic-1 with Mβ3 increased the abundance of Mβ3-ubiquitin conjugates by 100 ± 6.8% compared with control cells transfected with empty vector (Fig. 3B). However, Plic-1 did not appear to significantly alter the level of ubiquitination of individual GABAA receptorβ3 subunits, as the ubiquitin/β3 signal ratio remained the same (Fig. 3C). Thus, co-expression of Plic-1 with GABAARs increases the accumulation of β3-ubiquitin conjugates that previous studies have illustrated are enriched within the ER (11).


Figure 3
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 3.
Plic-1 increases the abundance of ubiquitinated GABAAR β3 subunits. A, HEK-293 cells were transfected (nucleofection) with equimolar amounts of Mβ3, Plic-1, and HA-tagged ubiquitin. After 24 h cells were lysed in 1% SDS plus 20 mM Tris-HCl, pH 7.4, and then diluted 10-fold with modified RIPA buffer lacking SDS. Mβ3 was immunoprecipitated with rabbit anti-Myc IgGs. Top panel, immunoblot of ubiquitinated β3 conjugates. Middle panel, blots were stripped and reprobed with anti-IgGs. Lower panel, immunoblot of 10% of total lysate. B, quantification of the ubiquitin signal from A. Data represent ± S.E. percentage increase in ubiquitin signal compared with control. *, significantly different from control; p < 0.01, t test, n = 4. C, the ubiquitin/Mβ3ratios were calculated and normalized to control ubiquitin/Mβ3 (Ub/Mβ3) signal. Data represent means ± S.E. of control Ub/Mβ3 ratio.

 
Plic-1 Enhances the Cell Surface Expression Levels of Neuronal GABAARs—To examine the relevance of our studies of recombinant receptors in HEK cells to endogenous GABAARs in neurons, we examined the effects of increased Plic-1 expression on the levels of GABAARs in cultured neurons. E18 cortical neurons were transfected with either pRK5-GFP (control) or with pRK5-Plic-1 using nucleofection and then incubated for 3–4 days in vitro. Neurons were then lysed in RIPA buffer, and equal amounts of protein were subjected to SDS-PAGE and immunoblotting with anti-Plic-1 and anti-β3 IgGs. In cultures nucleofected with pRK5-Plic-1 an increase in the level of Plic-1 expression was evident compared with control (Fig. 4A). Furthermore, Plic-1 increased the total pool of endogenousβ3 subunits by 26.7 ± 2% compared with that seen in control neurons expressing GFP (Fig. 4A). In contrast, the levels of the ionotropic glutamate receptor GluR1 subunit were unaltered (Fig. 4A).

To determine whether Plic-1 expression regulated the cell surface abundance of GABAARs in neurons, we used a biotinylation assay to label and isolate surface receptors. In these experiments cortical neurons were nucleofected with control pRK5-GFP or Plic-1 and incubated for 3–4 days in vitro followed by biotinylation and isolation of cell surface receptors. Plic-1 increased the cell surface expression levels of GABAARs incorporating β3 subunits by 27.6% compared with those in control neurons without altering the cell surface levels of the GluR1 subunit (Fig. 4B). We also examined the influence of Plic-1 on the cell surface degradation of GABAARs. Nucleofected neurons expressing pRK5-GFP or Plic-1 (3–4 days in vitro) were biotinylated to label cell surface receptors and then incubated for 0 or 24 h at 37 °C. Consistent with our recombinant experiments in HEK cells, Plic-1 did not appear to influence the degradation of cell surface β3-containing GABAARs (Fig. 4, C and D). Together these results suggest that Plic-1 acts to increase the steady state cell surface levels of GABAAR by selectively increasing their insertion into the plasma membrane.


