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• W1997207809 abstract "GABAB receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. The dynamic control of the cell surface stability of GABAB receptors is likely to be of fundamental importance in the modulation of receptor signaling. Presently, however, this process is poorly understood. Here we demonstrate that GABAB receptors are remarkably stable at the plasma membrane showing little basal endocytosis in cultured cortical and hippocampal neurons. In addition, we show that exposure to baclofen, a well characterized GABAB receptor agonist, fails to enhance GABAB receptor endocytosis. Lack of receptor internalization in neurons correlates with an absence of agonist-induced phosphorylation and lack of arrestin recruitment in heterologous systems. We also demonstrate that chronic exposure to baclofen selectively promotes endocytosis-independent GABAB receptor degradation. The effect of baclofen can be attenuated by activation of cAMP-dependent protein kinase or co-stimulation of β-adrenergic receptors. Furthermore, we show that increased degradation rates are correlated with reduced receptor phosphorylation at serine 892 in GABABR2. Our results support a model in which GABABR2 phosphorylation specifically stabilizes surface GABAB receptors in neurons. We propose that signaling pathways that regulate cAMP levels in neurons may have profound effects on the tonic synaptic inhibition by modulating the availability of GABAB receptors. GABAB receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. The dynamic control of the cell surface stability of GABAB receptors is likely to be of fundamental importance in the modulation of receptor signaling. Presently, however, this process is poorly understood. Here we demonstrate that GABAB receptors are remarkably stable at the plasma membrane showing little basal endocytosis in cultured cortical and hippocampal neurons. In addition, we show that exposure to baclofen, a well characterized GABAB receptor agonist, fails to enhance GABAB receptor endocytosis. Lack of receptor internalization in neurons correlates with an absence of agonist-induced phosphorylation and lack of arrestin recruitment in heterologous systems. We also demonstrate that chronic exposure to baclofen selectively promotes endocytosis-independent GABAB receptor degradation. The effect of baclofen can be attenuated by activation of cAMP-dependent protein kinase or co-stimulation of β-adrenergic receptors. Furthermore, we show that increased degradation rates are correlated with reduced receptor phosphorylation at serine 892 in GABABR2. Our results support a model in which GABABR2 phosphorylation specifically stabilizes surface GABAB receptors in neurons. We propose that signaling pathways that regulate cAMP levels in neurons may have profound effects on the tonic synaptic inhibition by modulating the availability of GABAB receptors. G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; PKA, cAMP activated protein kinase; GST, glutathione S-transferase; GRK, G protein receptor kinase; PBS, phosphate-buffered saline; EGFP, enhanced green fluorescent protein; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole; EEA1, early endosomal-antigen 1; Br-cAMP, 8-bromo-cAMP. 1The abbreviations used are: GPCR, G protein-coupled receptor; PKA, cAMP activated protein kinase; GST, glutathione S-transferase; GRK, G protein receptor kinase; PBS, phosphate-buffered saline; EGFP, enhanced green fluorescent protein; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole; EEA1, early endosomal-antigen 1; Br-cAMP, 8-bromo-cAMP. mediate responses to a wide variety of stimuli such as light, odorants, hormones, and neurotransmitters, and can be divided into three families (A, B, and C) on the basis of their sequence and structural similarity (1Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. 2002; 3: 639-650Crossref Scopus (2097) Google Scholar). Termination of GPCR signaling is a key process that defines the overall properties of the response to a particular stimulus and extensive observations have allowed the proposal of a conserved series of events, divided into three distinct stages, that take place during receptor inactivation (2Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar). In the first stage, agonist occupancy triggers GPCR phosphorylation by G protein-coupled receptor kinases (GRKs) causing desensitization over a time scale of seconds (3Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1068) Google Scholar). In the second, phosphorylation results in the binding to proteins of the arrestin family, which mediate recruitment of GPCRs to clathrin-coated vesicles, subsequent internalization, and sorting to endosomes or lysosomes after seconds or minutes of stimulation (4Luttrell L.M. Lefkowitz R.J. J. Cell Sci. 2002; 115: 455-465Crossref PubMed Google Scholar). Finally, prolonged agonist exposure causes down-regulation, a phenomenon defined as the reduction in the total number of receptors and usually correlated with reduced receptor mRNA (5Tsao P. von Zastrow M. Curr. Opin. Neurobiol. 2000; 10: 365-369Crossref PubMed Scopus (134) Google Scholar). GABAB receptors belong to family C and unlike other GPCRs they require the formation of a heterodimer composed of two subunits, namely GABABR1 and GABABR2 (6Bouvier M. Nat. Rev. Neurosci. 2001; 4: 274-286Crossref Scopus (580) Google Scholar). Both subunits display high homology to metabotropic glutamate, Ca2+-sensing, vomeronasal, and putative pheromone receptors (7Couve A. Moss S.J. Pangalos M.N. Mol. Cell. Neurosci. 2000; 16: 296-312Crossref PubMed Scopus (240) Google Scholar). Recombinant heterodimeric GABABR1/GABABR2 receptors reproduce most of the characteristics and pharmacology of native receptors (7Couve A. Moss S.J. Pangalos M.N. Mol. Cell. Neurosci. 2000; 16: 296-312Crossref PubMed Scopus (240) Google Scholar), i.e. they inhibit adenylyl cyclase, activate inwardly rectifying K+ channels, and inactivate voltage-gated Ca2+ channels (8Mott D.D. Lewis D.V. Int. Rev. Neurobiol. 1994; 36: 97-223Crossref PubMed Scopus (227) Google Scholar, 9Jones K.A. Borowski B. Tamm J.A. Craig D.A. Durkin M.M. Dai M. Yao W. Johnson M. Gunwaldsen C. Huang L. Tang C. Shen Q. Salon J.A. Morse K. Laz T. Smith K.E. Nagarathnam D. Noble S.A. Branchek T.A. Gerald C. Nature. 1998; 396: 674-679Crossref PubMed Scopus (923) Google Scholar, 10Kaupmann K. Malitschek B. Schuler V. Heid J. Froestl W. Beck P. Mosbacher J. Bischoff S. Kulik A. Shigemoto R. Karschin A. Bettler B. Nature. 1998; 396: 683-687Crossref PubMed Scopus (1015) Google Scholar, 11White 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. Nature. 1998; 396: 679-682Crossref PubMed Scopus (1011) Google Scholar, 12Kuner R. Kohr G. Grunewald S. Eisenhartdt G. Bach A. Kornau H.C. Science. 1999; 283: 74-77Crossref PubMed Scopus (501) Google Scholar, 13Filippov A.K. Couve A. Pangalos M.N. Walsh F.S. Brown D.A. Moss S.J. J. Neurosci. 2000; 20: 2867-2874Crossref PubMed Google Scholar). GABAB receptors mediate the slow and prolonged component of synaptic inhibition (8Mott D.D. Lewis D.V. Int. Rev. Neurobiol. 1994; 36: 97-223Crossref PubMed Scopus (227) Google Scholar) and their importance has been highlighted by the pro-convulsant phenotype displayed by GABABR1 knockout mice (14Proser H.M. Gill C.H. Hirst W.D. Grau E. Robbins M. Calver A. Soffin E.M. Farmer C.E. Lanneau C. Gray J. Schneck E. Warmerdam B.S. Clapham C. Reavill C. Rogers D.C. Stean T. Upton N. Humphreys K. Randall A. Geppert M. Davies C.H. Pangalos M.N. Mol. Cell. Neurosci. 2001; 17: 1059-1070Crossref PubMed Scopus (244) Google Scholar, 15Schuler V. Lüscher C. Blanchet C. Klix N. Sansig G. Klebs K. Schmutz M. Heid J. Gentry C. Urban L. Fox A. Spooren W. Jaton A. Vigouret J.M. Pozza M. Kelly P.H. Mosbacher J. Froestl W. Käslin E. Korn R. Bischoff S. Kaupmann K. van der Putten H. Bettler B. Neuron. 2001; 31: 47-58Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar). They have also been implicated in pain, depression, and cognition (7Couve A. Moss S.J. Pangalos M.N. Mol. Cell. Neurosci. 2000; 16: 296-312Crossref PubMed Scopus (240) Google Scholar) and their activation may be beneficial in reducing withdrawal symptoms from addictive drugs (16Roberts D.C. Andrews M.M. Psychopharmacology. 1997; 131: 271-277Crossref PubMed Scopus (148) Google Scholar, 17Xi Z.X. Ramamoorthy S. Shen H. Lake R. Samuvel D.J. Kalivas P.W. J. Neurosci. 