Lateral Diffusion of the GABAB Receptor Is Regulated by the GABAB2 C Terminus
2007; Elsevier BV; Volume: 282; Issue: 35 Linguagem: Inglês
10.1074/jbc.m702358200
ISSN1083-351X
AutoresAmy M. Pooler, R. A. Jeffrey McIlhinney,
Tópico(s)Nicotinic Acetylcholine Receptors Study
ResumoGABAB (γ-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. GABAB (γ-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. GABAB receptors are metabotropic receptors for the inhibitory neurotransmitter γ aminobutyric acid (GABA). 2The abbreviations used are:GABAγ-aminobutyric acidFRAPfluorescence recovery after photobleachingYFPyellow fluorescent proteinCFPcyan fluorescent proteinHAhemagglutininPBSphosphate-buffered salineAMPAα-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid. Pre- and post-synaptic GABAB receptors are coupled to inhibitory G-proteins and can regulate neurotransmission via several mechanisms, including modulation of adenylyl cyclase (1Knight A.R. Bowery N.G. Neuropharmacology. 1996; 35: 703-712Crossref PubMed Scopus (75) Google Scholar), inhibition of voltage-gated Ca2+ channels (2Poncer J.C. McKinney R.A. Gahwiler B.H. Thompson S.M. Neuron. 1997; 18: 463-472Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), and modulation of K+ channels (3Sodickson D.L. Bean B.P. J. Neurosci. 1996; 16: 6374-6385Crossref PubMed Google Scholar, 4Ng 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. Evans Jr., J.F. Bonner T.I. O'Neill G.P. J. Biol. Chem. 1999; 274: 7607-7610Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Formation of a functional receptor requires the heterodimerization of two subunits, GABAB1 and GABAB2 (5White J.H. Wise A. Main M.J. Green A. Fraser N.J. Disney G.H. Barnes A.A. Emson P. Foord S.M. Marshall F.H. Nature. 1998; 396: 679-682Crossref PubMed Scopus (1015) Google Scholar). 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 (6Kammerer R.A. Frank S. Schulthess T. Landwehr R. Lustig A. Engel J. Biochemistry. 1999; 38: 13263-13269Crossref PubMed Scopus (84) Google Scholar). 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 (7Calver 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. Pangalos M.N. J. Neurosci. 2001; 21: 1203-1210Crossref PubMed Google Scholar). The GABAB2 subunit contains intracellular loops that couple the receptor to the G-protein (8Duthey B. Caudron S. Perroy J. Bettler B. Fagni L. Pin J.P. Prezeau L. J. Biol. Chem. 2002; 277: 3236-3241Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 9Uezono Y. Kanaide M. Kaibara M. Barzilai R. Dascal N. Sumikawa K. Taniyama K. Am. J. Physiol. 2006; 290: C200-C207Crossref PubMed Scopus (17) Google Scholar, 10Margeta-Mitrovic M. Jan Y.N. Jan L.Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14649-14654Crossref PubMed Scopus (156) Google Scholar). γ-aminobutyric acid fluorescence recovery after photobleaching yellow fluorescent protein cyan fluorescent protein hemagglutinin phosphate-buffered saline α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid. 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 (5White J.H. Wise A. Main M.J. Green A. Fraser N.J. Disney G.H. Barnes A.A. Emson P. Foord S.M. Marshall F.H. Nature. 1998; 396: 679-682Crossref PubMed Scopus (1015) Google Scholar, 7Calver 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. Pangalos M.N. J. Neurosci. 2001; 21: 1203-1210Crossref PubMed Google Scholar). 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 (11Borgdorff A.J. Choquet D. Nature. 2002; 417: 649-653Crossref PubMed Scopus (421) Google Scholar). 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 α-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 (12Andrasfalvy B.K. Magee J.C. J. Physiol. Pt. 2. 2004; 559: 543-554Crossref PubMed Scopus (61) Google Scholar, 13Ashby M.C. Maier S.R. Nishimune A. Henley J.M. J. Neurosci. 2006; 26: 7046-7055Crossref PubMed Scopus (240) Google Scholar, 14Yang G. Huang A. Zhu S. Xiong W. J. Neurosci. 2006; 26: 9082-9083Crossref PubMed Scopus (5) Google Scholar). Furthermore, N-methyl-d-aspartate receptors readily exchange between synaptic and extrasynaptic compartments via movement within the plasma membrane (15Tovar K.R. Westbrook G.L. Neuron. 2002; 34: 255-264Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). However, the behavior of inhibitory neurotransmitter receptors is less well understood. The dynamics of the glycine receptor (16Jacob T.C. Bogdanov Y.D. Magnus C. Saliba R.S. Kittler J.T. Haydon P.G. Moss S.J. J. Neurosci. 2005; 25: 10469-10478Crossref PubMed Scopus (210) Google Scholar) and the ionotropic GABAA receptor (17Meier J. Vannier C. Serge A. Triller A. Choquet D. Nat. Neurosci. 