Figure 4
View larger version (21K):
[in this window]
[in a new window]

  		FIGURE 4.
Plic-1 increases expression levels of GABAAR β3 subunits in neurons. A, cortical neurons (E18) were transfected (nucleofection) with pRK5-GFP (Control) or pRK5-Plic-1 plasmids. After expression for 3 days in vitro, neurons were lysed and equal amounts of solubilized protein were immunoblotted with anti-β3, anti-GluR1, and anti-Plic-1 IgGs as indicated. Graph represents quantification of totalβ3 levels in transfected neurons. Data represent mean ± S.E. percentage of control values. *, significantly different from control; p < 0.01, n = 4, t test. B, Plic-1 increases cell surface expression of GABAAR β3 subunits. Cortical neurons (E18) were transfected (nucleofection) with pRK5-GFP (Control) or pRK5-Plic-1 plasmids. After expression for 3 days in vitro, cell surface proteins were biotinylated with NHS-SS-Biotin. Biotinylated receptors were isolated using immobilized avidin and immunoblotted with rabbit anti-β3 and rabbit anti-GluR-1 IgGs as indicated. Graph represents quantification of cell surface β3 levels in transfected neurons. Data represent mean ± S.E. percentage of control values. *, significantly different from control; p < 0.01, n = 4, t test. C, cell surface degradation of GABAAR in neurons. Cortical neurons were transfected with Plic-1 or GFP and incubated for 3 days in vitro. Neurons were then biotinylated and incubated at 37 °C for 0 or 24 h. Upper panel, immunoblot showing cell surface β3. Lower panel, immunoblot showing increased Plic-1 expression in total lysates from transfected neurons. D, graph represents percent β3 remaining at 24 h with (gray) or without (black) Plic-1. Data represent means ± S.E. percent of β3 at time zero, n = 4.

 
Plic-1 Enhances the Cell Surface Accumulation of Recombinant β3 Subunits in Neurons—To corroborate our biochemical experiments we examined the influence of Plic-1 on the cell surface accumulation of GABAARs using fluorescent labeling of live hippocampal neurons. For these experiments hippocampal neurons (9 DIV) were transfected with pRK5-Plic-1 or empty control vector and the GABAAR β3 subunit engineered with an {alpha}-bungarotoxin binding site (BBS) and a pHluorin reporter incorporated into the N terminus (BBSβ3) (11, 23). Critically, these modifications do not alter GABAAR assembly or their functional properties (11, 23, 32). Following transfection, neurons were incubated for 24 h and then labeled with {alpha}-bungarotoxin conjugated to rhodamine (Rd-Bgt) for 15 min at 15 °C in order to label cell surface BBSβ3 (Fig. 5A). Cell surface BBSβ3 labeling with Rd-Bgt was performed at 15 °C to block both GABAAR endo- and exocytosis. Quantitative analysis of cell surface Rd-Bgt fluorescence intensity showed a significant increase (64 ± 12.2%) in BBSβ3 in neurons cotransfected with Plic-1 compared with control (Fig. 5B). Furthermore, not only an increase in cell surface BBSβ3 was observed but also an increase in total BBSβ3 (65 ± 12.1%) pHluorin fluorescence (Fig. 5C). Thus, these experiments demonstrate that Plic-1 can enhance the cell surface accumulation of recombinant GABAAR receptors when co-expressed in cultured neurons.


Figure 5
View larger version (58K):
[in this window]
[in a new window]

  		FIGURE 5.
Plic-1 increases the cell surface expression of recombinant GABAARs when co-expressed in neurons. A, following transfection with BBSβ3 and Plic-1 or empty vector (pRK5), hippocampal neurons were incubated with rhodamine-Bgt (Rd-Bgt) to label cell surface BBSβ3. Boxed areas in main image are magnified in panels below. Quantification of cell surface BBSβ3 (B) and the total pool of BBSβ3 levels (C) from images in A. Data represent means ± S.E. percentage of control BBSβ3. *, significantly different from control; p < 0.01, t test, n = 10–12 neurons, n = 3 independent cultures).

