The Availability of Surface GABAB Receptors Is Independent of γ-Aminobutyric Acid but Controlled by Glutamate in Central Neurons

Abstract

The efficacy of synaptic transmission depends on the availability of ionotropic and metabotropic neurotransmitter receptors at the plasma membrane, but the contribution of the endocytic and recycling pathways in the regulation of γ-aminobutyric acid type B (GABAB) receptors remains controversial. To understand the mechanisms that regulate the abundance of GABAB receptors, we have studied their turnover combining surface biotin labeling and a microscopic immunoendocytosis assay in hippocampal and cortical neurons. We report that internalization of GABAB receptors is agonist-independent. We also demonstrate that receptors endocytose in the cell body and dendrites but not in axons. Additionally, we show that GABAB receptors endocytose as heterodimers via clathrin- and dynamin-1-dependent mechanisms and that they recycle to the plasma membrane after endocytosis. More importantly, we show that glutamate decreases the levels of cell surface receptors in a manner dependent on an intact proteasome pathway. These observations indicate that glutamate and not GABA controls the abundance of surface GABAB receptors in central neurons, consistent with their enrichment at glutamatergic synapses. The efficacy of synaptic transmission depends on the availability of ionotropic and metabotropic neurotransmitter receptors at the plasma membrane, but the contribution of the endocytic and recycling pathways in the regulation of γ-aminobutyric acid type B (GABAB) receptors remains controversial. To understand the mechanisms that regulate the abundance of GABAB receptors, we have studied their turnover combining surface biotin labeling and a microscopic immunoendocytosis assay in hippocampal and cortical neurons. We report that internalization of GABAB receptors is agonist-independent. We also demonstrate that receptors endocytose in the cell body and dendrites but not in axons. Additionally, we show that GABAB receptors endocytose as heterodimers via clathrin- and dynamin-1-dependent mechanisms and that they recycle to the plasma membrane after endocytosis. More importantly, we show that glutamate decreases the levels of cell surface receptors in a manner dependent on an intact proteasome pathway. These observations indicate that glutamate and not GABA controls the abundance of surface GABAB receptors in central neurons, consistent with their enrichment at glutamatergic synapses. The efficacy of synaptic transmission depends on the mechanisms of intracellular trafficking, which modify the availability of neurotransmitter receptors (1Collingridge G.L. Isaac J.T. Wang Y.T. Nat. Rev. Neurosci. 2004; 5: 952-962Crossref PubMed Scopus (820) Google Scholar). At the molecular level, the trafficking of glutamate receptors and ionotropic GABA 5The abbreviations used are:GABAγ-aminobutyric acidGABABγ-aminobutyric acid, type BGABABRGABAB receptorFITCfluorescein isothiocyanateHAhemagglutininCy5cyanineTRTexas Reddivdays in vitro. 5The abbreviations used are:GABAγ-aminobutyric acidGABABγ-aminobutyric acid, type BGABABRGABAB receptorFITCfluorescein isothiocyanateHAhemagglutininCy5cyanineTRTexas Reddivdays in vitro. receptors has been extensively studied (2Inoue A. Okabe S. Curr. Opin. Neurobiol. 2003; 13: 332-340Crossref PubMed Scopus (49) Google Scholar, 3Moss S.J. Smart T.G. Nat. Rev. Neurosci. 2001; 2: 240-250Crossref PubMed Scopus (399) Google Scholar). For example, differential trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors modifies synaptic strength and influences experience-dependent plasticity in vivo (4Clem R.L. Barth A. Neuron. 2006; 49: 663-670Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). However, the molecular mechanisms that govern the trafficking of metabotropic GABAB receptors (GABABRs) remain less clear. γ-aminobutyric acid γ-aminobutyric acid, type B GABAB receptor fluorescein isothiocyanate hemagglutinin cyanine Texas Red days in vitro. γ-aminobutyric acid γ-aminobutyric acid, type B GABAB receptor fluorescein isothiocyanate hemagglutinin cyanine Texas Red days in vitro. GABABRs mediate the slow component of synaptic inhibition by acting on pre- and postsynaptic targets (5Couve A. Calver A.R. Fairfax B. Moss S.J. Pangalos M.N. Biochem. Pharmacol. 2004; 68: 1527-1536Crossref PubMed Scopus (41) Google Scholar, 6Sakaba T. Neher E. Nature. 2003; 424: 775-778Crossref PubMed Scopus (196) Google Scholar). They have been implicated in epilepsy, anxiety, stress, sleep disorders, nociception, depression, and cognition (7Bettler B. Kaupmann K. Mosbacher J. Gassmann M. Physiol. Rev. 2004; 84: 835-867Crossref PubMed Scopus (683) Google Scholar). They also represent attractive targets for the treatment of withdrawal symptoms from drugs of addiction such as cocaine (8Cousins M.S. Roberts D.C. de Wit H. Drug Alcohol Depend. 