Advertisement
JBC

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


     

Originally published In Press as doi:10.1074/jbc.M702358200 on June 27, 2007

J. Biol. Chem., Vol. 282, Issue 35, 25349-25356, August 31, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/35/25349    most recent
M702358200v1
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 Pooler, A. M.
Right arrow Articles by McIlhinney, R. A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pooler, A. M.
Right arrow Articles by McIlhinney, R. A. 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?

Lateral Diffusion of the GABAB Receptor Is Regulated by the GABAB2 C Terminus*Formula

Amy M. Pooler and R. A. Jeffrey McIlhinney1

From the Medical Research Council Anatomical Neuropharmacology Unit, Mansfield Road, Oxford OX1 3TH, United Kingdom

Received for publication, March 19, 2007 , and in revised form, June 26, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GABAB ({gamma}-aminobutyric acid, type B) is a heterodimeric G-protein-coupled receptor. The GABAB1 subunit, which contains an endoplasmic reticulum retention sequence, is only transported to the cell surface when it is associated with the GABAB2 subunit. Fluorescence recovery after photobleaching studies in transfected COS-7 cells and hippocampal neurons revealed that GABAB2 diffuses slowly within the plasma membrane whether expressed alone or with the GABAB1 subunit. Treatment of cells with brefeldin A revealed that GABAB2 moves freely within the endoplasmic reticulum, suggesting that slow movement of GABAB2 is a result of its plasma membrane insertion. Disruption of the cytoskeleton did not affect the mobility of GABAB2, indicating that its restricted diffusion is not due to direct interactions with actin or tubulin. To determine whether the C terminus of GABAB2 regulates its diffusion, this region of the subunit was attached to the lymphocyte membrane protein, CD2, which then exhibited a slower rate of lateral diffusion. Furthermore, co-expression of a cytoplasmically expressed soluble form of the GABAB2 C terminus increased movement of the GABAB2 subunit. We constructed forms of GABAB2 with various C-terminal truncations. Truncation of GABAB2 after residue 862, but not residue 886, caused a dramatic increase in its mobility, suggesting that the region between these two residues is critical for restricting GABAB2 diffusion. Finally, we investigated whether activation of GABAB might modulate its movement. Treatment of COS-7 cells with the GABAB receptor agonist baclofen significantly increased its mobile fraction. These data show that the restricted movement of GABAB at the cell surface is regulated by a region within its C terminus.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GABAB receptors are metabotropic receptors for the inhibitory neurotransmitter {gamma} aminobutyric acid (GABA).2 Pre- and post-synaptic GABAB receptors are coupled to inhibitory G-proteins and can regulate neurotransmission via several mechanisms, including modulation of adenylyl cyclase (1), inhibition of voltage-gated Ca2+ channels (2), and modulation of K+ channels (3, 4). Formation of a functional receptor requires the heterodimerization of two subunits, GABAB1 and GABAB2 (5). Previous work has demonstrated that the stable assembly of these subunits occurs, to some extent, via association of coiled-coil domains within their C termini (6). The subunits appear to serve different functions within the fully formed receptor. GABAB1 contains the agonist binding site on its large extracellular N terminus, and the affinity of this site for agonists is increased following heterodimerization with GABAB2 (7). The GABAB2 subunit contains intracellular loops that couple the receptor to the G-protein (8–10).

Heterodimerization of the GABAB subunits is important not only for proper receptor function but also for forward trafficking of the receptor to the cell surface (5, 7). In the absence of GABAB2, the GABAB1 subunit is retained within the endoplasmic reticulum due to the presence of a C-terminal RSRR retention motif on its C terminus. The interaction of GABAB2 with GABAB1 apparently masks this motif, allowing the fully formed receptor to traffic to the cell surface, where it may be targeted to the synapse.

The number of neurotransmitter receptors within post-synaptic membranes is dependent not only on insertion of new receptors but also on lateral diffusion of extrasynaptic receptors into the synaptic compartment (11). Regulation of receptor movement within the plasma membrane is therefore likely to be important for plasticity at individual synapses. At excitatory synapses, lateral diffusion of {alpha}-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors has been suggested to regulate synaptic strength following either long-term potentiation or long-term depression (12–14). Furthermore, N-methyl-D-aspartate receptors readily exchange between synaptic and extrasynaptic compartments via movement within the plasma membrane (15). However, the behavior of inhibitory neurotransmitter receptors is less well understood. The dynamics of the glycine receptor (16) and the ionotropic GABAA receptor (17) have recently been examined. Extrasynaptically, both receptors diffuse freely, but within the synaptic compartment interactions with the synaptic scaffolding protein gephyrin significantly slow their movement. To date, however, membrane dynamics of the GABAB receptor have yet to be investigated.

In the present study we explored the movement of the GABAB receptor within the plasma membrane. We found lateral diffusion of GABAB at the cell surface to be slow, due to restricted mobility of the GABAB2 subunit. Disruption of the cytoskeleton did not affect GABAB2 diffusion; however, truncation of the GABAB2 C-terminal region allowed GABAB2 to diffuse more rapidly. We show, therefore, that GABAB exhibits distinct cell surface dynamics that can be regulated by a region within the C terminus of the GABAB2 subunit.