2001; 4: 253-260Crossref PubMed Scopus (225) Google Scholar) 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. 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: GABAB2Δ920, 5′-GCT AAG CTT GAC GCA GGG GCT GAC ACA GCT GGC-3′; GABAB2Δ886, 5′-GCT AAG CTT TGG GAG AGT TTA TAT CTT CTA TAC G-3′; GABAB2 Δ862, 5′-TGT GTT CCA CTG AAG CTT GGG ATT TTG ATC GAG-3′; GABAB2Δ841, 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 (18Chan W.Y. Soloviev M.M. Ciruela F. McIlhinney R.A.J. Mol. Cell. Neurosci. 2001; 17: 577-588Crossref PubMed Scopus (52) Google Scholar) 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. FRAP—We subjected transfected COS-7 cells or hippocampal neurons to FRAP analysis to assess the lateral diffusion of expressed proteins (19Axelrod D. Koppel D.E. Schlessinger J. Elson E. Webb W.W. Biophys. J. 1976; 16: 1055-1069Abstract Full Text PDF PubMed Scopus (2032) Google Scholar). Cells expressing YFP-tagged proteins were maintained at 37 °C and imaged on a Zeiss LSM510 inverted confocal microscope with a ×40 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 (20Phair R.D. Gorski S.A. Misteli T. Methods Enzymol. 2004; 375: 393-414Crossref PubMed Scopus (263) Google Scholar). The mobile fraction and time to half-maximal recovery (t½ 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-β-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 ×40 oil objective. 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 (21Coleman S.K. Cai C. Mottershead D.G. Haapalahti J.P. Keinanen K. J. Neurosci. 2003; 23: 798-806Crossref PubMed Google Scholar, 22Shen L. Liang F. Walensky L.D. Huganir R.L. J. Neurosci. 2000; 20: 7932-7940Crossref PubMed Google Scholar, 23Zhou Q. Xiao M. Nicoll R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1261-1266Crossref PubMed Scopus (200) Google Scholar), whereas in the inhibitory synapse GABAA interacts with the anchoring protein gephyrin (16Jacob T.C. Bogdanov Y.D. Magnus C. Saliba R.S. Kittler J.T. Haydon P.G. Moss S.J. J. Neurosci. 2005; 25: 10469-10478Crossref PubMed Scopus (210) Google Scholar, 17Meier J. Vannier C. Serge A. Triller A. Choquet D. Nat. Neurosci. 2001; 4: 253-260Crossref PubMed Scopus (225) Google Scholar). 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. 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 (24Pagano 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. Bettler B. J. Neurosci. 2001; 21: 1189-1202Crossref PubMed Google Scholar) and stabilization (25Grunewald S. Schupp B.J. Ikeda S.R. Kuner R. Steigerwald F. Kornau H.C. Kohr G. Mol. Pharmacol. 2002; 61: 1070-1080Crossref PubMed Scopus (44) Google Scholar, 26Fairfax B.P. Pitcher J.A. Scott M.G. Calver A.R. Pangalos M.N. Moss S.J. Couve A. J. Biol. Chem. 2004; 279: 12565-12573Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) 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 (t½ 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 t½ 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, Δ920-YFP, Δ886-YFP, Δ862-YFP, or Δ841-YFP. Although plasma membrane diffusion rates of Δ920-YFP and Δ886-YFP were similar to the diffusion of GABAB2-YFP, both Δ862-YFP and Δ841-YFP exhibited faster movement as measured by FRAP analysis than GABAB2-YFP (Fig. 5B). The mobile fraction of Δ862-YFP was significantly (p < 0.01) higher compared with the mobile fractions of either GABAB2-YFP or Δ886-YFP (67.91 ± 7.74 versus 43.24% ± 3.21 or 43.15% ± 4.39) (Fig. 5C). Correspondingly, the t½ maximal recovery of Δ862-YFP was also significantly reduced relative to recovery of either GABAB2-YFP or Δ886-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 Δ841, compared with co-expression with GABAB2, significantly increased its mobile fraction (71.62 ± 3.23 versus 48.83% ± 2.72) and decreased its t½ 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. 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. 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 (27Sharma K. Fong D.K. Craig A.M. Mol. Cell. Neurosci. 2006; 31: 702-712Crossref PubMed Scopus (126) Google Scholar) and in extrasynaptic regions of neurons (13Ashby M.C. Maier S.R. Nishimune A. Henley J.M. J. Neurosci. 2006; 26: 7046-7055Crossref PubMed Scopus (240) Google Scholar). Similarly, the GABAA receptor has been shown to diffuse rapidly within the extrasynaptic membrane (28Charrier C. Ehrensperger M.V. Dahan M. Levi S. Triller A. J. Neurosci. 