2002; 65: 209-220Crossref PubMed Scopus (228) Google Scholar). GABABRs are heteromers composed of two subunits, namely GABABR1 and GABABR2. GABABR1 contains an endoplasmic reticulum retention motif in the intracellular C-terminal domain (9Couve A. Filippov A.K. Connolly C.N. Bettler B. Brown D.A. Moss S.J. J. Biol. Chem. 1998; 273: 26361-26367Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 10Margeta-Mitrovic M. Jan Y.N. Jan L.Y. Neuron. 2000; 27: 97-106Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar). The endoplasmic reticulum retention sequence is masked upon assembly with GABABR2, which results in the appearance of the functional receptor at the plasma membrane. GABABR1 contains the ligand-binding site, whereas G protein signaling is exclusively mediated by GABABR2 (11Pin J.P. Kniazeff J. Binet V. Liu J. Maurel D. Galvez T. Duthey B. Havlickova M. Blahos J. Prezeau L. Rondard P. Biochem. Pharmacol. 2004; 68: 1565-1572Crossref PubMed Scopus (124) Google Scholar). GABABRs are located in dendrites and axons, but it has been difficult to establish what determines their pre- versus postsynaptic localization. Recent evidence suggests that the extracellular domains of the GABABR1a and -1b isoforms, which differ in two sushi domains (12Hawrot E. Xiao Y. Shi Q.L. Norman D. Kirkitadze M. Barlow P.N. FEBS Lett. 1998; 432: 103-108Crossref PubMed Scopus (67) Google Scholar), may specify axonal/dendritic targeting (13Vigot R. Barbieri S. Brauner-Osborne H. Turecek R. Shigemoto R. Zhang Y.P. Lujan R. Jacobson L.H. Biermann B. Fritschy J.M. Vacher C.M. Muller M. Sansig G. Guetg N. Cryan J.F. Kaupmann K. Gassmann M. Oertner T.G. Bettler B. Neuron. 2006; 50: 589-601Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Unexpectedly, postsynaptic GABABRs are enriched at glutamatergic synapses, frequently adjacent to the postsynaptic density in the CA1 region of the hippocampus and Purkinje cells of the cerebellum, thus constituting perisynaptic receptors (14Kulik A. Nakadate K. Nyíri G. Notomi T. Malitschek B. Bettler B. Shigemoto R. Eur. J. Neurosci. 2002; 15: 291-307Crossref PubMed Scopus (142) Google Scholar, 15Kulik A. Vida I. Lujan R. Haas C.A. Lopez-Bendito G. Shigemoto R. Frotscher M. J. Neurosci. 2003; 23: 11026-11035Crossref PubMed Google Scholar, 16Luján R. Shigemoto R. Eur. J. Neurosci. 2006; 23: 1479-1490Crossref PubMed Scopus (74) Google Scholar). They are also found adjacent to presynaptic release sites of glutamatergic and gabaergic synapses (15Kulik A. Vida I. Lujan R. Haas C.A. Lopez-Bendito G. Shigemoto R. Frotscher M. J. Neurosci. 2003; 23: 11026-11035Crossref PubMed Google Scholar). The existence of a classical endocytic pathway for GABABRs has been the cause of a recent controversy. Whereas most reports agree that receptors do not internalize in response to agonist (17Couve A. Thomas P. Calver A.R. Hirst W.D. Pangalos M.N. Walsh F.S. Smart T.G. Moss S.J. Nat. Neurosci. 2002; 5: 415-424Crossref PubMed Scopus (106) Google Scholar, 18Perroy J. Adam L. Qanbar R. Chénier S. Bouvier M. EMBO J. 2003; 22: 3816-3824Crossref PubMed Scopus (105) Google Scholar, 19Fairfax 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, 20Ramoino P. Gallus L. Beltrame F. Diaspro A. Fato M. Rubini P. Stigliani S. Bonanno G. Usai C. J. Cell Sci. 2006; 119: 2056-2064Crossref PubMed Scopus (17) Google Scholar, 21Grampp T. Sauter K. Markovic B. Benke D. J. Biol. Chem. 2007; 282: 24157-24165Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), at least two studies suggest the existence of agonist-induced internalization (22Gonzalez-Maeso J. Wise A. Green A. Koenig J.A. Eur. J. Pharmacol. 2003; 481: 15-23Crossref PubMed Scopus (24) Google Scholar, 23Laffray S. Tan K. Dulluc J. Bouali-Benazzouz R. Calver A.R. Nagy F. Landry M. Eur. J. Neurosci. 2007; 25: 1402-1416Crossref PubMed Scopus (29) Google Scholar). Likewise, although several studies argue that constitutive receptor endocytosis occurs via clathrin-dependent mechanisms (20Ramoino P. Gallus L. Beltrame F. Diaspro A. Fato M. Rubini P. Stigliani S. Bonanno G. Usai C. J. Cell Sci. 2006; 119: 2056-2064Crossref PubMed Scopus (17) Google Scholar, 21Grampp T. Sauter K. Markovic B. Benke D. J. Biol. Chem. 2007; 282: 24157-24165Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 22Gonzalez-Maeso J. Wise A. Green A. Koenig J.A. Eur. J. Pharmacol. 2003; 481: 15-23Crossref PubMed Scopus (24) Google Scholar, 23Laffray S. Tan K. Dulluc J. Bouali-Benazzouz R. Calver A.R. Nagy F. Landry M. Eur. J. Neurosci. 