Figure 1
View larger version (37K):
[in this window]
[in a new window]

  		FIGURE 1.
GABAB diffuses slowly at the cell surface. A, COS-7 cells were transfected with either GABAB2-YFP (top) or CD2-YFP(bottom). A circular region(arrow) was bleached with a high intensity laser, and recovery of the YFP signal was imaged over time. B, diffusion of GABAB2-YFP into the bleached region is very slow, whether expressed alone or together with the GABA1b subunit. C, COS-7 cells expressing GABAB2-YFP were treated with brefeldin A, which causes retention of proteins in the endoplasmic reticulum. Within the endoplasmic reticulum GABAB2-YFP diffused rapidly, indicating that GABAB2 only exhibits restricted movement within the plasma membrane.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transfection—COS-7 cells were maintained in minimal essential medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and incubated at 37 °C, 5% CO2. For experiments, cells were seeded onto glass coverslips and transfected 24 h later using JetPEI (Autogen Bioclear, Calne, UK) according to the manufacturer's instructions. Cells were incubated for 48 h post-transfection before FRAP (fluorescence recovery after photobleaching) analysis. Primary neuron cultures were prepared from E18 Sprague-Dawley rat embryos. Briefly, dissected hippocampi were mechanically dissociated in Hanks' balanced salt solution lacking calcium and magnesium, supplemented with 1 mM pyruvate and 10 mM HEPES. Cells were plated immediately onto glass coverslips coated with poly-D-lysine (5 µg/ml) and cultured in Neurobasal medium containing 2% B-27 serum-free supplement, 0.5 mM L-glutamine, 25 µM glutamate, 0.05% gentamicin. To restrict the proliferation of non-neuronal cells, after 4 days the medium were changed to Neurobasal medium supplemented with 2% B27, 0.5 mM L-glutamine, 3 µM cytosine arabinofuranoside, 0.05% gentamicin. Neurons were transfected with Lipofectamine (Invitrogen) 5 days in vitro according to the manufacturer's instructions and imaged 48 h later. Approximately 1–3% of cells were transfected per coverslip; 8–10 dendrites from distinct cells were analyzed in each independent experiment.

Plasmids—HA-tagged GABAB2 and Myc-tagged GABAB1b were obtained from GlaxoSmithKline, as were the GABAB1b-YFP and GABAB2-YFP and their CFP derivatives. All GABAB2 truncations and chimeras were generated by PCR from the HA-tagged construct in pCDNA3.1(-). The truncated forms of GABAB2 were produced by excising the EcoRV/HindIII fragment from the HA-tagged GABAB2 in pCDNA3.1(-). The different regions of interest were obtained using polymerase chain reaction (PCR) to amplify them using the common forward primer 5'-GCA GGA CGG GAT ATC TCC ATC CGC CCT CTC C-3' that covers the EcoRV site positioned in the third extracellular loop of GABAB2. The reverse primers covered the C terminus from amino acids 920, 886, 862, or 841 and contained codons at these positions that permitted ligation inframe into eYFP-N1 (Clontech). These were as follows: GABAB2{Delta}920, 5'-GCT AAG CTT GAC GCA GGG GCT GAC ACA GCT GGC-3'; GABAB2{Delta}886, 5'-GCT AAG CTT TGG GAG AGT TTA TAT CTT CTA TAC G-3'; GABAB2 {Delta}862, 5'-TGT GTT CCA CTG AAG CTT GGG ATT TTG ATC GAG-3'; GABAB2{Delta}841, 5'-GCA AGC TTT CCC AGG TTG AGG ATG TCA TTG AGC-3'. The PCR products were then ligated into the EcoRV/HindIII-digested HA-tagged GABAB2 to produce the protein of interest. For production of the YFP-tagged truncated forms of GABAB2 the plasmids containing the ligated PCR products were excised from pCDNA3.1 using NheI and HindIII, and this fragment was then ligated into NheI/HindIII-digested eYFP-N1.

The plasmid containing CD2 fused to the C-terminal tail of mGluR1a previously described (18) was digested with BamHI and NotI to remove the mGluR1a sequence. YFP (Clontech) was amplified by PCR with the primers 5'-GAC TCA GAT CTC GAG CTA AGC TTC GAA TTC-3' and 5'-GAT CTA GAG TCG CGG CCG CTT TAC TTG TAC-3' containing 5'-BglII and 3'-NotI sites. The PCR product was gel-purified, digested with the appropriate enzymes, and ligated into the digested CD2 plasmid to give the construct CD2-YFP. This in turn was digested with XhoI and BamHI and ligated with the PCR product obtained from amplifying the C-terminal tail of the GABABB2 subunit using the primers 5'-GTG CCG AAG CTC GAG ACC CTG AGA ACA AAC-3' and 5'-CCA CGG ATC CAG GCC CGA GAC CAT GAC TCG-3', after digestion with the same enzymes, to give CD2-R2-YFP. This contained the N-terminal and transmembrane domain of CD2 followed by the C terminus of the GABAB2 subunit and then YFP. To give a soluble form of the GABAB2 subunit C terminus that could be detected in cells, the C terminus of the subunit was amplified by PCR using the primers 5'-CTC ATC ACC CTG AGA TCT AAC CCA GAT GCA GC-3' and 5'-CGT ATC TAG ATT ACA GGC CCG AGA CCA TGA CTC G-3'. The product was digested with BglII and XbaI and the product inserted into similarly digested pECFP-C1 (Clontech). All PCR reactions were carried out using the proofreading KOD polymerase (Invitrogen) and the conditions recommended by the manufacturer. DNA alterations to all constructs were verified by DNA sequencing.


Figure 2
View larger version (33K):
[in this window]
[in a new window]

  		FIGURE 2.
GABAB2 diffuses slowly in hippocampal neurons. A and B, cultured rat hippocampal neurons were transfected with either GABAB2-YFP or CD2-YFP. Protein diffusion was monitored in dendrites using FRAP analysis. GABAB2-YFP diffused slowly compared with diffusion of the control protein CD2-YFP. C, co-expression of GABAB2-YFP with GABAB1 did not alter the rate of diffusion of GABAB2-YFP. Values represent means ± S.E.

 
FRAP—We subjected transfected COS-7 cells or hippocampal neurons to FRAP analysis to assess the lateral diffusion of expressed proteins (19). Cells expressing YFP-tagged proteins were maintained at 37 °C and imaged on a Zeiss LSM510 inverted confocal microscope with a x40 oil objective. Regions of interest (ROIs) in COS-7 cells were circular with a diameter of 7 µm; in hippocampal neurons, the ROIs were 7-µm lengths of dendritic processes. ROIs were scanned for 5 cycles with an Argon 514 laser at 1% maximal power to determine initial fluorescence intensity before being bleached by 15 cycles at 90% maximal laser power. The fluorescence intensity of the whole cell was captured for 2 min at 1% laser power; recovery is presented as percentage of original fluorescence, corrected for any bleaching due to repetitive scanning. To examine the effects of brefeldin A (5 µg/ml; Sigma-Aldrich), baclofen (100 µM; Sigma), lactrunculin (5 µM; Sigma), or colchicine (5 µM; Sigma), the cells were incubated with the appropriate compound at 37 °C for 1 h prior to FRAP analysis. For antagonist treatments, cells were incubated with the GABAB antagonist CGP-54626 (4.2 µM; Tocris Cookson, Ellisville, MO) for 10 min and then co-treated with antagonist (4.2 µM) and baclofen (100 µM) for 1 h prior to FRAP analysis. For treatment with antagonist only, cells were incubated with 4.2 µM CGP-54626 for 1 h.

For diffusion analysis of HA-tagged proteins, we incubated COS-7 cells expressing these constructs with an anti-HA antibody (kindly provided by GlaxoSmithKline) conjugated with the fluorophore Alexa 488 (Alexa Fluor 488 Protein Labeling kit; Molecular Probes, Eugene, OR) for 20 min at 37 °C. The cells were washed twice with Hanks' balanced salt solution and then imaged as described above, except using an Argon 488 laser instead of the 514 laser.

Data analyses were performed using Igor Pro 5.05 software (Wavemetrics, Lake Oswego, OR) with FRAP plug-in written by K. Miura (EMBL Heidelberg, Germany). Data are presented as means ± S.E., representing at least three independent experiments/group, each group containing a minimum of 10 cells. For the region of interest of each cell, the fluorescence recovery curve was best fitted with a double exponential function with Phair normalization (20). The mobile fraction and time to half-maximal recovery (t1/2 maximal) was calculated from each curve.

Immunocytochemistry—Following the transfection period, COS-7 cells were treated for 1 h at 37°C with either lactrunculin, to block actin polymerization, or colchicine, to block tubulin polymerization. After treatment, cells were washed once with PBS, fixed for 5 min at in 4% paraformaldehyde at room temperature, and then washed twice with PBS and twice with Tris-saline. To detect intracellular proteins, cells were permeabilized with 0.2% Triton X-100 for 5 min. Cells were incubated for 1 h at room temperature in blocking solution containing 1% bovine serum albumin and 1% normal goat serum. To visualize actin, cells were incubated in PBS containing fluorescein isothiocyanate-labeled phalloidin (Molecular Probes) for 1 h, washed with PBS, and mounted on slides. For tubulin detection, the cells were incubated for 2 h at room temperature in PBS containing mouse anti-beta-tubulin (1:1000; Sigma), washed three times with PBS, and incubated for 1 h with anti-mouse ALEXA 546 (Molecular Probes). After three washes with PBS, the coverslips were mounted and viewed with a Zeiss LSM510 inverted confocal microscope with a x40 oil objective.