 


Figure 6
View larger version (40K):
[in this window]
[in a new window]

  		FIGURE 6.
Plic-1 enhances the membrane insertion of GABAARs in cultured neurons. A, increase in BBSβ3 insertion over time. Hippocampal neurons (9 DIV) were transfected with BBSβ3 and 24 h later were preincubated with unlabeled Bgt to block existing surface BBSβ3. Neurons were then incubated at 37 °C for various time points (as indicated) in the presence of rhodamine-Bgt to label newly inserted BBSβ3. B, graph represents fluorescence intensity of newly inserted BBSβ3 over time (n = 10 neurons/time point). C, hippocampal neurons (9 DIV) were transfected with equimolar amounts of BBSβ3 and Plic-1 or pRK5 control vector as indicated. After 24 h of expression, neurons were preincubated with unlabeled Bgt and then incubated with rhodamine-Bgt (Rd-Bgt) for 4 min at 37 °C. Boxed areas of dendrites in images are magnified in panels below. D, graph shows quantification of fluorescence intensity of newly inserted BBSβ3. Data represent means ± S.E. percentage of control BBSβ3. *, significantly different from control; p < 0.01, t test, n = 10–12 neurons, n = 3 independent cultures.

 
Plic-1 Increases the Insertion of GABAARs into the Neuronal Plasma Membrane—Finally, we used labeling of hippocampal neurons with fluorescent Bgt to directly assess the role that Plic-1 plays in regulating GABAAR membrane insertion. First, hippocampal neurons (9 DIV) were transfected with BBSβ3, and 24 h later live neurons were labeled with 10 µg/ml unlabeled Bgt for 15 min at 15 °C in order to block existing cell surface GABAARs containing BBSβ3 subunits. Then, to measure insertion of BBSβ3, neurons were incubated with 1 µg/ml Rd-Bgt at 37 °C for varying time points (Fig. 6A). The insertion of BBSβ3 increased linearly over time, reaching a plateau at ~10 min (Fig. 6B). This increase in rhodamine fluorescence could be specifically blocked by co-incubation with 10 µg/ml unlabeled Bgt (data not shown). We therefore used a time point of 4 min to assess the influence of Plic-1 on the insertion of BBSβ3. Hippocampal neurons were transfected with BBSβ3 and Plic-1 or control empty vector (pRK5). 24 h later neurons were incubated with Bgt to block existing cell surface BBSβ3 and then incubated with Rd-Bgt for 4 min to label newly inserted BBSβ3. These experiments revealed that in the presence of Plic-1 the level of BBSβ3 insertion increased by 50 ± 10.6% compared with control (Fig. 6, C and D).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Plic-1, an established negative regulator of proteasome activity (30), has been previously documented to be associated with GABAARs. This interaction is mediated between the C-terminal ubiquitin-associated domain of Plic-1 and a conserved motif within the major intracellular domains of GABAAR receptor {alpha} and β subunits (14). Although this interaction is critical for modulating GABAAR receptor functional expression, the underlying mechanisms remain to be established.

Here we have examined the role that Plic-1 plays in regulating the membrane trafficking of GABAARs. To do so we compared the expression levels of heterotrimeric GABAARs composed of {alpha}1β3 and {gamma}2 subunits in the presence or absence of a Plic-1 expression construct in HEK-293 cells. Co-expression with Plic-1 increased the cell surface and total expression levels of GABAARs in these cells. Consistent with this result, overexpression of Plic-1 in cortical neurons also significantly enhanced the cell surface expression of endogenous GABAARs containingβ3 subunits. In both systems this modulatory effect of Plic-1 was independent of alterations in receptor endocytic sorting. Thus, these experiments suggest that Plic-1 acts to increase GABAAR receptor cell surface expression primarily by altering receptor trafficking or stability within the secretory pathway.

To analyze further the significance of Plic-1 for GABAAR receptor stability within the secretory pathway, we assessed its potential role in regulating the abundance of ER-retained GABAARs. We focused on this compartment because it represents the site where hetero-oligomers of GABAARs are assembled from their constituent subunits (17, 33, 34). As revealed by Endo H digestion, Plic-1 increased the stability of ER-retained GABAARs. In support of our findings, immunofluorescence and immunoelectron microscopy have revealed that Plic-1 is associated with subsynaptic cisternae, the ER, and the Golgi apparatus in fibroblasts and neurons (14). It is emerging that the residence time of GABAAR subunits within the ER is determined by their rates of ubiquitination and subsequent proteasomal degradation (6, 11, 14). In agreement with this mechanism we have established that Plic-1 increases the accumulation of ubiquitinated GABAAR subunits. Finally, we were also able to directly demonstrate that increasing the expression of Plic-1 in neurons specifically enhanced the insertion of GABAA receptors into the plasma membrane.

Collectively, our results suggest that Plic-1 increases GABAAR cell surface expression levels by increasing their stability within the ER. Given that the assembly of GABAARs from their constituent subunits in the ER is inefficient (6), this enhanced stability may increase the production of transport-competent heteromeric receptors for insertion into the plasma membrane. In agreement with our findings, inhibiting proteasome activity has been shown to increase the abundance of assembled nicotinic acetylcholine receptors, which leads to enhanced insertion into the plasma membrane (35). Significantly, previous studies have revealed that Plic-1 can interact with the GABAAR receptor {alpha}1–3, {alpha}6, and β1–3 subunits (14), which is suggestive of a conserved role for this protein in regulating the cell surface stability of this structurally diverse family of inhibitory receptors. Consistent with our results with GABAAR, Plic-1 and its yeast homolog Dsk2 are associated with the proteasome and are implicated in regulating ER-associated degradation and protein targeting to aggresomes (29, 30, 36, 37). In addition, Plic proteins appear to have a role in regulating the endocytosis of G protein-coupled receptors and function of heterotrimeric G proteins (25, 38).

Finally, chronic perturbation of neuronal activity leads to bidirectional changes in ubiquitin-dependent degradation of GABAARs (11). Given the role of Plic-1 in ubiquitin-dependent proteasomal degradation of the GABAAR β3 subunit, it is tempting to speculate that activity-dependent ubiquitination, and thus turnover, may be regulated by binding of Plic-1 to the β3 subunit in response to changes in neuronal activity.


   FOOTNOTES
 
* This work was supported, in whole or in part, by National Institutes of Health Grants NS046478, NS048045, NS051195, NS056359, and P01NS054900 (to S. J. M.). This work was also supported by the Medical Research Council (to S. J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by a postdoctoral fellowship from the Epilepsy Foundation. Back

2 To whom correspondence should be addressed. E-mail: sjmoss{at}mail.med.upenn.edu.

3 The abbreviations used are: GABAAR, {gamma}-aminobutyric acid type A receptor; HEK, human embryonic kidney; PBS, phosphate-buffered saline; Bgt, {alpha}-bungarotoxin; Rd-Bgt, rhodamine Bgt; Endo H, endoglycosidase H; HA, hemagglutinin; DIV, days in vitro; GFP, green fluorescent protein; ER, endoplasmic reticulum; RIPA, radioimmune precipitation assay buffer; BBS, bungarotoxin binding site. Back


   ACKNOWLEDGMENTS
 
We thank Margie Maronski from the Dichter laboratory for preparation of cultured hippocampal neurons and Yolande Haydon for manuscript preparation.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sieghart, W., and Sperk, G. (2002) Curr. Top Med. Chem. 2, 795–816[CrossRef][Medline] [Order article via Infotrieve]
  2. Rudolph, U., and Mohler, H. (2006) Curr. Opin. Pharmacol. 6, 18–23[CrossRef][Medline] [Order article via Infotrieve]
  3. Rudolph, U., and Mohler, H. (2004) Annu. Rev. Pharmacol. Toxicol. 44, 475–498[CrossRef][Medline] [Order article via Infotrieve]
  4. Farrant, M., and Nusser, Z. (2005) Nat. Rev. Neurosci. 6, 215–229[CrossRef][Medline] [Order article via Infotrieve]
  5. Luscher, B., and Keller, C. A. (2001) Nat. Cell Biol. 3, E232–E233[CrossRef][Medline] [Order article via Infotrieve]
  6. Gorrie, G. H., Vallis, Y., Stephenson, A., Whitfield, J., Browning, B., Smart, T. G., and Moss, S. J. (1997) J. Neurosci. 17, 6587–6596[Abstract/Free Full Text]
  7. Kittler, J. T., and Moss, S. J. (2003) Curr. Opin. Neurobiol. 13, 341–347[CrossRef][Medline] [Order article via Infotrieve]
  8. Luscher, B., and Keller, C. A. (2004) Pharmacol. Ther. 102, 195–221[CrossRef][Medline] [Order article via Infotrieve]
  9. Jacob, T. C., Moss, S. J., and Jurd, R. (2008) Nat. Rev. Neurosci. 9, 331–343[CrossRef][Medline] [Order article via Infotrieve]
  10. Glickman, M. H., and Ciechanover, A. (2002) Physiol. Rev. 82, 373–428[Abstract/Free Full Text]
  11. Saliba, R. S., Michels, G., Jacob, T. C., Pangalos, M. N., and Moss, S. J. (2007) J. Neurosci. 27, 13341–13351[Abstract/Free Full Text]
  12. Hicke, L., Schubert, H. L., and Hill, C. P. (2005) Nat. Rev. Mol. Cell. Biol. 6, 610–621[CrossRef][Medline] [Order article via Infotrieve]
  13. Wu, A. L., Wang, J., Zheleznyak, A., and Brown, E. J. (1999) Mol. Cell 4, 619–625[CrossRef][Medline] [Order article via Infotrieve]
  14. Bedford, F. K., Kittler, J. T., Muller, E., Thomas, P., Uren, J. M., Merlo, D., Wisden, W., Triller, A., Smart, T. G., and Moss, S. J. (2001) Nat. Neurosci. 4, 908–916[CrossRef][Medline] [Order article via Infotrieve]
  15. Brandon, N. J., Jovanovic, J. N., Colledge, M., Kittler, J. T., Brandon, J. M., Scott, J. D., and Moss, S. J. (2003) Mol. Cell. Neurosci. 22, 87–97[CrossRef][Medline] [Order article via Infotrieve]
  16. Jovanovic, J. N., Thomas, P., Kittler, J. T., Smart, T. G., and Moss, S. J. (2004) J. Neurosci. 24, 522–530[Abstract/Free Full Text]
  17. Connolly, C. N., Krishek, B. J., McDonald, B. J., Smart, T. G., and Moss, S. J. (1996) J. Biol. Chem. 271, 89–96[Abstract/Free Full Text]
  18. Moss, S. J., Gorrie, G. H., Amato, A., and Smart, T. G. (1995) Nature 377, 344–348[CrossRef][Medline] [Order article via Infotrieve]
  19. Kittler, J. T., Delmas, P., Jovanovic, J. N., Brown, D. A., Smart, T. G., and Moss, S. J. (2000) J. Neurosci. 20, 7972–7977[Abstract/Free Full Text]
  20. Couve, A., Restituito, S., Brandon, J. M., Charles, K. J., Bawagan, H., Freeman, K. B., Pangalos, M. N., Calver, A. R., and Moss, S. J. (2004) J. Biol. Chem. 279, 13934–13943[Abstract/Free Full Text]
  21. Kittler, J. T., Thomas, P., Tretter, V., Bogdanov, Y. D., Haucke, V., Smart, T. G., and Moss, S. J. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 12736–12741[Abstract/Free Full Text]