2007; 25: 1402-1416Crossref PubMed Scopus (29) Google Scholar), others suggest the existence a nonclassical mechanism (18Perroy J. Adam L. Qanbar R. Chénier S. Bouvier M. EMBO J. 2003; 22: 3816-3824Crossref PubMed Scopus (105) Google Scholar, 19Fairfax 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). These discrepancies are likely to originate from the different experimental systems utilized, the different methodology, and the varying times of observation. Here we characterize the turnover of GABABRs in cultured cortical and hippocampal neurons combining surface biotin labeling and a microscopic immunoendocytosis assay. We report that internalization of GABABRs is agonist-independent and that the mechanism depends on clathrin and dynamin-1. We also demonstrate that receptors endocytose as heterodimers in the cell body and dendrites but not in axons, and that they recycle to the plasma membrane. More importantly, we show that membrane availability is regulated by glutamate in a proteasome-dependent manner. These results support the notion that GABABR function is intimately linked to glutamatergic neurotransmission in the brain (15Kulik A. Vida I. Lujan R. Haas C.A. Lopez-Bendito G. Shigemoto R. Frotscher M. J. Neurosci. 2003; 23: 11026-11035Crossref PubMed Google Scholar). Reagents—Glutamate, baclofen, GABA, saclofen, and MG132 were purchased from Sigma. Plasmids—The constructs containing MYC-GABABR1, HA-GABABR2, and FLAG-GABABR2 in pRK5 have been described previously and contain their epitope tags on the extracellular N-terminal domains (9Couve A. Filippov A.K. Connolly C.N. Bettler B. Brown D.A. Moss S.J. J. Biol. Chem. 1998; 273: 26361-26367Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 17Couve A. Thomas P. Calver A.R. Hirst W.D. Pangalos M.N. Walsh F.S. Smart T.G. Moss S.J. Nat. Neurosci. 2002; 5: 415-424Crossref PubMed Scopus (106) Google Scholar). EGFP-wt-dynamin-1, EGFP-K44A-dynamin-1, EGFP-wt-dynamin-2, and EGFP-K44A-dynamin-2 were kindly provided by M. McNiven. EGFP-wt-Rab11 was kindly provided by F. Bronfman. Antibodies—GABABR1 and GABABR2 antibodies have been described previously (24Kuramoto N. Wilkins M.E. Fairfax B.P. Revilla-Sanchez R. Terunuma M. Tamaki K. Iemata M. Warren N. Couve A. Calver A. Horvath Z. Freeman K. Carling D. Huang L. Gonzales C. Cooper E. Smart T.G. Pangalos M.N. Moss S.J. Neuron. 2007; 53: 233-247Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Anti-MYC antibodies were purchased from Sigma. HA antibodies were purchased from Roche Applied Science. The secondary anti-mouse and anti-rabbit antibodies conjugated to Texas Red (TR), fluorescein isothiocyanate (FITC), cyanine (Cy5), and horseradish peroxidase were purchased from Jackson ImmunoResearch (West Grove, PA). Animals—Adult pregnant female Sprague-Dawley rats were purchased from the Central Animal Facility at Universidad Católica de Chile and killed by asphyxia in a CO2 chamber according to the Guide for Care and Use of Laboratory Animals. Neuronal Cultures and Transfection—Primary cultures of cortical and hippocampal neurons were obtained from E18 rats and transfected by calcium phosphate as reported earlier (25Goslin K. Banker G. Banker G. Goslin K. Culturing Nerve Cells. MIT Press, Cambridge, MA1991: 251-281Google Scholar, 26Jiang M. Chen G. Nat. Protoc. 2006; 1: 695-700Crossref PubMed Scopus (229) Google Scholar). Biotinylation—Labeling of surface proteins for steady-state and internalization assays were performed as reported previously in 5 div cortical neurons (19Fairfax 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). Antibody Feeding—14–21 div rat hippocampal neurons grown on poly-l-lysine-coated coverslips were transfected with MYC-GABABR1a and HA-GABABR2. Live cells were washed twice with phosphate-buffered saline and incubated for 15 min in media containing MYC and/or HA antibodies. Neurons were left untreated or stimulated with 100 μm baclofen or 100 μm GABA for 60 min. Control neurons were kept at 4 °C. Treated cells were returned to the 37 °C incubator for 60 min. Cells were then fixed and processed for immunofluorescence. The surface GABABR1 and/or GABABR2 pools were detected with MYC, HA, and secondary antibodies prior to permeabilization. The internalized receptor pool was detected using secondary antibodies after permeabilization. For experiments using low K+, cells were incubated in hypotonic medium (Dulbecco's modified Eagle's medium diluted 1:1 with water) for 5 min at 37 °C, followed by isotonic KCl-free medium for 30 min at 37 °C prior to immunoendocytosis. Control treatments were carried out in isotonic KCl medium. Images were acquired with a Zeiss LSM-5 Pascal 5 Axiovert 200 confocal microscope and a Plan-Apochromat 63x/1.4 Oil differential interference