Figure 3
View larger version (33K):
[in this window]
[in a new window]

  		FIGURE 3.
A,top,in COS-7 cells,latrunculin treatment does not significantly increase the recovery rate of GABAB2-YFP compared with recovery in untreated cells. Bottom, phalloidin staining reveals that latrunculin treatment disrupts actin polymerization on COS-7 cells. B, top, treatment of COS-7 cells with colchicine did not alter GABAB2-YFP diffusion. Bottom, colchicine treatment disrupts tubulin polymerization, as visualized using an antibody directed against beta-tubulin. C, top, in hippocampal neurons, latrunculin treatment did not affect the diffusion rate of GABAB2-YFP, although the treatment did disrupt dendritic actin filaments as visualized by phalloidin staining (bottom). Values represent means ± S.E.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
GABAB Diffuses Slowly within the Plasma Membrane, as Measured by FRAP Analysis—To determine the diffusional mobility of GABAB2 at the cell surface, we performed FRAP experiments on COS-7 cells transiently transfected with a YFP-tagged GABAB2 construct. We monitored subunit movement within the plasma membrane by focusing the laser excitation at the cell surface. Fluorescent proteins within a defined region of the cell were photobleached by high intensity laser, and the diffusion of unbleached proteins into the bleached region was monitored for 2 min. GABAB2 diffused very slowly at the cell surface compared with the diffusion rate of YFP-tagged CD2, a membrane-targeted protein known to freely move within the plasma membrane (Fig. 1A). To examine the mobility of the GABAB receptor, cells were co-transfected with GABAB1b-CFP and GABAB2-YFP. In the absence of GABAB2, GABAB1b did not reach the cell surface (data not shown). Movement of the GABAB1b-GABAB2 heterodimer was similar to the diffusion rate of GABAB2 (Fig. 1B), suggesting that the restricted movement of GABAB is limited by the diffusion of the GABAB2 subunit. Slow diffusion of GABAB2 is specific to the plasma membrane, because retention of GABAB2 in the endoplasmic reticulum following brefeldin A treatment increased its diffusion rate (Fig. 1C). To confirm that the YFP tag was not affecting subunit diffusion, we also analyzed movement of HA-GABAB2 by live labeling of surface receptors with fluorescein isothiocyanate-labeled antibodies directed against HA (supplemental Fig. S1). No difference in mobility was detected between YFP-tagged GABAB2 and HA-tagged GABAB2.

Diffusion of GABAB2 was also restricted in plasma membrane of cultured rat hippocampal neurons. As expected, transfection of primary neurons using Lipofectamine yielded transfection rates of ~1–3%. FRAP analysis of neurons transiently transfected with either GABAB2-YFP or CD2-YFP revealed that movement of GABAB2 was constrained within dendrites relative to movement of CD2-YFP (Figs. 2, A and B). The mobility of GABAB2-YFP was not affected by co-expression with GABAB1 (Fig. 2C).

Disruption of the Cytoskeleton Does Not Significantly Affect GABAB2 Movement—Previous studies investigating receptor movement suggest that receptors may interact directly or indirectly with the cytoskeleton. The AMPA receptor binds actin directly (21–23), whereas in the inhibitory synapse GABAA interacts with the anchoring protein gephyrin (16, 17). To determine whether slow diffusion of the GABAB receptor might be due to interaction with the cytoskeleton, we transiently transfected COS-7 cells with GABAB2-YFP and treated them with either latrunculin (5 µM, 1 h), to inhibit actin polymerization, or colchicine (5 µM, 1 h), to inhibit tubulin polymerization. Using FRAP analysis, we found that neither latrunculin (Fig. 3A) nor colchicine (Fig. 3B) treatment substantially affected GABAB2 lateral diffusion within the plasma membrane. Furthermore, lactrunculin treatment did not affect diffusion of GABAB2-YFP in the dendrites of hippocampal neurons (Fig. 3C). These results suggest that slow movement of GABAB2 is unlikely due to direct interaction between the subunit and the cytoskeleton.


Figure 4
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 4.
The C terminus of GABAB2 regulates protein diffusion. A and B, addition of the GABAB2 C-terminal to CD2-YFP significantly slows diffusion of this protein in the plasma membrane of COS-7 cells, as indicated by a decrease in the mobile fraction and an increase in time to half-maximal recovery. C and D, co-expression of a soluble GABAB2 C-terminal construct with GABAB2-YFP significantly increases diffusion of the subunit, evidenced by an increase in the mobile fraction and a decrease in time to half-maximal recovery. R2C, GABAB2 C terminus; R2, GABAB2-YFP. Values represent means ± S.E. *, p < 0.05; ***, p < 0.001 versus control.