  22. Jacob, T. C., Bogdanov, Y. D., Magnus, C., Saliba, R. S., Kittler, J. T., Haydon, P. G., and Moss, S. J. (2005) J. Neurosci. 25, 10469–10478[Abstract/Free Full Text]
  23. Bogdanov, Y., Michels, G., Armstrong-Gold, C., Haydon, P. G., Lindstrom, J., Pangalos, M., and Moss, S. J. (2006) EMBO J. 25, 4381–4389[CrossRef][Medline] [Order article via Infotrieve]
  24. Sekine-Aizawa, Y., and Huganir, R. L. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 17114–17119[Abstract/Free Full Text]
  25. N'Diaye, E. N., Hanyaloglu, A. C., Kajihara, K. K., Puthenveedu, M. A., Wu, P., von Zastrow, M., and Brown, E. J. (2008) Mol. Biol. Cell 19, 1252–1260[Abstract/Free Full Text]
  26. Helenius, A., and Aebi, M. (2001) Science 291, 2364–2369[Abstract/Free Full Text]
  27. Helenius, A., and Aebi, M. (2004) Annu. Rev. Biochem. 73, 1019–1049[CrossRef][Medline] [Order article via Infotrieve]
  28. Buchstaller, A., Fuchs, K., and Sieghart, W. (1991) Neurosci. Lett. 129, 237–241[CrossRef][Medline] [Order article via Infotrieve]
  29. Kleijnen, M. F., Alarcon, R. M., and Howley, P. M. (2003) Mol. Biol. Cell 14, 3868–3875[Abstract/Free Full Text]
  30. Kleijnen, M. F., Shih, A. H., Zhou, P., Kumar, S., Soccio, R. E., Kedersha, N. L., Gill, G., and Howley, P. M. (2000) Mol. Cell 6, 409–419[CrossRef][Medline] [Order article via Infotrieve]
  31. Huh, K. H., Delorey, T. M., Endo, S., and Olsen, R. W. (1995) Mol. Pharmacol. 48, 666–675[Abstract]
  32. Kittler, J. T., Wang, J., Connolly, C. N., Vicini, S., Smart, T. G., and Moss, S. J. (2000) Mol. Cell. Neurosci. 16, 440–452[CrossRef][Medline] [Order article via Infotrieve]
  33. Taylor, P. M., Connolly, C. N., Kittler, J. T., Gorrie, G. H., Hosie, A., Smart, T. G., and Moss, S. J. (2000) J. Neurosci. 20, 1297–1306[Abstract/Free Full Text]
  34. Taylor, P. M., Thomas, P., Gorrie, G. H., Connolly, C. N., Smart, T. G., and Moss, S. J. (1999) J. Neurosci. 19, 6360–6371[Abstract/Free Full Text]
  35. Christianson, J. C., and Green, W. N. (2004) EMBO J. 23, 4156–4165[CrossRef][Medline] [Order article via Infotrieve]
  36. Medicherla, B., Kostova, Z., Schaefer, A., and Wolf, D. H. (2004) EMBO Rep. 5, 692–697[CrossRef][Medline] [Order article via Infotrieve]
  37. Heir, R., Ablasou, C., Dumontier, E., Elliott, M., Fagotto-Kaufmann, C., and Bedford, F. K. (2006) EMBO Rep. 7, 1252–1258[CrossRef][Medline] [Order article via Infotrieve]
  38. N'Diaye, E. N., and Brown, E. J. (2003) J. Cell Biol. 163, 1157–1165[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Neurosci.Home page
K. Rezvani, Y. Teng, Y. Pan, J. A. Dani, J. Lindstrom, E. A. Garcia Gras, J. M. McIntosh, and M. De Biasi
UBXD4, a UBX-Containing Protein, Regulates the Cell Surface Number and Stability of {alpha}3-Containing Nicotinic Acetylcholine Receptors
J. Neurosci., May 27, 2009; 29(21): 6883 - 6896.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/27/18538    most recent
M802077200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Saliba, R. S.
Right arrow Articles by Moss, S. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Saliba, R. S.
Right arrow Articles by Moss, S. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2008 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement