γ-Aminobutyric Acid Type B Receptors Are Constitutively Internalized via the Clathrin-dependent Pathway and Targeted to Lysosomes for Degradation
2007; Elsevier BV; Volume: 282; Issue: 33 Linguagem: Inglês
10.1074/jbc.m702626200
ISSN1083-351X
AutoresThomas Grampp, Kathrin Sauter, Branko Marković, Dietmar Benke,
Tópico(s)Receptor Mechanisms and Signaling
ResumoReceptor internalization is recognized as an important mechanism for rapidly regulating cell surface numbers of receptors. However, there are conflicting results on the existence of rapid endocytosis of γ-aminobutyric acid, type B (GABAB) receptors. Therefore, we analyzed internalization of GABAB receptors expressed in HEK 293 cells qualitatively and quantitatively using immunocytochemical, cell surface enzyme-linked immunosorbent assay, and biotinylation methods. The data indicate the existence of rapid constitutive receptor internalization, with the first endocytosed receptors being observed in proximity of the plasma membrane after 10 min. After 120 min, a loss of about 40–50% of cell surface receptors was detected. Stimulation of GABAB receptors with GABA or baclofen did not enhance endocytosis of receptors, indicating the lack of agonist-induced internalization. The data suggest that GABAB receptors were endocytosed via the classical dynamin- and clathrin-dependent pathway and accumulated in an endosomal sorting compartment before being targeted to lysosomes for degradation. No evidence for recycling of receptors back to the cell surface was found. In conclusion, the results indicate the presence of constitutive internalization of GABAB receptors via clathrin-coated pits, which resulted in lysosomal degradation of the receptors. Receptor internalization is recognized as an important mechanism for rapidly regulating cell surface numbers of receptors. However, there are conflicting results on the existence of rapid endocytosis of γ-aminobutyric acid, type B (GABAB) receptors. Therefore, we analyzed internalization of GABAB receptors expressed in HEK 293 cells qualitatively and quantitatively using immunocytochemical, cell surface enzyme-linked immunosorbent assay, and biotinylation methods. The data indicate the existence of rapid constitutive receptor internalization, with the first endocytosed receptors being observed in proximity of the plasma membrane after 10 min. After 120 min, a loss of about 40–50% of cell surface receptors was detected. Stimulation of GABAB receptors with GABA or baclofen did not enhance endocytosis of receptors, indicating the lack of agonist-induced internalization. The data suggest that GABAB receptors were endocytosed via the classical dynamin- and clathrin-dependent pathway and accumulated in an endosomal sorting compartment before being targeted to lysosomes for degradation. No evidence for recycling of receptors back to the cell surface was found. In conclusion, the results indicate the presence of constitutive internalization of GABAB receptors via clathrin-coated pits, which resulted in lysosomal degradation of the receptors. GABAB 3The abbreviations used are: GABAB, γ-aminobutyric acid, type B; HEK 293, human embryonic kidney 293; NSF, N-ethylmaleimide-sensitive fusion protein; ELISA, enzyme-linked immunosorbent assay. receptors are G protein-coupled receptors that play an important role in the control of neurotransmission. They are widely expressed in the nervous system and have been implicated as potential targets for neurological diseases, such as epilepsy, pain, spasticity, addiction, schizophrenia, depression, and anxiety (for a review, see Ref. 1Enna S.J. Bowery N.G. Biochem. Pharmacol. 2004; 68: 1541-1548Crossref PubMed Scopus (67) Google Scholar). GABAB receptors mediate slow inhibitory neurotransmission by either activating postsynaptically K+ channels or inhibiting presynaptically the release of neurotransmitters by modulation of Ca2+ channels. On the structural level, functional GABAB receptors require the heterodimerization of two distinct seven-transmembrane proteins, termed GABAB1 and GABAB2 (2Jones K.A. Borowsky B. Tamm J.A. Craig D.A. Durkin M.M. Dai M. Yao W-J. Johnson M. Gunwaldsen C. Huang L.-Y. 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 (932) Google Scholar, 3Kaupmann K. Malitschek B. Schuler V. Heid J. Froestl W. Beck P. Mosbacher J. Bischoff S. Kulik A. Shigemoto R. Karshin A. Bettler B. Nature. 1998; 396: 683-687Crossref PubMed Scopus (1022) Google Scholar, 4Kuner R. Koöhr G. Gruönewald S. Eisenhardt G. Bach A. Kornau H-C. Science. 1999; 283: 74-77Crossref PubMed Scopus (503) Google Scholar, 5Martin S.C. Russek S.J. Farb D.H. Mol. Cell. Neurosci. 1999; 13: 180-191Crossref PubMed Scopus (102) Google Scholar, 6Ng G.Y.K. Clark J. Coulombe N. Ethier N. Hebert T.E. Sullivan R. Kargman S. Chateauneuf A. 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Heterodimerization of GABAB1a or GABAB1b with GABAB2 leads to two main GABAB receptor subtypes, GABAB1a/GABAB2 and GABAB1b/GABAB2, which are abundantly expressed in all major brain structures (10Benke D. Honer M. Michel C. Bettler B. Mohler H. J. Biol. Chem. 1999; 274: 27323-27330Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 11Fritschy J.M. Meskenaite V. Weinmann O. Honer M. Benke D. Mohler H. Eur. J. Neurosci. 1999; 11: 761-768Crossref PubMed Scopus (283) Google Scholar, 12Fritschy J.M. Sidler C. Parpan F. Gassmann M. Kaupmann K. Bettler B. Benke D. J. Comp. Neurol. 2004; 477: 235-252Crossref PubMed Scopus (53) Google Scholar, 13Malitschek B. Ruegg D. Heid J. Kaupmann K. Bittiger H. Frostl W. Bettler B. Kuhn R. Mol. Cell. Neurosci. 1998; 12: 56-64Crossref PubMed Scopus (81) Google Scholar). An important aspect in the regulation of G protein-coupled receptors is their internalization or endocytosis. To protect cells against receptor overstimulation, the vast majority of G protein-coupled receptors desensitize upon prolonged agonist exposure, followed by rapid internalization. Many G protein-coupled receptors undergo phosphorylation upon agonist exposure by a G protein receptor kinase and subsequently recruit an arrestin protein (14Gainetdinov R.R. Premont R.T. Bohn L.M. Lefkowitz R.J. Caron M.G. Annu. Rev. Neurosci. 2004; 27: 107-144Crossref PubMed Scopus (691) Google Scholar). Arrestins often enhance phosphorylation, sterically interfere with binding of the G protein, and function as a signal for receptor endocytosis (15Luttrell L.M. Lefkowitz R.J. J. Cell Sci. 2002; 115: 455-465Crossref PubMed Google Scholar). Once internalized, receptors are targeted to specialized compartments, where they are dephosphorylated and recycled back to the plasma membrane or targeted to lysosomes for degradation. The processes of GABAB receptor desensitization and internalization are currently poorly understood. Initial studies revealed unexpected differences from the common G protein-coupled receptor pathway. Although G protein receptor kinases 4 and 5 directly associate with GABAB receptors and are indispensable for receptor desensitization in certain cells (16Perroy J. Adam L. Qanbar R. Chénier S. Bouvier M. EMBO J. 2003; 22: 3816-3824Crossref PubMed Scopus (106) Google Scholar, 17Kanaide M. Uezono Y. Matsumoto M. Hojo M. Ando Y. Sudo Y. Sumikawa K. Taniyama K. J. Cell. Physiol. 2007; 210: 237-245Crossref PubMed Scopus (35) Google Scholar), kinase activity is not required (16Perroy J. Adam L. Qanbar R. Chénier S. Bouvier M. EMBO J. 2003; 22: 3816-3824Crossref PubMed Scopus (106) Google Scholar). A second mechanism proposed involves the interaction of the GABAB receptor heterodimer with N-ethylmaleimide-sensitive fusion protein (NSF), which primes the receptor for phosphorylation by protein kinase C upon agonist stimulation and leads to desensitization (18Pontier S.M. Lahaie N. Ginham R. St-Gelais F. Bonin H. Bell D.J. Flynn H. Trudeau L.E. McIlhinney J. White J.H. Bouvier M. EMBO J. 2006; 25: 2698-2709Crossref PubMed Scopus (41) Google Scholar). Thus, although the mechanism of GABAB receptor desensitization is still poorly understood, it is clearly mediated by processes distinct from the generally accepted model of G protein receptor kinase phosphorylation-induced desensitization and subsequent internalization of the receptors. Consistent with the atypical mode of desensitization, recent findings suggest that GABAB receptors are not internalized and do not recruit arrestin in response to agonist exposure (16Perroy J. Adam L. Qanbar R. Chénier S. Bouvier M. EMBO J. 2003; 22: 3816-3824Crossref PubMed Scopus (106) 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, 20Mutneja M. Berton F. Suen K.F. Luöscher C. Slesinger P.A. Pfluögers Arch. 2005; 450: 61-73Crossref PubMed Scopus (42) Google Scholar). Interestingly, 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) hypothesized that GABAB receptors may be targeted directly from the surface to the proteasome for degradation. However, agonist-induced internalization of GABAB receptors has also been reported (21Gonzalez-Maeso J. Wise A. Green A. Koenig J.A. Eur. J. Pharmacol. 2003; 481: 15-23Crossref PubMed Scopus (25) Google Scholar). In view of these contradictory reports, it remains unclear whether GABAB receptors undergo noticeable constitutive endocytosis and whether an agonist-induced internalization of GABAB receptors exists. In addition, if appreciable internalization of GABAB receptors occurs, the underlying pathways are completely unknown. To clarify this issue, we analyzed endocytosis of GABAB receptors expressed in HEK 293 cells and the involved pathways using antibody-based and biotinylation assays. The results suggest that GABAB receptors undergo constitutive internalization via the classical dynamin and clathrin-dependent pathway and are targeted to lysosomes for degradation. Antibodies—The following primary antibodies were used: rabbit GABAB1b(N) directed against the N terminus of GABAB1b (affinity-purified, 1:50 for immunofluorescence (10Benke D. Honer M. Michel C. Bettler B. Mohler H. J. Biol. Chem. 1999; 274: 27323-27330Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar)), rabbit GABAB2(N) directed against the N terminus of GABAB2 (serum, 1:250–1:5000 for immunofluorescence (22Benke D. Michel C. Mohler H. J. Recept. Signal Transduction. 2002; 22: 253-266Crossref PubMed Scopus (22) Google Scholar)), rabbit GABAB1a,b directed against the C terminus of GABAB1 (coupled to protein A-agarose for immunoprecipitation (10Benke D. Honer M. Michel C. Bettler B. Mohler H. J. Biol. Chem. 1999; 274: 27323-27330Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar)), rabbit GABAB2(C) directed against the C terminus of GABAB2 (coupled to protein A-agarose for immunoprecipitation (22Benke D. Michel C. Mohler H. J. Recept. Signal Transduction. 2002; 22: 253-266Crossref PubMed Scopus (22) Google Scholar)), guinea pig GABAB2 directed against the C terminus of GABAB2 (1:1000–1:2000 for immunofluorescence and Western blotting; Chemicon International), mouse Lamp-1 (1:100 for immunofluorescence; Axxora), mouse β1/β2 adaptins (1:250 for immunofluorescence and Western blotting; Sigma), mouse caveolin 1 and mouse caveolin 2 (both 1:250 for immunofluorescence; BD Biosciences). Plasmids—Expression plasmids containing GABAB1b and GABAB2 were described previously (23Sauter K. Grampp T. Fritschy J.M. Kaupmann K. Bettler B. Mohler H. Benke D. J. Biol. Chem. 2005; 280: 33566-33572Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Dynamin-enhanced green fluorescent protein and dynamin K44A-enhance green fluorescent protein were kindly provided by U. Greber (Institute of Zoology, University of Zurich, Switzerland), and arrestin 3-green fluorescent protein was a gift from H. Hatt (Cell Physiology, University Bochum, Germany). Cell Culture—HEK 293 cells were maintained in MEM containing 10% fetal calf serum, 2 mm glutamine and transfected with appropriate plasmids by calcium phosphate precipitation as described previously (23Sauter K. Grampp T. Fritschy J.M. Kaupmann K. Bettler B. Mohler H. Benke D. J. Biol. Chem. 2005; 280: 33566-33572Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Cells were used 2 days after transfection for internalization studies. Immunofluorescence-based Internalization Assay—Living HEK 293 cells transiently transfected with GABAB1b and GABAB2 were incubated with the GABAB1b(N) or GABAB2(N) antibody in buffer A (25 mm HEPES, pH 7.4, 119 mm NaCl, 5 mm KCl, 2 mm CaCl2, 2 mm MgCl2, 30 mm glucose) containing 10% normal goat serum for 30 min at 4 °C. After washing the cells extensively with ice-cold buffer A, the cells were incubated for 10–120 min at 37 °C in the presence or absence of drugs. Control cultures were left at 4 °C, a condition that is nonpermissive for internalization. After washing the cells with ice-cold buffer A, cell surface receptors were labeled with an Alexa Fluor 488-conjugated secondary antibody (1:500; Invitrogen) for 60 min at 4 °C. Cells were then fixed with 4% paraformaldehyde in phosphate-buffered saline containing 4% sucrose, permeabilized for 5 min with 0.5% Triton X-100 in buffer A, followed by staining of internalized receptors with Cy3-conjugated secondary antibodies (1:500; Jackson ImmunoResearch) for 60 min at room temperature. After washing, the cells were embedded in fluorescence mounting medium (DakoCytomation) and analyzed by confocal laser-scanning microscopy (LSM 510 Meta; Zeiss). Images were processed using Imaris (version 4.2, Bitplane, Zurich, Switzerland). Cell Surface ELISA—For labeling of cell surface receptors, living HEK 293 cells expressing GABAB1b/GABAB2 receptors plated on 24-well plates were incubated with the GABAB1b(N) or GABAB2N antibody in buffer A containing 10% normal goat serum for 30 min at 4 °C. After washing the cells extensively with buffer A, they were incubated for 120 min at 37 °C in the presence or absence of 100 μm GABA, 100 μm baclofen, or 100 μm GABA plus 10 μm CGP 56999A in buffer A. Control cells were kept on ice to prevent internalization of receptors (100% controls). Internalization was stopped by washing the cells with ice-cold buffer A, followed by incubation with horseradish peroxidase-conjugated anti-rabbit antibodies for 60 min at 4 °C. After extensive washes with buffer A, horseradish peroxidase activity was determined using tetramethylbenzidine as substrate (0.24 mg/ml tetramethylbenzidine, 0.2 m sodium citrate, pH 3.95, 0.03% H2O2). The color reaction was terminated after 2–5 min by the addition of an equal volume of 1 m H2SO4, and the optical density was recorded at 450 nm in a microplate reader (Synergy HT; Biotek). A nonspecific antibody reaction was determined in parallel cultures of nontransfected HEK 293 cells. Biotinylation Assay—Transfected HEK 293 cells cultured in 6-cm dishes were placed on ice and washed two times with ice-cold buffer A, followed by biotinylation of cell surface proteins with ice-cold sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-Biotin) (Pierce) in buffer A (0.5 mg/ml) for 15 min in the presence of 100 μm chloroquine (to block lysosomal degradation of proteins). After three washes with buffer A containing 100 μm chloroquine and 1% bovine serum albumin, the cells were incubated for 120 min at 37 °C in the presence of 100 μm chloroquine. Cultures for determination of total cell surface and background labeling were left on ice. In all samples, except for total surface labeling, cell surface biotin was cleaved off with glutathione solution (75 mm glutathione, 75 mm NaCl, 10 mm EDTA, 1% bovine serum albumin) two times for 15 min each on ice. Cells were harvested in buffer A, transferred to Eppendorf tubes, and pelleted by centrifugation. Cells were resuspended in 40 μl of 10 mm Tris, pH 8, 150 mm NaCl containing protease inhibitors (complete Mini; Roche Applied Science), supplemented with 1% SDS and heated for 10 min at 80 °C. After dilution with 400 μl of solubilization buffer (10 mm Tris, pH 8, 150 mm NaCl, complete Mini, 1% Triton X-100), the samples were sonified and centrifuged for 30 min at 100,000 × g and 4 °C. Supernatants containing equal amounts of protein were incubated with 60 μl of streptavidin-Sepharose (GE Healthcare) overnight at 4 °C to precipitate biotinylated proteins. The Sepharose beads were washed two times with solubilization buffer, two times with solubilization buffer containing 0.6 m NaCl, and again two times with solubilization buffer. Bound proteins were eluted by incubation in SDS sample buffer for 5 min at 80 °C followed by SDS-polyacrylamide gel electrophoresis and Western blotting using guinea pig GABAB2 antibodies. Chemoluminescence (Super Signal West Pico Chemoluminescence Substrate; Pierce) signals were captured using a Fuji LAS-1000 plus imaging system (Fujifilm, Tokyo, Japan), and immunoreactive bands were quantified with the AIDA software (version 3.25; Raystest, Pforzheim, Germany). Immunoprecipitation—Immunoprecipitation of GABAB receptors from rat brain tissue was performed essentially as described previously (10Benke D. Honer M. Michel C. Bettler B. Mohler H. J. Biol. Chem. 1999; 274: 27323-27330Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). HEK 293 cells transiently expressing GABAB1 and GABAB2 were harvested in 10 mm Tris, pH 8, 150 mm NaCl, protease inhibitor mixture (complete Mini; Roche Applied Science), homogenized, and solubilized using 0.5% sodium deoxycholate for 1 h on ice followed by centrifugation at 100,000 × g for 30 min. For immunoprecipitation, GABAB1a.b or GABAB2(C) antibodies covalently coupled to protein A-agarose were added to the supernatant and incubated overnight at 4 °C. Immune complexes were collected by centrifugation and extensively washed with 10 mm Tris, pH 8, 150 mm NaCl, protease inhibitor mixture, 1% Triton X-100. Bound proteins were released by incubation with 2× sample buffer for SDS polyacrylamide gel electrophoresis for 15 min at 65 °C and analyzed by Western blotting. GABAB Receptors Undergo Constitutive but Not Agonist-induced Internalization—In order to visualize internalization of GABAB receptors transiently expressed in HEK 293 cells, surface receptors of living cells were labeled with an antibody directed against the N terminus of GABAB1b at 4 °C, a condition that is nonpermissive for internalization. After labeling, the cells were incubated for different time intervals at 37 °C in the presence or absence of GABAB receptor agonists. Cell surface and internalized receptors were then differentially visualized. Cell surface receptors were stained with a secondary antibody coupled to a green fluorescent fluorophor and, after fixation and permeabilization of the cells, internalized receptors were labeled with a secondary antibody carrying a red fluorescent fluorophor. Small clusters of internalized receptors, which were predominantly localized in the vicinity of the cell surface, were already detected after 10 min (Fig. 1A). After 30, 60, and 120 min, an increasing intracellular accumulation of GABAB receptors in large clusters was observed. Stimulation of receptors with either 100 μm GABA or baclofen did not result in an appreciable increase of receptor internalization (Fig. 1A). Thus, GABAB receptors were rapidly constitutively internalized, but there was no evidence for agonist-induced internalization. The extent of internalization was determined by cell surface ELISA. Surface GABAB receptors on living HEK 293 cells cultured in 24-well plates were labeled with GABAB1b(N) or GABAB2(N) antibodies, respectively, and subsequently incubated for 120 min at 37 °C in the presence or absence of agonists. The amount of cell surface receptors was then quantified with a microplate reader using a secondary antibody coupled to horseradish peroxidase. In line with the results from the immunofluorescence-based internalization assay, a substantial fraction of cell surface staining was lost after 120 min (47 ± 7%), indicating considerable constitutive endocytosis of GABAB receptors (Fig. 1B). Activation of GABAB receptors with GABA or baclofen did not lead to further loss of cell surface receptors (GABA, 48 ± 4% internalization; baclofen, 48 ± 7% internalization). The loss of cell surface staining was blocked by the addition of hypertonic concentrations of sucrose, a condition that is known to inhibit clathrin-mediated endocytosis (Fig. 1B). To further exclude the presence of agonist-induced internalization of GABAB receptors, cells were stimulated with GABA or baclofen for 120 min prior to labeling and quantifying cell surface receptors. In case of agonist-induced internalization, a loss of cell surface staining would be expected in the cultures stimulated with agonist. However, no loss of cell surface receptors was observed (Fig. 1C). Most G protein-coupled receptors displaying agonist-induced internalization recruit an arrestin protein, which serves as a signal for endocytosis (24Marchese A. Chen C. Kim Y.M. Benovic J.L. Trends Biochem. Sci. 2003; 28: 369-376Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). However, co-expression of arrestin 3-green fluorescent protein with GABAB receptors in HEK 293 cells did not result in a redistribution of arrestin 3-green fluorescent protein from the cytoplasm to the plasma membrane in response to GABA stimulation, being in line with the lack of agonist-promoted internalization (not shown). Internalization of GABAB Receptors Is Not Induced by Antibodies—In an antibody-dependent internalization assay, binding of the antibody to the receptors may induce their endocytosis. To determine whether the antibodies used in the internalization assay affect endocytosis of GABAB receptors, we analyzed the effect of antibody concentration on the extent of internalization. If the antibodies used for labeling would induce internalization of GABAB receptors, we expected a greater extent of internalization with increasing antibody concentration. Surprisingly, high antibody concentrations appeared to inhibit internalization in the immunofluorescence based assay. At an antibody dilution of 1:250, only a few small clusters of internalized receptors were observed in proximity to the cell membrane after 120 min, whereas at higher antibody dilutions (1:1000 and 1:5000), large clusters of internalized receptors abundantly accumulated within the cells (Fig. 2A). Quantification of this effect by cell surface ELISA revealed at high antibody concentration (1:250) an internalization of 21 ± 4% of cell surface receptors after 120 min and at low antibody concentrations an internalization of about 40% (1:1000, 36 ± 6%; 1:5000, 45 ± 9%) (Fig. 2B). Thus, at high concentrations, the antibodies inhibited endocytosis of GABAB receptors. To further exclude that the antibodies used induce internalization of GABAB receptors, we analyzed endocytosis of receptors with an antibody-independent cell surface biotinylation assay. For this assay, all surface proteins were biotinylated for 15 min at 4 °C and incubated then for 120 min at 37 °C in the presence or absence of GABAB receptor agonists to allow endocytosis. Biotin on cell surface proteins was then cleaved off with glutathione, leaving internalized proteins biotinylated. After solubilization of cells, internalized biotinylated proteins were purified with streptavidin-Sepharose, and the amount of biotinylated GABAB receptor was determined by Western blotting with GABAB2 antibodies. As in the antibody-based assays, we observed robust constitutive but no agonist-induced internalization of GABAB receptors in the biotinylation assay (Fig. 3A). Quantification of the blots revealed a similar extent of receptor endocytosis as in the antibody-dependent assay (about 40% after 120 min) (Fig. 3B). These results show that a substantial fraction of cell surface GABAB receptors expressed in HEK 293 cells constitutively internalize. GABAB Receptors Internalize via the Dynamin- and Clathrin-dependent Pathway—To determine the mechanism by which GABAB receptors internalize, we tested different inhibitors for their ability to block endocytosis. First, we analyzed whether GABAB receptors internalize via a dynamin-dependent mechanism by overexpressing a GTP-binding and hydrolysis-defective dynamin mutant (dynamin K44A) that has been shown to restrain invaginated pits from pinching off (25Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1048) Google Scholar). Although overexpression of wild type dynamin did not affect internalization of GABAB receptors (not shown), dynamin K44A completely blocked endocytosis of the receptors (Fig. 4). Next, we analyzed whether GABAB receptors internalized via a clathrin- or caveolin-dependent mechanism. Hypertonic concentrations of sucrose and chlorpromazine both inhibit the formation of clathrin-coated pits (26Wang L.H. Rothberg K.G. Anderson R.G. J. Cell Biol. 1993; 123: 1107-1117Crossref PubMed Scopus (968) Google Scholar, 27Heuser J.E. Anderson R.G. J. Cell Biol. 1989; 108: 389-400Crossref PubMed Scopus (791) Google Scholar) and thus have been widely used to inhibit clathrin-dependent endocytosis. In the presence of sucrose (450 mm) or chlorpromazine (100 μg/ml), internalization was completely blocked (Fig. 4A). However, treatment of cells with nystatin (50 μg/ml) or filipin (5 μg/ml), inhibitors of caveolae/raft-dependent endocytosis (28Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y.S. Glenney J.R. Anderson R.G. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1899) Google Scholar, 29Schnitzer J.E. Oh P. Pinney E. Allard J. J. Cell Biol. 1994; 127: 1217-1232Crossref PubMed Scopus (787) Google Scholar), did not affect internalization of GABAB receptors (Fig. 4A). To further substantiate the finding that GABAB receptors may predominantly internalize via the clathrin-dependent pathway, we analyzed their potential co-localization with the AP2 complex, which has been implicated in the recruitment of plasma membrane proteins into clathrin-coated pits (30Schmid S.L. Annu. Rev. Biochem. 1997; 66: 511-548Crossref PubMed Scopus (675) Google Scholar). The antibody used recognized the β2-adaptin subunit of the AP2 complex located predominantly at the plasma membrane and in addition also the β1-adaptin subunit of the AP1 complex, which is restricted to clathrin-coated membranes of the trans-Golgi network (30Schmid S.L. Annu. Rev. Biochem. 1997; 66: 511-548Crossref PubMed Scopus (675) Google Scholar). In order to detect co-localization of GABAB receptors specifically with β2-adaptin, we labeled exclusively cell surface GABAB receptors with GABAB2N antibodies and let them internalize for 60 min, followed by fixation and permeabilization of the cells and staining with the β-adaptin antibody. This experimental set-up prevented the co-detection of a potential co-localization with β1-adaptin of intracellular GABAB receptors in the exocytotic pathway. Under these experimental conditions, we observed a frequent co-localization of β2-adaptin with cell surface and also with internalized GABAB receptors (Fig. 4B). Under the same experimental conditions, we found only a rare co-localization of GABAB receptors with caveolin 1 or caveolin 2, supporting the view that GABAB receptors in HEK 293 cells predominantly internalize via clathrin-coated pits (Fig. 4B). The immunofluorescence data suggested the association of GABAB receptors with the AP2 complex and thus endocytosis via clathrin-coated pits. We therefore tested for a direct interaction of GABAB receptors with β-adaptin by immunoprecipitation using GABAB1a,b or GABAB2(C) antibodies coupled to protein A-agarose. As expected, β-adaptin immunoreactivity was detected in both GABAB receptor immunoprecipitates (Fig. 4C). Although we cannot exclude a possible association also with β1-adaptin, the immunoprecipitation experiment in combination with the co-localization study indicates a direct association of GABAB receptors with the AP2 complex and thus an endocytosis via clathrin-coated pits. GABAB Receptors Are Targeted to Lysosomes for Degradation—Internalized receptors can be principally directed to two distinct destinations: to lysosomes for degradation or recycled back to the cell surface. To analyze the fate of internalized GABAB receptors, we first tested whether they are able to recycle back to the cell surface. Cell surface receptors were labeled with antibodies and allowed to internalize for 90 min at 37 °C. Subsequently, antibodies bound to cell surface GABAB receptors were stripped off with two consecutive washes with 0.2 m glycine, pH 2.5, 0.5 m NaCl for 3 min. The stripped cells were then further incubated at 37 °C for 10–30 min to allow receptors to recycle back to the cell surface. Putative recycled receptors were stained with a green fluorescent secondary antibody, whereas internalized receptors were stained, after fixation and permeabilization of the cells, with a red fluorescent secondary antibody. Under these conditions, no recycling of GABAB receptors was observed (Fig. 5A). To ensure that the antibody strippin
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