 
The C Terminus of GABAB2 Regulates Its Membrane Diffusion—Because recent studies suggest that the C terminus of GABAB2 might contain interaction regions important for trafficking (24) and stabilization (25, 26) of the fully formed GABAB receptor, we next investigated whether the C-terminal region of GABAB2 might also be involved in regulating diffusion of the subunit at the cell surface. To determine whether the presence of the GABAB2 C terminus is sufficient to slow diffusion of a surface protein, we ligated the GABAB2 C terminus (R2C) to the C terminus of CD2 to generate CD2-R2C-YFP. FRAP analysis of COS-7 cells expressing either this construct or CD2-YFP revealed that the mobile fraction of CD2-R2C-YFP was significantly (p < 0.001) smaller compared with the mobile fraction of CD2-YFP (52.82 ± 2.63 versus 80.63% ± 1.43) (Fig. 4, A and B). Conversely, CD2-R2C-YFP took significantly (p < 0.05) more time to reach half-maximal (t1/2 maximal) recovery than CD2-YFP (9.04 ± 1.42 versus 4.99 s ± 0.41 s) (Fig. 4B). CD2-R2C-YFP also diffused significantly more slowly than CD2-YFP in the dendrites of cultured hippocampal neurons (supplemental Fig. S2). Next, we constructed a soluble version of the GABAB2 C terminus. Co-expression of COS-7 cells with GABAB2-YFP and soluble R2C significantly (p < 0.001) increased the mobile fraction of GABAB2-YFP relative to GABAB2-YFP expressed alone (72.01 ± 3.14 versus 47.53% ± 3.88) and decreased t1/2 maximal recovery (24.28 ± 5.01 versus 53.23 s ± 5.96 s) (Fig. 4, C and D). These data suggest that the GABAB2 C terminus regulates diffusion of GABAB at the cell surface.

Lateral Diffusion of GABAB2 Is Regulated by a 24-Amino Acid Region within the C Terminus—To determine which region of the GABAB2 C terminus might be involved in regulating its lateral diffusion rate, we constructed a series of C-terminal-truncated forms of GABAB2 with truncations at residues 841, 862, 886, or 920 (Fig. 5A). We then transfected COS-7 cells with one of the following constructs: GABAB2-YFP, {Delta}920-YFP, {Delta}886-YFP, {Delta}862-YFP, or {Delta}841-YFP. Although plasma membrane diffusion rates of {Delta}920-YFP and {Delta}886-YFP were similar to the diffusion of GABAB2-YFP, both {Delta}862-YFP and {Delta}841-YFP exhibited faster movement as measured by FRAP analysis than GABAB2-YFP (Fig. 5B). The mobile fraction of {Delta}862-YFP was significantly (p < 0.01) higher compared with the mobile fractions of either GABAB2-YFP or {Delta}886-YFP (67.91 ± 7.74 versus 43.24% ± 3.21 or 43.15% ± 4.39) (Fig. 5C). Correspondingly, the t1/2 maximal recovery of {Delta}862-YFP was also significantly reduced relative to recovery of either GABAB2-YFP or {Delta}886-YFP (7.80 ± 0.55 versus 47.47 ± 4.69 or 52.89 s ± 4.69 s) (Fig. 5C). Co-expression of GABAB1-YFP with {Delta}841, compared with co-expression with GABAB2, significantly increased its mobile fraction (71.62 ± 3.23 versus 48.83% ± 2.72) and decreased its t1/2 maximal recovery (15.6 ± 4.32 versus 31.15 s ± 4.26 s) (Fig. 5D). These data suggest that diffusion of GABAB1 is therefore regulated by the GABAB2 subunit. Thus, the region of the GABAB2 C terminus between amino acids 862–886 may be important for regulating mobility of GABAB at the cell surface.


Figure 5
View larger version (21K):
[in this window]
[in a new window]

  		FIGURE 5.
The region of GABAB2 between residues 862 and 886 regulates mobility of the subunit. A, schematic diagram of the GABAB2 subunit constructs. Truncations of the subunit were made at the indicated amino acid residues. The YFP tag was placed at the C terminus of each construct. B, {Delta}862-YFP and {Delta}841-YFP diffused rapidly within the plasma membrane of COS-7 cells compared with diffusion of R2-YFP, {Delta}920-YFP, and {Delta}886-YFP. C, the {Delta}862-YFP construct is significantly more mobile than either {Delta}886-YFP or R2-YFP as it exhibits a larger mobile fraction and a shorter time to half-maximal recovery. D, GABAB1-YFP displays a significantly higher mobile fraction and significantly lower time to half-maximal recovery when co-expressed with {Delta}841 than when co-expressed with GABAB2. N, N terminus; C, C terminus; CCD, coiled-coil domain; TMD, seven-transmembrane domain; R2, GABAB2. Values represent means ± S.E.; *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus R2 and {Delta}886 groups.