2006; 26: 8502-8511Crossref PubMed Scopus (114) Google Scholar). Rapid diffusion is not restricted to receptor ion channels, as the G-protein-coupled serotonin 5-HT1a (29Pucadyil T.J. Chattopadhyay A. Glycoconj. J. 2007; 24: 25-31Crossref PubMed Scopus (15) Google Scholar) and dopamine D1 (30Scott L. Zelenin S. Malmersjo S. Kowalewski J.M. Markus E.Z. Nairn A.C. Greengard P. Brismar H. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 762-767Crossref PubMed Scopus (99) Google Scholar) 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 (28Charrier C. Ehrensperger M.V. Dahan M. Levi S. Triller A. J. Neurosci. 2006; 26: 8502-8511Crossref PubMed Scopus (114) Google Scholar) 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 (21Coleman S.K. Cai C. Mottershead D.G. Haapalahti J.P. Keinanen K. J. Neurosci. 2003; 23: 798-806Crossref PubMed Google Scholar, 22Shen L. Liang F. Walensky L.D. Huganir R.L. J. Neurosci. 2000; 20: 7932-7940Crossref PubMed Google Scholar, 23Zhou Q. Xiao M. Nicoll R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1261-1266Crossref PubMed Scopus (200) Google Scholar). 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 (31Allison D.W. Gelfand V.I. Spector I. Craig A.M. J. Neurosci. 1998; 18: 2423-2436Crossref PubMed Google Scholar) and diffusion of the dopamine D1 receptor is unaffected by microtubule disruption (30Scott L. Zelenin S. Malmersjo S. Kowalewski J.M. Markus E.Z. Nairn A.C. Greengard P. Brismar H. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 762-767Crossref PubMed Scopus (99) Google Scholar). 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 (16Jacob T.C. Bogdanov Y.D. Magnus C. Saliba R.S. Kittler J.T. Haydon P.G. Moss S.J. J. Neurosci. 2005; 25: 10469-10478Crossref PubMed Scopus (210) Google Scholar) and the D1 receptor is anchored via a direct interaction with the N-methyl-d-aspartate receptor (30Scott L. Zelenin S. Malmersjo S. Kowalewski J.M. Markus E.Z. Nairn A.C. Greengard P. Brismar H. Aperia A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 762-767Crossref PubMed Scopus (99) Google Scholar). These findings suggested that GABAB2 might possess an intracellular region capable of similar protein interactions. 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 (32Peran M. Hooper H. Boulaiz H. Marchal J.A. Aranega A. Salas R. Cell. Motil. Cytoskeleton. 2006; 63: 747-757Crossref PubMed Scopus (8) Google Scholar). Previous studies have demonstrated that the C terminus of the GABAB2 subunit contains several important regions, such as those involved in dimerization with GABAB1 (6Kammerer R.A. Frank S. Schulthess T. Landwehr R. Lustig A. Engel J. Biochemistry. 1999; 38: 13263-13269Crossref PubMed Scopus (84) Google Scholar), G-protein binding (8Duthey B. Caudron S. Perroy J. Bettler B. Fagni L. Pin J.P. Prezeau L. J. Biol. Chem. 2002; 277: 3236-3241Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 9Uezono Y. Kanaide M. Kaibara M. Barzilai R. Dascal N. Sumikawa K. Taniyama K. Am. J. Physiol. 2006; 290: C200-C207Crossref PubMed Scopus (17) Google Scholar, 10Margeta-Mitrovic M. Jan Y.N. Jan L.Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14649-14654Crossref PubMed Scopus (156) Google Scholar), and mediating interactions with scaffolding proteins (33Balasubramanian S. Fam S.R. Hall R.A. J. Biol. Chem. 2007; 282: 4162-4171Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). 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 (33Balasubramanian S. Fam S.R. Hall R.A. J. Biol. Chem. 2007; 282: 4162-4171Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar); 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 (29Pucadyil T.J. Chattopadhyay A. Glycoconj. J. 2007; 24: 25-31Crossref PubMed Scopus (15) Google Scholar, 34Pucadyil T.J. Kalipatnapu S. Harikumar K.G. Rangaraj N. Karnik S.S. Chattopadhyay A. Biochemistry. 2004; 43: 15852-15862Crossref PubMed Scopus (66) Google Scholar). 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 (13Ashby M.C. Maier S.R. Nishimune A. Henley J.M. J. Neurosci. 2006; 26: 7046-7055Crossref PubMed Scopus (240) Google Scholar, 27Sharma K. Fong D.K. Craig A.M. Mol. Cell. Neurosci. 2006; 31: 702-712Crossref PubMed Scopus (126) Google Scholar, 28Charrier C. Ehrensperger M.V. Dahan M. Levi S. Triller A. J. Neurosci. 2006; 26: 8502-8511Crossref PubMed Scopus (114) Google Scholar, 35Dahan M. Levi S. Luccardini C. Rostaing P. Riveau B. Triller A. Science. 2003; 302: 442-445Crossref PubMed Scopus (1348) Google Scholar). 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. 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. Download .pdf (.51 MB) Help with pdf files
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