2003; 23: 3498-3505Crossref PubMed Google Scholar). We have recently shown that phosphorylation of the GABABR2 subunit by the cAMP-dependent protein kinase (PKA) promotes potentiation but not desensitization of GABAB receptors in dissociated cultured neurons (18Couve A. Thomas P. Calver A.R. Hirst W.D. Pangalos M.N. Walsh F.S. Moss S.J. Nat. Neurosci. 2002; 5: 415-424Crossref PubMed Scopus (106) Google Scholar). These results conflict with the classical model of GPCR inactivation. To further understand the regulation of GABAB receptors we have investigated their modulation by phosphorylation and cell surface stability. Our results demonstrate that GABAB receptors do not undergo agonist-induced phosphorylation or internalization and are very stable at the neuronal plasma membrane. In addition, they show that over long time scales GABAB receptors are subject to degradation at or near the cell surface in a PKA-dependent manner. Together our observations suggest that unique mechanisms have evolved to regulate the activity of GABAB receptors reflecting their critical role in the tonic control of neuronal activity. Materials—Baclofen, forskolin, and isoproterenol were purchased from Sigma. [35S]Methionine and [32P]orthophosphate were obtained from Amersham Biosciences. EZ Link Sulfo-NHS-SS Biotin, EZ Link Sulfo-NHS Biotin, and UltraLink NeutrAvidin beads were purchased from Pierce (Rockford, IL). GRK2 was purified from Sf9 insect cells. The PKA catalytic subunit was purchased from Promega (Southampton, United Kingdom). Plasmids—The plasmid pEGFP-C1 was purchased from BD Biosciences. The MYC-GABABR1 and FLAG-GABABR2 expression vectors and the GST fusion protein vectors pGEX-CR1 (CR1, containing the carboxyl-terminal domain of GABABR1) and pGEX-CR2 (CR2, containing the carboxyl-terminal domain of GABABR2) have been described previously (18Couve A. Thomas P. Calver A.R. Hirst W.D. Pangalos M.N. Walsh F.S. Moss S.J. Nat. Neurosci. 2002; 5: 415-424Crossref PubMed Scopus (106) Google Scholar, 19Couve 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). The cDNAs for THRH, arrestin 2-EGFP, and arrestin 3-EGFP have been described previously (20Scott M.G. Benmerah A. Muntaner O. Marullo S. J. Biol. Chem. 2002; 277: 3552-3559Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). All DNA manipulations and fidelity of DNA constructs were verified by DNA sequencing. Antibodies—Monoclonal FLAG and VSV antibodies were obtained from Sigma. Monoclonal Myc antibodies were obtained from a 9E10 hybridoma. The polyclonal anti-myc antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The guinea pig anti-GABABR1 and -GABABR2 antibodies and the rabbit anti-GluR1 antibody were purchased from Chemicon International (Harrow, UK). The early endosomal-antigen 1 (EEA1) antibody was purchased from BD Biosciences. The rabbit anti-GABABR1, anti-GABABR2, and anti-GABABR2-P-Ser892 (UCL71) have been described previously (18Couve A. Thomas P. Calver A.R. Hirst W.D. Pangalos M.N. Walsh F.S. Moss S.J. Nat. Neurosci. 2002; 5: 415-424Crossref PubMed Scopus (106) Google Scholar, 21Calver A. Medhurst A.D. Robbins M.J. Charles K.J. Evans M.L. Harrison D.C. Stammers M. Hughes S.A. Hervieu G. Couve A. Moss S.J. Middlemiss D.N. Pangalos M.N. Neuroscience. 2000; 100: 155-170Crossref PubMed Scopus (131) Google Scholar, 22Calver 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 anti-neurofilament antibody was purchased from Affiniti (Nottingham, UK). The secondary anti-mouse, anti-rabbit, and anti-guinea pig antibodies conjugated to Cy5, Texas Red, and fluorescein isothiocyanate were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). The secondary anti-rabbit antibody conjugated to [125I] was purchased from Amersham Biosciences. Cell Culture and cDNA Expression—COS-7 and HEK293 cells were maintained and transfected as described previously (19Couve 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). Ca2+ Mobilization Assay and a Fluorimetric Imaging Plate Reader— This was performed as described previously (23Robbins M.J. Calver A.R. Filippov A.K. Hirst W.D. Russell R.B. Wood M.D. Nasir S. Couve A. Brown D.A. Moss S.J. Pangalos M.N. J. Neurosci. 2001; 21: 8043-8052Crossref PubMed Google Scholar). Metabolic [35S]Methionine, [32P]Orthophosphate Labeling, and Immunoprecipitations—For metabolic labeling, COS-7 cells were washed twice with methionine-free Dulbecco's modified Eagle's medium or phosphate-free Dulbecco's modified Eagle's medium and incubated in methionine-free Dulbecco's modified Eagle's medium containing 0.5 mCi of [35S]methionine or in phosphate-free Dulbecco's modified Eagle's medium containing 0.5 mCi of [32P]orthophosphate for 4 h at 37 °C. After the incubation periods, cells were washed twice with phosphate-buffered saline (PBS) and lysed in 0.6 ml of RIPA buffer (50 mm Tris-Cl, 5 mm EGTA, 5 mm EDTA, 50 mm NaF, 10 mm sodium pyrophosphate, 1 mm sodium orthovanadate, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 0.1% phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml antipain). Nuclei were removed from cell lysates by centrifugation at 22,000 × g for 5 min at 4 °C in a microcentrifuge. Cell lysates were preabsorbed with 25 μl of protein A- or protein G-Sepharose, previously equilibrated in RIPA buffer, for 1 h at 4 °C. Protein A/G-Sepharose beads were then removed by centrifugation at 22,000 × g for 1 min at 4 °C, and lysates were rotated with 5 μg of antibodies for 1 h at 4 °C. Immune complexes were then precipitated with 25 μl of protein A/G-Sepharose for 1 h at 4 °C. Sepharose beads were washed twice in RIPA buffer containing 500 mm NaCl and once in RIPA buffer containing 150 mm NaCl. Immunoprecipitated proteins were eluted in 40 μl of SDS-PAGE loading buffer, boiled for 3 min, resolved by SDS-PAGE, and visualized by phosphorimager (Bio-Rad). Quantification of radioactive bands was performed using the Quantity One software (Bio-Rad). In Vitro Kinase Assays—10 μg of GST fusion proteins or rod outer segments were mixed with purified GRK2 or purified PKA in kinase buffer (20 mm Tris-Cl, pH 7.2, 5 mm MgCl2, 2 mm EDTA, 1 mm dithiothreitol; for GRK reactions the buffer was supplemented with Gβγ and PIP2) in a final volume of 25 μl. Agarose beads were pre-warmed in kinase buffer for 1 min at 37 °C, ATP was then added to the reaction to a final concentration of 0.2 mm containing 1/10 volume of [γ-32P]ATP (Amersham Biosciences, 10 mCi/ml) and the reactions were continued for 15 min at 37 °C. Reactions were stopped by the addition of 12.5 μl of 3× SDS-PAGE loading buffer, boiled for 3 min, resolved by SDS-PAGE, and visualized by phosphorimager (Bio-Rad). Quantification of radioactive bands was performed using the Quantity One software (Bio-Rad). Cortical and Hippocampal Neuron Primary Cultures—Cortical and hippocampal neurons were obtained from embryonic day 18 rats as described previously (24Kittler J.T. Delmas P. Jovanovic J.N. Brown D.A. Smart T.G. Moss S.J. J. Neurosci. 2000; 20: 7972-7977Crossref PubMed Google Scholar). Corticals neurons were used between 5 and 7 days in vitro and hippocampal neurons were maintained for 21 days in vitro before use. Biotinylation—5 days in vitro rat cortical neurones grown on poly-llysine-coated dishes were incubated for 1 h in culture media containing 100 μg/ml leupeptin. Dishes were placed on ice and washed twice with ice-cold PBS/Ca2+/Mg2+ (PBS containing 1 mm CaCl2 and 0.5 mm MgCl2). The biotin reagent was freshly dissolved at 1 mg/ml in ice-cold PBS/Ca2+/Mg2+ and cultures were incubated with biotin for 12 min on ice. The biotin solution was aspirated and the dishes were blocked by washing 3× 5 min in PBS/Ca2+/Mg2+ containing 0.1% bovine serum albumin and 2× 5 min in ice-cold PBS/Ca2+/Mg2+. Neurons were returned to the 37 °C incubator for the appropriate periods of time (10 min to 60 h) in the presence of 100 μg/ml leupeptin. Dishes were placed on ice, the media was removed, and 2–5 ml of ice-cold cleaving buffer was added (50 mm glutathione in 75 mm NaCl, 10 mm EDTA, 1% bovine serum albumin, and 0.075 n NaOH). Dishes were incubated 2× 15 min at 4 °C with constant shaking. Finally neurons were washed twice in ice-cold PBS/Ca2+/Mg2+, lysed in 300 μl of ice-cold RIPA buffer, and solubilized by rotating 1 h at 4 °C. Nuclear and cellular debris was removed by centrifugation at 14,000 × g for 5 min at 4 °C and the supernatants were precipitated with 100 μl of UltraLink NeutrAvidin slurry for 2 h at 4 °C. Beads were washed twice in RIPA buffer containing 500 mm NaCl and once in RIPA buffer containing 150 mm NaCl. Beads were resuspended in 60 μl of SDS sample buffer and resolved on SDS-PAGE and transferred to Hybond-C membranes. Immunoblot Analysis—Immunoblots of brain membranes, transfected cells, cultured neurons, and biotinylation procedures were performed as described previously by incubating with the corresponding primary antibodies overnight at 4 °C and secondary antibodies conjugated to [125I] for 1 h at room temperature as described previously (18Couve A. Thomas P. Calver A.R. Hirst W.D. Pangalos M.N. Walsh F.S. Moss S.J. Nat. Neurosci. 2002; 5: 415-424Crossref PubMed Scopus (106) Google Scholar). Immunoblots were visualized via phosphorimager (Bio-Rad) and the radioactive bands were quantified using the Quantity One software (Bio-Rad). Imaging—Transfected cells were plated onto 6-cm dishes containing 10 μg/ml poly-l-lysine-coated 13-mm coverslips. Cells were washed twice with PBS, fixed for 10 min in 4% paraformaldehyde, and blocked for 10 min in immunofluorescence solution (0.25% bovine serum albumin, 10% horse serum in PBS). Cells were permeabilized in immunofluorescence solution containing 0.5% Nonidet P-40 for 10 min at room temperature and blocked for another 10 min in immunofluorescence solution containing 0.1% Nonidet P-40. Samples were incubated sequentially with primary and secondary antibodies for 1 h at room temperature in blocking solution. Coverslips were examined using a confocal microscope (MRC1000, Bio-Rad). GABAB Receptors Do Not Undergo Agonist-induced Phosphorylation—Classically, monomeric GPCRs undergo agonist-induced phosphorylation by GRKs as a first step during their desensitization. The role of this process for the heterodimeric GABAB receptor remains poorly understood. To investigate the effect of agonist on GABAB receptor phosphorylation, incorporation of [32P]orthophosphate into GABABR1 and GABABR2 was followed in COS-7 cells. Myc-tagged GABABR1 and FLAG-tagged GABABR2 cDNAs were transiently transfected and immunoprecipitated from cell lysates after metabolic labeling. In agreement with recent observations, a single band of ∼120 kDa, corresponding to basally phosphorylated GABABR1 (Fig. 1b, lanes 1 and 3), and a single band of ∼110 kDa, corresponding to basally phosphorylated GABABR2 (Fig. 1b, lanes 2 and 3) were obtained in immunoprecipitations from lysates derived from GABABR1-, GABABR2 or GABABR1-, GABABR2-transfected cells, respectively. Control immunoprecipitations from [35S]methionine metabolic labeled receptors indicated that the visualized bands corresponded indeed to GABABR1 and GABABR2 (Fig. 1a, lanes 1–4). Unexpectedly, neither GABABR1 nor GABABR2 phosphorylation levels were increased in response to the GABAB-specific agonist baclofen at all times up to 30 min of receptor stimulation (Fig. 1c, lanes 1–5). In contrast, phosphorylation of GABABR2 was significantly increased after treatment with forskolin, a known activator of PKA (Fig. 1c, lane 6). To explore the effects of various members of the GRK family in agonist-mediated receptor phosphorylation, the phosphorylation levels of GABAB receptors were examined in cells cotransfected with GABABR1, GABABR2, GRK2, or GRK3. Interestingly, the levels of phosphorylation of GABAB receptors remained unchanged after baclofen treatment even in the presence of the additional kinases (Fig. 1d, lanes 1–4). The same results were obtained when cells were co-transfected with GRK5 and GRK6 (data not shown). The intracellular loops of class C GPCRs are generally small, whereas the carboxyl-terminal domains are relatively large and the most likely targets for kinase-dependent modulation. To further examine the roles of GRKs as regulators of GABAB receptor phosphorylation in vitro, phosphorylation assays were performed using purified GRK2 and the carboxyl-terminal domains of GABABR1 (CR1) and GABABR2 (CR2) expressed as GST fusion proteins. In agreement with our metabolic labeling experiments, GRK2 did not phosphorylate the carboxyl-terminal domains of either receptor subunit (Fig. 1e, lanes 1–4). This was in marked contrast with the in vitro phosphorylation of CR2 by PKA (Fig. 1e, lane 5; the major band of 45 kDa) (18Couve A. Thomas P. Calver A.R. Hirst W.D. Pangalos M.N. Walsh F.S. Moss S.J. Nat. Neurosci. 2002; 5: 415-424Crossref PubMed Scopus (106) Google Scholar), or the phosphorylation of rod outer segment by GRK2 (Fig. 1e, lane 6). These observations indicate that GABAB receptors are not phosphorylated in an agonist-dependent manner and rule out the participation of agonist-induced phosphorylation as a key element in the inactivation of the GABAB receptor signal. GABAB Receptors Do Not Undergo Rapid Agonist-induced Internalization—GPCRs that undergo endocytosis in response to agonist occupancy do so by recruiting proteins of the arrestin family in a phosphorylation-dependent manner (2Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar). To explore the agonist-induced internalization of GABAB receptors, an endocytosis and arrestin recruitment assay was performed. Arrestins 2 and 3 (β-arrestin 1 and β-arrestin 2) have been shown to mediate GPCR internalization. Therefore, GABAB receptor internalization was evaluated in the presence of arrestin 2 or arrestin 3 via a microscopic analysis. COS-7 cells were transfected with GABABR1/GABABR2 or with the control thyrotropic-related hormone receptor in the presence of arrestin 2-EGFP or arrestin 3-EGFP. After transfections both the GABAB receptor heterodimer and the thyrotropic-related hormone receptor were present at the cell surface of COS cells (Fig. 2, b and f). In addition, there was an abundant expression of arrestin 3-EGFP, which did not significantly co-localize with the surface receptor population (Fig. 2 a, d and e, h). Activation of thyrotropic-related hormone receptor resulted in a rapid removal of receptors from the plasma membrane (Fig. 2, n, o and v, w). Moreover, agonist stimulation produced a robust recruitment of receptors to an intracellular sorting compartment that co-localized extensively with arrestin 3-EGFP (Fig. 2, m, o, p and u, w, x). In contrast, exposure of GABAB expressing cells to baclofen did not result in receptor internalization (Fig. 2, j, k and r, s) or arrestin recruitment (Fig. 2, i, k, l and q, s, t). Identical results were obtained when the experiment was performed in the presence of arrestin 2-EGFP (data not shown). These results indicate that arrestins do not facilitate GABAB receptor internalization and strongly suggest that GABAB receptors are stable at the plasma membrane. Agonist Exposure Does Not Promote Receptor Endocytosis in Cortical Neurons—To investigate the relevance of these recombinant studies in a neuronal context, we examined the fate of cell surface GABAB receptors after periods of agonist exposure in primary cultures of rat cortical neurons. Cell surface receptors were labeled using a cell-impermeable active biotin reagent that binds covalently to surface proteins (25Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (897) Google Scholar). Cells were then treated with agonists and the internalized biotinylated fraction of GABAB receptors and control proteins was evaluated by quantitative immunoblotting. As expected, a significant GABABR1a reactivity was observed in the surface-biotinylated pool, demonstrating the presence of GABABR1 at the cell surface in cultured cortical neurons (Fig. 3a, lane 1). In addition, a very small fraction was detected in intracellular compartments after the surface proteins were allowed to undergo internalization (Fig. 3a, lanes 2 and 3). Interestingly, the small internalized pool of GABABR1a did not increase after 10 or 60 min of agonist stimulation (60 min: control, 2.63 ± 0.97%; baclofen, 3.50 ± 0.99%, n = 3). A time course analysis indicated that no difference existed between the internalized pools of GABABR1a in the absence and presence of baclofen at all times ranging from 5 min to 6 h (data not shown). Similar results were obtained when the GABABR2 subunit was analyzed and no enhancement of internalized subunits was observed after agonist treatment (Fig. 3b). The biotin reagent did not interfere with GABAB receptor agonist potency suggesting that the lack of GABAB receptor internalization did not result from an interfering effect of the labeling procedure (Table I). In contrast, 43% of the GluR1 subunit of the AMPA receptor internalized efficiently after 60 min of AMPA treatment (Fig. 3c, lanes 1–4) indicating that the membrane dynamics remain intact during our experimental procedure.Table IHEK293 cells were transfected with GABABR1 and GABABR2 Cells were left untreated or labelled with biotin prior to stimulation with GABAB agonists (GABA or baclofen). The activity of the GABAB receptor was determined using a Ca2+ mobilization assay and a fluorimetric imaging plate reader. The data represents the average and S.E. of three independent experiments.AgonistBiotinEC50nμmGABA–6.95 ± 0.263GABA+6.87 ± 0.193Baclofen–6.06 ± 0.373Baclofen+6.14 ± 0.123 Open table in a new tab To confirm these results an imaging analysis was performed in dissociated cultures of primary hippocampal neurons. Neurons were treated with baclofen or AMPA for 15 min and subsequently fixed to evaluate the distribution of GABABR2 and AMPA receptors relative to the EEA1, an established endosomal marker. 15 min after agonist exposure the distribution of AMPA receptors changed dramatically and most of the receptors clustered in intracellular compartments were closely associated with EEA1 (Fig. 3d, bottom panels). In contrast, exposure to baclofen for 15 min had no effect on GABAB receptor distribution and the receptors and EEA1 remained in different subcellular compartments. This situation was indistinguishable from the control condition (Fig. 3d, top panels). Similar results were obtained for 30 and 60 min of agonist treatment (data not shown). Taken together these results conclusively demonstrate that GABAB receptors fail to internalize in response to short term agonist exposure in dissociated cortical and hippocampal neurons and suggest that non-classical mechanisms of inactivation must take place after receptor stimulation. Chronic Agonist Exposure Results in a Specific Loss of Cell Surface GABAB Receptors—The lack of receptor internalization and lack of endosomal accumulation suggest that a novel mechanism may exist to regulate surface receptor numbers. To further analyze this, the fate of cell surface GABAB receptors was examined after chronic baclofen treatment using the biotinylation assay. In untreated neurons the levels of the biotinylated fraction of surface GABABR1a receptors decreased progressively over time (Fig. 4a, lanes 1, 2, 4, 6, and the duplicate lanes 8 and 9). Interestingly, treatment of the neurons with baclofen accelerated the decrease in the biotinylated population of surface GABAB receptors (Fig. 4a, lanes 1, 3, 5, 7, and the duplicate lanes 10 and 11). After 60 h the remaining fraction of GABAB receptors was significantly lower in neurons that received baclofen stimulation (basal, 24.33 ± 0.4% n = 7; baclofen, 14.75 ± 0.5%, n = 7, p < 0.0001). The specificity of this effect was demonstrated by following the fate of the biotinylated β3 subunit of the GABAA receptor. Importantly, baclofen had no effect on the levels of the β3 subunit remaining after 60 h of treatment compared with the levels remaining after basal degradation (baclofen, 97.10 ± 3.3% control, n = 6). Multiple labeling experiments were used to estimate the halflife of the GABAB receptor under these conditions. The half-life of the control population of cell surface GABAB receptors was calculated to be 31.0 h (Fig. 4b, τ basal) and was significantly reduced to 23.5 h after baclofen treatment (Fig. 4b, τ baclofen). To verify that biotinylated receptors recovered after 60 h corresponded to a surface population, a cleavable biotin reagent was used in the same experiment. When neurons were exposed to cleavage media no GABAB receptor immunoreactivity could be detected after purification with streptavidin (Fig. 4c, lanes 1 and 2). These results indicate that prolonged agonist treatment destabilizes cell s" @default.
• W1997207809 created "2016-06-24" @default.
• W1997207809 creator A5000338259 @default.
• W1997207809 creator A5010982846 @default.
• W1997207809 creator A5015795152 @default.
• W1997207809 creator A5020338130 @default.
• W1997207809 creator A5087879092 @default.
• W1997207809 creator A5091256958 @default.
• W1997207809 creator A5091465169 @default.
• W1997207809 date "2004-03-01" @default.
• W1997207809 modified "2023-09-28" @default.
• W1997207809 title "Phosphorylation and Chronic Agonist Treatment Atypically Modulate GABAB Receptor Cell Surface Stability" @default.
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