 
Agonist Binding Increases Lateral Diffusion of GABAB2—To determine whether lateral diffusion of GABAB2 could be dynamically regulated, we treated COS-7 cells co-transfected with GABAB1b-CFP and GABAB2-YFP with the GABAB agonist baclofen. After 1 h of baclofen treatment (100 µM), cells were subjected to FRAP analysis of GABAB2-YFP. Treatment with baclofen increased lateral diffusion of GABAB2-YFP (Fig. 6A) and significantly (p < 0.001) increased its mobile fraction relative to untreated controls (63.49 ± 6.92 versus 39.62% ± 2.47) (Fig. 6B). Co-treatment of cells with the GABAB antagonist CGP-54626 (4.2 µM) prevented the effect of baclofen on the mobile fraction (35.69% ± 4.77; p < 0.01); treatment with the antagonist alone had no effect on the mobile fraction relative to the control group (Fig. 6B). These results suggest that, although GABAB2 movement is restricted under basal conditions, stimulation of the receptor might alter its membrane dynamics.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we examined lateral diffusion of the GABAB receptor within the plasma membrane using FRAP analysis of fluorescently tagged GABAB2 subunits. We found that the GABAB receptor and the GABAB2 subunit diffuse slowly at the cell surface in both hippocampal neurons and heterologous cells. Our data suggest that the restricted diffusion of the receptor is regulated by a region within the C terminus of the GABAB2 subunit.

The diffusion characteristics of GABAB differ from those reported for other neurotransmitter receptors. We consistently found GABAB to exhibit a mobile fraction of ~40%. By contrast, AMPA receptors are reported to have a mobile fraction of >80% both in COS-7 cells (27) and in extrasynaptic regions of neurons (13). Similarly, the GABAA receptor has been shown to diffuse rapidly within the extrasynaptic membrane (28). Rapid diffusion is not restricted to receptor ion channels, as the G-protein-coupled serotonin 5-HT1a (29) and dopamine D1 (30) receptors also move freely in extrasynaptic membrane. Taken together, the data indicate that GABAB receptors exhibit distinctive diffusion dynamics.

To assess the regulation of GABAB diffusion, we examined the effect of disrupting the cytoskeleton on receptor movement. We observed that the restricted diffusion of GABAB2 was not altered following latrunculin or colchicine treatment. In contrast, disruption of microtubules increases the diffusion rate of glycine receptors (28) and disturbance of the actin cytoskeleton by latrunculin has been shown to disrupt AMPA clustering due to interaction between the GluR1 subunit and the actin complex (21–23). Our data indicate that direct interactions between GABAB2 and either actin or tubulin are unlikely to be responsible for anchoring GABAB at the cell surface. Similarly, diffusion of the GABAA receptor appears to be unaffected by latrunculin treatment (31) and diffusion of the dopamine D1 receptor is unaffected by microtubule disruption (30). Diffusion of these receptors is likely regulated by interactions with intracellular proteins other than actin, as movement of GABAA is restricted when bound to gephyrin (16) and the D1 receptor is anchored via a direct interaction with the N-methyl-D-aspartate receptor (30). These findings suggested that GABAB2 might possess an intracellular region capable of similar protein interactions.


Figure 6
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 6.
Stimulation of the GABAB receptor increases its diffusion rate. A, COS-7 cells transfected with GABAB1b-CFP and GABAB2-YFP were treated with the GABAB agonist baclofen. FRAP analysis of receptor movement was performed by bleaching the YFP signal and recording its recovery. B, baclofen treatment significantly increased the mobile fraction of the GABAB receptor. Co-treatment of cells with baclofen and the GABAB antagonist CGP-54626 prevented the baclofen-induced increase in the mobile fraction. Treatment of the cells with the antagonist alone had no effect. Values represent means ± S.E.; **, p < 0.01; ***, p < 0.001.

 
We next investigated whether the C terminus of GABAB2 might regulate its diffusion. Intracellular regions are likely to be involved in movement of receptor at the cell surface, as a recent study has demonstrated that an intracellular loop of GABAA regulates its diffusion (32). Previous studies have demonstrated that the C terminus of the GABAB2 subunit contains several important regions, such as those involved in dimerization with GABAB1 (6), G-protein binding (8–10), and mediating interactions with scaffolding proteins (33). In the present study we found that attachment of the GABAB2 C terminus to a freely diffusing protein significantly slowed diffusion of that protein within the plasma membrane. Furthermore, when GABAB2 was co-expressed with a soluble form of its C terminus, the diffusion rate of the subunit was increased. Taken together, these findings suggest that the C-terminal region of GABAB2 does regulate its dynamics at the cell surface.

To determine whether a specific domain within the C terminus controls GABAB2 diffusion, we constructed a series of truncated subunits. FRAP analysis of these constructs revealed that the region of the GABAB2 C terminus between residues 862 and 886 is critical for restricting movement of the subunit at the cell surface, as removal of this region significantly increased its diffusion rate. Furthermore, our data on diffusion of GABAB1-YFP indicate that the C terminus of GABAB2 regulates movement of the heterodimerized receptor. MUPP1, a scaffolding protein, binds to a motif within the GABAB2 C terminus (33); however, this interaction is unlikely to regulate GABAB diffusion because the motif (residues 938–941) is distal to the domain identified in the present study. In addition, truncation of the C-terminal region of GABAB2 containing the MUPP1 binding motif did not affect GABAB diffusion. In the present study, we have identified a domain involved in regulation of GABAB movement, although the protein or proteins that might interact with GABAB at this site remain to be determined. Nevertheless, these data indicate interaction sites exist on the GABAB2 C terminus that are important for trafficking, stability, and movement of GABAB at the cell surface.