contrast objective, using the LSM 5 3.2 image capture and analysis software. Raw confocal images were deconvolved by Huygens Scripting software (Scientific Volume Imaging, Hilversum, Netherlands). Quantification of internalization was performed using the MetaMorph software. The amount of internalized receptor was expressed as the percent of the internalized intensity (internalized intensity of one channel excluding the surface intensity of both channels) relative to the total intensity (internalized intensity of one channel plus the surface intensity of both channels). GABABRs Endocytose in Neurons—We used microscopy and biochemistry to determine whether GABABR endocytosis occurs in cultured hippocampal and cortical neurons, two model systems widely used to study neurotransmitter receptor trafficking (27Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (897) Google Scholar), and which express abundant GABABRs (19Fairfax 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, 28Correa S.A. Munton R. Nishimune A. Fitzjohn S. Henley J.M. Neuropharmacology. 2004; 47: 475-484Crossref PubMed Scopus (20) Google Scholar). GABABRs were transfected into hippocampal neurons, and the fate of surface receptors was subsequently followed by immunoendocytosis. Abundant GABABR1 concentrated at the plasma membrane after transfection, and a significant proportion accumulated in intracellular compartments after 60 min of endocytosis (Fig. 1, A and B, arrows). The accumulation occurred in vesicle-like structures that located throughout the cell body and often converged to a perinuclear compartment. Stimulation with baclofen, a GABAB agonist, or GABA for 60 min failed to increase the intracellular buildup of GABABR1 (Fig. 1, C and D, arrows, and E). To corroborate these observations, we carried out experiments of surface biotinylation and internalization for endogenous GABABRs in cultured cortical neurons. As expected GABABR1 was efficiently detected at the cell surface, and a significant fraction accumulated in intracellular stores under basal conditions (Fig. 1F, lanes 1–3). However, the intracellular accumulation of GABABR1 was not stimulated by baclofen or GABA, in agreement with our microscopic observations (Fig. 1F, lanes 4 and 5). A quantitative analysis of multiple experiments confirmed these observations (Fig. 1G). Saclofen, a competitive antagonist for GABABRs, had no effect on the levels of internalized receptors (Fig. 1H). Consistent with these results the steady-state abundance of GABABR1 and GABABR2 at the cell surface was not significantly modified by baclofen or GABA (supplemental Fig. 1, A and B). Taken together these results indicate that a proportion of GABAB receptors undergo agonist-independent endocytosis in neurons. GABABRs Endocytose in the Cell Body and Dendrites but Not in Axons—To determine whether GABABR endocytosis differs in pre- and postsynaptic compartments, we carried out the same immunoendocytosis analysis in dendrites and axons. GABABR1 was expressed abundantly at the cell surface of dendrites (Fig. 2A). More importantly, under basal conditions a significant proportion of GABABR1 endocytosed constitutively in vesicle-like structures that accumulated along the dendritic shaft (Fig. 2, A and B, arrows). GABABR1 was also abundant at the surface of the axon, but no intracellular accumulation was visible throughout the length of the axonal shaft, as indicated by the absence of exclusively red vesicle-like structures (Fig. 2, D and E, arrows). This was not because of the resolution limit of our technique as dendrites of small diameter also showed clear punctate accumulation of endocytosed GABABR1 (Fig. 2C, arrow). GABABRs Endocytose as Dimers—GABABRs are heteromeric at the plasma membrane (29Bettler B. Tiao J.Y. Pharmacol. Ther. 2006; 110: 533-543Crossref PubMed Scopus (133) Google Scholar), but the fate of the heteromer during endocytosis has received little attention. Thus, to determine whether GABABR1 and GABABR2 subunits are endocytosed as monomers or heteromers, we simultaneously labeled GABABR1 and GABABR2 prior to internalization. Abundant GABABR1 accumulated at the cell surface indicating that heteromeric receptors effectively reached the plasma membrane (Fig. 3A, left panel). Both GABABR1 and GABABR2 endocytosed efficiently after double labeling (Fig. 3A, central panels). Interestingly, the great majority of endocytosed GABABR1 and GABABR2 subunits co-localized in round vesicle-like structures in the cell body (Fig. 3A, right panel, arrow). Identical observations were obtained in dendrites (Fig. 3B, arrow). As predicted, when GABABR1 and GABABR2 were detected at the somatic or dendritic cell surface, they showed a high degree of co-localization (Fig. 3C, arrows). These results clearly indicate that the dimeric structure of functional GABABRs is maintained during endocytosis. GABABR Endocytosis Is Clathrin- and Dynamin-dependent—Next we determined whether endocytosis occurred via classical clathrin- and dynamin-dependent mechanisms. First, neurons were exposed to low K+, an established treatment that inhibits clathrin-dependent endocytosis (30Bayer N. Schober D. Hüttinger M. Blaas D. Fuchs R. J. Biol. Chem. 2001; 276: 3952-3962Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Under control conditions GABABRs endocytosed constitutively, and a significant accumulation of internalized receptors was observed after 60 min (Fig. 4A, top panels). Importantly, GABABR endocytosis was strongly inhibited by low K+ (Fig. 4, A, bottom panels, and B). Interestingly, occasional intracellular structures were still visible near the cell surface (Fig. 4A, bottom panels, arrows). A similar inhibition was obtained with hypertonic sucrose, another treatment that inhibits clathrin-dependent endocytosis (not shown). Clathrin-dependent endocytosis may be dynamin-dependent or -independent (31Praefcke G.J. McMahon H.T. Nat. Rev. Mol. Cell Biol. 2004; 5: 133-147Crossref PubMed Scopus (1112) Google Scholar). To discriminate between these possibilities, we evaluated the role of dynamin-1 and dynamin-2 in GABABR endocytosis. Neurons were co-transfected with GABABR subunits and EGFP-wt-dynamin-1 or a dominant negative version of dynamin-1 that inhibits endocytosis, EGFP-K44A-dynamin-1 (32Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (397) Google Scholar). GABABRs were internalized efficiently in the cell body and dendrites of neurons transfected with EGFP-wt-dynamin-1 (Fig. 5A, arrows). However, endocytosis was severely impaired in the presence of EGFP-K44A-dynamin-1 (Fig. 5B, arrows). No impairment was observed with EGFP-wt-dynamin-2 or EGFP-K44A-dynamin-2, indicating that the endocytosis of GABABRs is specifically dependent on dynamin-1 (Fig. 5, C and D, arrows). Quantification of multiple images indicated that EGFP-K44A-dynamin-1 significantly reduced the endocytosis of GABABRs, whereas EGFP-K44A-dynamin-2 had no effect (Fig. 5E). These results were corroborated by the identification of dynamin-1 in a mass spectrometry analysis of GABABR-associated proteins (not shown) and further confirmed by the co-immunoprecipitation of dynamin-1 with GABABRs from brain (supplemental Fig. 1C). Taken together they indicate that in hippocampal neurons GABABRs internalize via clathrin- and dynamin-1-dependent mechanisms. Dynamin internalization is dependent on the interaction of surface proteins to adaptor molecules. To determine whether adaptor proteins are involved in GABABR endocytosis, receptors were immunoprecipitated from rat brain lysates, and the presence of α-adaptin and β-adaptin in the receptor complex was evaluated by immunoblotting. Interestingly, α-adaptin and β-adaptin did not bind GABABR1 or GABABR2 (supplemental Fig. 1D). GABABRs Efficiently Recycle in Neurons—An essential mechanism to control the availability of neurotransmitter receptors at the cell surface is the recruitment of internalized receptors by reinsertion into the plasma membrane after endocytosis (27Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (897) Google Scholar). However, little is known regarding the recycling of GABABRs in neurons. To start addressing this issue we determined whether GABABRs accumulated in recycling endosomes by evaluating the distribution of the internalized pool relative to Rab11. Rab11 is a small GTPase that regulates recycling of internalized proteins back to the plasma membrane (33Seachrist J.L. Ferguson S.S. Life Sci. 2003; 74: 225-235Crossref PubMed Scopus (174) Google Scholar). Interestingly, the majority of internalized GABABRs accumulated in Rab11 positive vesicle-like structures in the cell body and dendrites of hippocampal neurons after 60 min (Fig. 6A, arrows). Next, we explored the fate of the internalized population of GABABRs by evaluating their reappearance at the plasma membrane after a round of internalization. Surface receptors were incubated with MYC antibodies and internalized using the conditions described above. Remaining receptors at the cell surface were removed by acid-stripping, and the reappearance of receptors, which reached the plasma membrane from intracellular stores, was evaluated by immunofluorescence. GABABRs were present at the cell surface and accumulated in intracellular stores after 90 min (Fig. 6B, arrow indicates cell surface and arrowhead indicates internalized). After acid-stripping, the great majority of surface receptors disappeared from the cell surface, but the treatment had no effect on the intracellular population (Fig. 6C, arrow and arrowhead). Importantly, a significant proportion of GABABRs reappeared at the plasma membrane after an additional 60 min (Fig. 6D, arrow indicates recycled and arrowhead indicates internalized). Together these results indicate that internalized GABABRs recycle back to the plasma membrane from Rab11 positive endosomes. Surface Availability of GABABRs Is Reduced by Glutamate and Is Dependent on an Intact Proteasome Pathway—It has been convincingly established that GABABRs are mainly located peripherally to glutamatergic synapses (14Kulik A. Nakadate K. Nyíri G. Notomi T. Malitschek B. Bettler B. Shigemoto R. Eur. J. Neurosci. 2002; 15: 291-307Crossref PubMed Scopus (142) Google Scholar, 15Kulik A. Vida I. Lujan R. Haas C.A. Lopez-Bendito G. Shigemoto R. Frotscher M. J. Neurosci. 2003; 23: 11026-11035Crossref PubMed Google Scholar, 16Luján R. Shigemoto R. Eur. J. Neurosci. 2006; 23: 1479-1490Crossref PubMed Scopus (74) Google Scholar). We reasoned that instead of GABA, glutamate release might modulate receptor endocytosis. To test this hypothesis, we carried out GABABR internalization assays in the absence and presence of glutamate stimulation. Interestingly, a significant decrease of steady-state levels of plasma membrane GABABR1 and GABABR2 was observed after glutamate stimulation (Fig. 7, A–D). However, we were not able to detect a significant increase in the accumulation of intracellular GABABRs after glutamate treatment even in the presence of lysosomal inhibitors (not shown). These results suggest that glutamate controls the surface levels of GABABRs and that it stimulates their degradation through a lysosome-independent mechanism. To determine the fate of cell surface GABABRs after glutamate treatment, neurons were incubated with MG132, an established inhibitor of the proteasome pathway. Incubation with MG132 alone did not change the steady-state cell surface levels of GABABRs (supplemental Fig. 2). However, MG132 completely blocked the glutamate-mediated disappearance of GABABRs (Fig. 8, A–D). These results indicate that glutamate stimulates the degradation of GABABRs via the proteasome. Importantly, MG132 also caused a significant reduction of the endocytosed pool of GABABRs (Fig. 8E). These observations indicate that the agonist-independent internalization and the glutamate-mediated disappearance of receptors are blocked by MG132, suggesting that both pathways share an initial step of internalization from the plasma membrane. In this study we contribute to clarifying the controversy concerning GABABR endocytosis in central neurons. We show that heteromeric GABABRs internalize basally, in an agonist-independent fashion, and that endocytosis is dependent on clathrin and dynamin-1. Our findings also establish the fate of internalized receptors by showing that after endocytosis GABABRs recycle back to the plasma membrane. Importantly, they demonstrate that glutamate significantly alters the steady-state levels of GABABRs by causing them to disappear from the plasma membrane. Finally, they demonstrate that there is a strong dependence between disappearance of surface receptors and proteasome activity. In view of the enrichment of GABABRs at glutamatergic synapses, this constitutes a relevant mechanism for controlling the availability of GABABRs in neurons. Agonist-dependent or -independent Endocytosis of GABABRs in Neurons?—Several studies have addressed the endocytosis of GABABRs (17Couve A. Thomas P. Calver A.R. Hirst W.D. Pangalos M.N. Walsh F.S. Smart T.G. Moss S.J. Nat. Neurosci. 2002; 5: 415-424Crossref PubMed Scopus (106) Google Scholar, 18Perroy J. Adam L. Qanbar R. Chénier S. Bouvier M. EMBO J. 2003; 22: 3816-3824Crossref PubMed Scopus (105) Google Scholar, 19Fairfax 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, 20Ramoino P. Gallus L. Beltrame F. Diaspro A. Fato M. Rubini P. Stigliani S. Bonanno G. Usai C. J. Cell Sci. 2006; 119: 2056-2064Crossref PubMed Scopus (17) Google Scholar, 21Grampp T. Sauter K. Markovic B. Benke D. J. Biol. Chem. 2007; 282: 24157-24165Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 22Gonzalez-Maeso J. Wise A. Green A. Koenig J.A. Eur. J. Pharmacol. 2003; 481: 15-23Crossref PubMed Scopus (24) Google Scholar, 23Laffray S. Tan K. Dulluc J. Bouali-Benazzouz R. Calver A.R. Nagy F. Landry M. Eur. J. Neurosci. 2007; 25: 1402-1416Crossref PubMed Scopus (29) Google Scholar). Two reports have shown that GABABR internalization is agonist-independent in neurons (18Perroy J. Adam L. Qanbar R. Chénier S. Bouvier M. EMBO J. 2003; 22: 3816-3824Crossref PubMed Scopus (105) Google Scholar, 19Fairfax 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), and at least one suggests the existence of agonist-induced internalization (23Laffray S. Tan K. Dulluc J. Bouali-Benazzouz R. Calver A.R. Nagy F. Landry M. Eur. J. Neurosci. 2007; 25: 1402-1416Crossref PubMed Scopus (29) Google Scholar). The discrepancy between these findings may originate in the different systems used. Although the studies by Perroy et al. (18Perroy J. Adam L. Qanbar R. Chénier S. Bouvier M. EMBO J. 2003; 22: 3816-3824Crossref PubMed Scopus (105) Google Scholar) and Fairfax et al. (19Fairfax 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) center on cerebellar, cortical, and hippocampal neurons, the work by Laffray et al. (23Laffray S. Tan K. Dulluc J. Bouali-Benazzouz R. Calver A.R. Nagy F. Landry M. Eur. J. Neurosci. 2007; 25: 1402-1416Crossref PubMed Scopus (29) Google Scholar) used a co-culture model of dorsal root ganglion and spinal cord. However, because GABABRs display a unique molecular composition throughout the nervous system, this difference is unlikely to explain the discrepancy. Thus, alternative explanations need to be considered. These include trace amounts of GABA in the media and quantification procedures. Our results strongly support an agonist-independent internalization of GABABRs in neurons taking into account their physiological context (cortical and hippocampal neurons), no free GABA in the internalization solutions, and an appropriate quantification methodology (biotinylation). Furthermore, they indicate that the GABABR antagonist saclofen has no effect on constitutive endocytosis corroborating that internalization is not the result of receptor activation by trace levels of GABA. Our study confirms that the pool of GABABRs that accumulates in intracellular compartments in neurons is small compared with cell lines (19Fairfax 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, 21Grampp T. Sauter K. Markovic B. Benke D. J. Biol. Chem. 2007; 282: 24157-24165Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). GABABR Endocytosis Is Clathrin- and Dynamin-1-dependent in Neurons—The clathrin dependence shown here is in agreement with other findings in neurons and the role of dynamin-1 expands previous findings regarding the generic participation of dynamins in GABABR endocytosis in cell lines (21Grampp T. Sauter K. Markovic B. Benke D. J. Biol. Chem. 2007; 282: 24157-24165Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 23Laffray S. Tan K. Dulluc J. Bouali-Benazzouz R. Calver A.R. Nagy F. Landry M. Eur. J. Neurosci. 2007; 25: 1402-1416Crossref PubMed Scopus (29) Google Scholar, 34Restituito S. Couve A. Bawagan H. Jourdain S. Pangalos M.N. Calver A.R. Freeman K.B. Moss S.J. Mol. Cell. Neurosci. 2005; 28: 747-756Crossref PubMed Scopus (37) Google Scholar). Although dynamin-1 is preferentially presynaptic, it has been found in pre- and postsynaptic sites (35Gray N.W. Fourgeaud L. Huang B. Chen J. Cao H. Oswald B.J. Hémar A. McNiven M.A. Curr. Biol. 2003; 13: 510-515Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Our studies do not uncover a preferential function of dynamin-1 on pre- or postsynaptic GABABRs, and more experiments are needed to address this issue, which may have significant physiological implications (13Vigot R. Barbieri S. Brauner-Osborne H. Turecek R. Shigemoto R. Zhang Y.P. Lujan R. Jacobson L.H. Biermann B. Fritschy J.M. Vacher C.M. Muller M. Sansig G. Guetg N. Cryan J.F. Kaupmann K. Gassmann M. Oertner T.G. Bettler B. Neuron. 2006; 50: 589-601Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Recycling—We have shown the recycling of GABABRs to the plasma membrane after accumulation in Rab11 positive endosomes. Recycling in neurons has been indirectly examined before using pharmacological blockers (23Laffray S. Tan K. Dulluc J. Bouali-Benazzouz R. Calver A.R. Nagy F. Landry M. Eur. J. Neurosci. 2007; 25: 1402-1416Crossref PubMed Scopus (29) Google Scholar). As the authors correctly indicate, monensin inhibits the recycling of internalized proteins back to the plasma membrane. However, monensin is an ion-selective ionophore that neutralizes the acidification of compartments and may have notorious effects on the Golgi apparatus, endosomes, and lysosomes (36Mollenhauer H.H. Morre D.J. Rowe L.D. Biochim. Biophys. Acta. 1990; 1031: 225-246Crossref PubMed Scopus (532) Google Scholar). To avoid these unrelated effects, we directly demonstrated the reinsertion of GABABRs at the plasma membrane of hippocampal neurons. Unexpectedly, our data disagree with findings in HEK-293 cells (21Grampp T. Sauter K. Markovic B. Benke D. J. Biol. Chem. 2007; 282: 24157-24165Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). At present we cannot explain this discrepancy, but the turnover of GABABRs is likely to contain additional steps and checkpoints in neurons that have been uncovered in our analyses (34Restituito S. Couve A. Bawagan H. Jourdain S. Pangalos M.N. Calver A.R. Freeman K.B. Moss S.J. Mol. Cell. Neurosci. 2005; 28: 747-756Crossref PubMed Scopus (37) Google Scholar). Heteromeric Endocytosis in Dendrites and Axons—We did not detect internalization of GABABRs in axons despite the fact that receptors were abundantly expressed at the plasma membrane. The existence of a molecular machinery that regulates endocytosis for G protein-coupled receptors in the axon has been established. For example, mGluR5 is efficiently endocytosed in the axon of cultured hippocampal neurons (37Fourgeaud L. Bessis A.S. Rossignol F. Pin J.P. Olivo-Marin J.C. Hémar A. J. Biol. Chem. 2003; 278: 12222-12230Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Therefore, we suggest that the axonal shaft is endocytosis-incompetent relative to GABABRs or that the rate/number of internalized receptors is significantly reduced compared with dendrites. Internalization in axon terminals remains to be explored. However, our data imply that if receptors are internalized at presynaptic terminals then retrograde trafficking must be slow for GABABRs. Finally, whether different mechanisms or rates of internalization operate for GABABR1a and GABABR1b in axons and dendrites remains to be explored (13Vigot R. Barbieri S. Brauner-Osborne H. Turecek R. Shigemoto R. Zhang Y.P. Lujan R. Jacobson L.H. Biermann B. Fritschy J.M. Vacher C.M. Muller M. Sansig G. Guetg N. Cryan J.F. Kaupmann K. Gassmann M. Oertner T.G. Bettler B. Neuron. 2006; 50: 589-601Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). It has been demonstrated convincingly that heteromeric GABABRs composed of GABABR1 and GABABR2 form the functional receptor at the neuronal plasma membrane. In this study we have shown that the receptor is internalized as a heteromer in the soma and dendrites of hippocampal neurons. Endocytosis of an assembled heteromer represents an efficient mechanism to reinsert receptors at the plasma membrane. In addition, co-endocytosis has been shown for other kinds of dimeric receptors, where it defines alternative trafficking and signaling routes (38Lenferink A.E. Pinkas-Kramarski R. van de Poll M.L. van Vugt M.J. Klapper L.N. Tzahar E. Waterman H. Sela M. van Zoelen E.J. Yarden Y. EMBO J. 1998; 17: 3385-3397Crossref PubMed Scopus (340) Google Scholar). Regulation of GABABR Surface Availability by Glutamate— Accumulated evidence from this study and other studies strongly suggests that surface availability of GABABRs is not modulated by GABA. Here we demonstrate that glutamate significantly reduces the levels of plasma membrane receptors. This finding has direct physiological implications, as GABABRs are localized in pre- and postsynaptic terminals of glutamatergic synapses in several regions of the brain, including the hippocampus. We hypothesize that the fluctuations of GABA concentration at these synapses may be relatively slow and small compared with the rapid and concentrated bursts of glutamate release. Thus, it is conceivable that the acute regulation of GABABR availability at the cell surface is controlled by the neurotransmitter which presents greater variability within the synapse. Functionally, the removal of GABABRs after glutamate exposure may act to facilitate synaptic transmission at glutamatergic terminals by reducing the postsynaptic hyperpolarizing effect produced by GABABRs and/or lowering the inhibition of neurotransmitter release at presynaptic sites. The molecular identity of the glutamate receptor and the second messenger pathway responsible for linking the glutamate signal to the GABABR remain to be explored. The inhibition caused by MG132 raises the possibility that endocytosis of GABABRs is mediated by ubiquitination. This interpretation of our results is supported by the fact that MG132 depletes free ubiquitin thus preventing endocytosis of ubiquitinated proteins (39Melikova M.S. Kondratov K.A. Kornilova E.S. Cell Biol. Int. 2006; 30: 31-43PubMed Google Scholar). However, the exact mechanism behind the loss of cell surface receptors after glutamate exposure remains to be explored. We were unable to detect a buildup of intracellular GABABRs after glutamate treatment, even in the presence of lysosomal or proteasomal inhibitors, but the glutamate-mediated disappearance of steady-state cell surface GABABRs was strongly inhibited by the proteasomal inhibitor MG132. Taken together our results suggest that GABABRs endocytose basally and that glutamate causes a rapid redirectioning of the endocytosed pool to the proteasome for degradation. Download .pdf (.2 MB) Help with pdf files