We investigated whether stimulation of GABAB might affect receptor movement, as previous studies have demonstrated that movement of other metabotropic receptors, such as the serotonin 5-HT1a receptor, is greater following agonist stimulation (29, 34). We treated cells expressing GABAB1 and GABAB2 with the agonist baclofen and found that baclofen stimulation significantly increased its diffusion rate; the effect of baclofen was prevented by co-treatment with a GABAB antagonist. Agonist binding may induce a conformational change in the receptor that alters its interactions with intracellular scaffolding proteins. Our data suggest a mechanism by which release of neurotransmitter into the synapse might increase diffusion of nearby extrasynaptic receptors, thereby allowing movement into the post-synaptic density.

Regulation of receptor diffusion, and therefore receptor number, is crucial for maintaining synaptic strength. Recently, the dynamics of several neurotransmitter receptors, including AMPA, GABAA, and glycine, have been described (13, 27, 28, 35). In the present study we have demonstrated for the first time that GABAB exhibits restricted movement within the plasma membrane; furthermore, we determined that diffusion of GABAB is regulated by a specific region within the C terminus of the GABAB2 subunit. These findings provide insight into the regulation of GABAB receptor movement at the cell surface and are consistent with the hypothesis that regulated lateral diffusion is a mechanism by which the cell controls synaptic strength.


   FOOTNOTES
 
* This work was supported by the Medical Research Council and the Blaschko European Visiting Fellowship. 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

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. Back

1 To whom correspondence should be addressed. Tel.: 44-1865-271-895; Fax: 44-1865-271-647; E-mail: jeff.mcilhinney{at}pharm.ox.ac.uk.

2 The abbreviations used are: GABA, {gamma}-aminobutyric acid; FRAP, fluorescence recovery after photobleaching; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; HA, hemagglutinin; PBS, phosphate-buffered saline AMPA, {alpha}-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Alan Wise and Dr. Julia White of GlaxoSmithKline for the gift of the GABAB2 YFP, CFP, and HA-tagged constructs and Dr. Neil Barclay for the gift of CD2 DNA.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Knight, A. R., and Bowery, N. G. (1996) Neuropharmacology 35, 703–712[CrossRef][Medline] [Order article via Infotrieve]
  2. Poncer, J. C., McKinney, R. A., Gahwiler, B. H., and Thompson, S. M. (1997) Neuron 18, 463–472[CrossRef][Medline] [Order article via Infotrieve]
  3. Sodickson, D. L., and Bean, B. P. (1996) J. Neurosci. 16, 6374–6385[Abstract/Free Full Text]
  4. Ng, G. Y., Clark, J., Coulombe, N., Ethier, N., Hebert, T. E., Sullivan, R., Kargman, S., Chateauneuf, A., Tsukamoto, N., McDonald, T., Whiting, P., Mezey, E., Johnson, M. P., Liu, Q., Kolakowski, L. F., Jr., Evans, J. F., Bonner, T. I., and O'Neill, G. P. (1999) J. Biol. Chem. 274, 7607–7610[Abstract/Free Full Text]
  5. White, J. H., Wise, A., Main, M. J., Green, A., Fraser, N. J., Disney, G. H., Barnes, A. A., Emson, P., Foord, S. M., and Marshall, F. H. (1998) Nature 396, 679–682[CrossRef][Medline] [Order article via Infotrieve]
  6. Kammerer, R. A., Frank, S., Schulthess, T., Landwehr, R., Lustig, A., and Engel, J. (1999) Biochemistry 38, 13263–13269[CrossRef][Medline] [Order article via Infotrieve]
  7. Calver, A. R., Robbins, M. J., Cosio, C., Rice, S. Q., Babbs, A. J., Hirst, W. D., Boyfield, I., Wood, M. D., Russell, R. B., Price, G. W., Couve, A., Moss, S. J., and Pangalos, M. N. (2001) J. Neurosci. 21, 1203–1210[Abstract/Free Full Text]
  8. Duthey, B., Caudron, S., Perroy, J., Bettler, B., Fagni, L., Pin, J. P., and Prezeau, L. (2002) J. Biol. Chem. 277, 3236–3241[Abstract/Free Full Text]
  9. Uezono, Y., Kanaide, M., Kaibara, M., Barzilai, R., Dascal, N., Sumikawa, K., and Taniyama, K. (2006) Am. J. Physiol. 290, C200–C207
  10. Margeta-Mitrovic, M., Jan, Y. N., and Jan, L. Y. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 14649–14654[Abstract/Free Full Text]
  11. Borgdorff, A. J., and Choquet, D. (2002) Nature 417, 649–653[CrossRef][Medline] [Order article via Infotrieve]
  12. Andrasfalvy, B. K., and Magee, J. C. (2004) J. Physiol. 559, Pt. 2, 543–554[Abstract/Free Full Text]
  13. Ashby, M. C., Maier, S. R., Nishimune, A., and Henley, J. M. (2006) J. Neurosci. 26, 7046–7055[Abstract/Free Full Text]
  14. Yang, G., Huang, A., Zhu, S., and Xiong, W. (2006) J. Neurosci. 26, 9082–9083[Free Full Text]
  15. Tovar, K. R., and Westbrook, G. L. (2002) Neuron 34, 255–264[CrossRef][Medline] [Order article via Infotrieve]
  16. 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]
  17. Meier, J., Vannier, C., Serge, A., Triller, A., and Choquet, D. (2001) Nat. Neurosci. 4, 253–260[CrossRef][Medline] [Order article via Infotrieve]
  18. Chan, W. Y., Soloviev, M. M., Ciruela, F., and McIlhinney, R. A. J. (2001) Mol. Cell. Neurosci. 17, 577–588[CrossRef][Medline] [Order article via Infotrieve]
  19. Axelrod, D., Koppel, D. E., Schlessinger, J., Elson, E., and Webb, W. W. (1976) Biophys. J. 16, 1055–1069[Medline] [Order article via Infotrieve]
  20. Phair, R. D., Gorski, S. A., and Misteli, T. (2004) Methods Enzymol. 375, 393–414[Medline] [Order article via Infotrieve]
  21. Coleman, S. K., Cai, C., Mottershead, D. G., Haapalahti, J. P., and Keinanen, K. (2003) J. Neurosci. 23, 798–806[Abstract/Free Full Text]
  22. Shen, L., Liang, F., Walensky, L. D., and Huganir, R. L. (2000) J. Neurosci. 20, 7932–7940[Abstract/Free Full Text]
  23. Zhou, Q., Xiao, M., and Nicoll, R. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 1261–1266[Abstract/Free Full Text]
  24. Pagano, A., Rovelli, G., Mosbacher, J., Lohmann, T., Duthey, B., Stauffer, D., Ristig, D., Schuler, V., Meigel, I., Lampert, C., Stein, T., Prezeau, L., Blahos, J., Pin, J., Froestl, W., Kuhn, R., Heid, J., Kaupmann, K., and Bettler, B. (2001) J. Neurosci. 21, 1189–1202[Abstract/Free Full Text]
  25. Grunewald, S., Schupp, B. J., Ikeda, S. R., Kuner, R., Steigerwald, F., Kornau, H. C., and Kohr, G. (2002) Mol. Pharmacol. 61, 1070–1080[Abstract/Free Full Text]
  26. Fairfax, B. P., Pitcher, J. A., Scott, M. G., Calver, A. R., Pangalos, M. N., Moss, S. J., and Couve, A. (2004) J. Biol. Chem. 279, 12565–12573[Abstract/Free Full Text]
  27. Sharma, K., Fong, D. K., and Craig, A. M. (2006) Mol. Cell. Neurosci. 31, 702–712[CrossRef][Medline] [Order article via Infotrieve]
  28. Charrier, C., Ehrensperger, M. V., Dahan, M., Levi, S., and Triller, A. (2006) J. Neurosci. 26, 8502–8511[Abstract/Free Full Text]
  29. Pucadyil, T. J., and Chattopadhyay, A. (2007) Glycoconj. J. 24, 25–31[CrossRef][Medline] [Order article via Infotrieve]
  30. Scott, L., Zelenin, S., Malmersjo, S., Kowalewski, J. M., Markus, E. Z., Nairn, A. C., Greengard, P., Brismar, H., and Aperia, A. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 762–767[Abstract/Free Full Text]
  31. Allison, D. W., Gelfand, V. I., Spector, I., and Craig, A. M. (1998) J. Neurosci. 18, 2423–2436[Abstract/Free Full Text]
  32. Peran, M., Hooper, H., Boulaiz, H., Marchal, J. A., Aranega, A., and Salas, R. (2006) Cell. Motil. Cytoskeleton 63, 747–757[CrossRef][Medline] [Order article via Infotrieve]
  33. Balasubramanian, S., Fam, S. R., and Hall, R. A. (2007) J. Biol. Chem. 282, 4162–4171[Abstract/Free Full Text]
  34. Pucadyil, T. J., Kalipatnapu, S., Harikumar, K. G., Rangaraj, N., Karnik, S. S., and Chattopadhyay, A. (2004) Biochemistry 43, 15852–15862[CrossRef][Medline] [Order article via Infotrieve]
  35. Dahan, M., Levi, S., Luccardini, C., Rostaing, P., Riveau, B., and Triller, A. (2003) Science 302, 442–445[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. Biol. Chem.Home page
M. E. Wilkins, X. Li, and T. G. Smart
Tracking Cell Surface GABAB Receptors Using an {alpha}-Bungarotoxin Tag
J. Biol. Chem., December 12, 2008; 283(50): 34745 - 34752.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/35/25349    most recent
M702358200v1
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 Pooler, A. M.
Right arrow Articles by McIlhinney, R. A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pooler, A. M.
Right arrow Articles by McIlhinney, R. A. 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 © 2007 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement