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Originally published In Press as doi:10.1074/jbc.M702626200 on June 20, 2007

J. Biol. Chem., Vol. 282, Issue 33, 24157-24165, August 17, 2007
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{gamma}-Aminobutyric Acid Type B Receptors Are Constitutively Internalized via the Clathrin-dependent Pathway and Targeted to Lysosomes for Degradation*

Thomas Grampp, Kathrin Sauter1, Branko Markovic, and Dietmar Benke2

From the Institute of Pharmacology and Toxicology, University of Zurich, Zurich 8057, Switzerland

Received for publication, March 27, 2007 , and in revised form, June 12, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {gamma}-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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
GABAB3 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. 1). 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 (2–7). Two main variants of GABAB1 have been reported (GABAB1a and GABAB1b (8)), which are generated by alternative promoter usage (9) and differ solely in their N-terminal domain. 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 (10–13).

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 (14). Arrestins often enhance phosphorylation, sterically interfere with binding of the G protein, and function as a signal for receptor endocytosis (15). 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 (16, 17), kinase activity is not required (16). 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 (18). 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 (16, 19, 20). Interestingly, Fairfax et al. (19) 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 (21). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—The following primary antibodies were used: rabbit GABAB1b(N) directed against the N terminus of GABAB1b (affinity-purified, 1:50 for immunofluorescence (10)), rabbit GABAB2(N) directed against the N terminus of GABAB2 (serum, 1:250–1:5000 for immunofluorescence (22)), rabbit GABAB1a,b directed against the C terminus of GABAB1 (coupled to protein A-agarose for immunoprecipitation (10)), rabbit GABAB2(C) directed against the C terminus of GABAB2 (coupled to protein A-agarose for immunoprecipitation (22)), 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 beta1/beta2 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 (23). 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 (23). 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 x 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 (10). 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 x 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 2x sample buffer for SDS polyacrylamide gel electrophoresis for 15 min at 65 °C and analyzed by Western blotting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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).


Figure 1
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  		FIGURE 1.
Constitutive internalization of GABAB receptors. A, immunofluorescence assay revealed rapid constitutive internalization of GABAB receptors in HEK 293 cells transiently expressing GABAB1b and GABAB2. Cell surface receptors of living HEK 293 cells were labeled for 30 min at 4 °C with an antibody directed against the N terminus of GABAB1b. Subsequently, the cells were incubated for different time intervals at 37 °C in the presence or absence of 100 µM GABA or baclofen (GABAB receptor agonists). Cell surface receptors were visualized by staining living cells with a secondary antibody coupled to a green fluorescent fluorophor and internalized receptors were stained after fixation and permeabilization of the cells with secondary antibody carrying a red fluorescent fluorophor. Already after 10 min, small clusters of internalized receptors close to the cell membrane were observed. Activation of receptors with 100 µM baclofen or 100 µM GABA did not affect receptor internalization. Bar, 10 µm. B, quantification of GABAB receptor internalization using cell surface ELISA. HEK 293 cells transiently expressing GABAB1b and GABAB2 were labeled for 30 min at 4 °C with antibodies and were subsequently incubated for 120 min at 37 °C in the presence or absence of 100 µM GABA, 100 µM GABA + 10 µM CGP 56999A (GABAB receptor antagonist), or 450 mM sucrose. After incubation of cells for 60 min at 4 °C with a secondary antibody coupled to horseradish peroxidase, tetramethylbenzidine-based color development was determined using a microplate reader. Controls were left at 4 °C to prevent internalization and were taken as 100% values. The data represent the mean ± S.D. of six independent experiments done in quadruplicate. Internalization was not promoted by GABA. Hypertonic sucrose, a blocker of clathrin-mediated endocytosis, completely prevented internalization. C, lack of agonist-induced internalization. HEK 293 cells transiently expressing GABAB1b and GABAB2 were incubated for 120 min at 37 °C in the presence or absence of either 100 µM GABA or 100 µM + 10 µM CGP 56999A. After fixation and labeling of cell surface receptors with GABAB1b(N) antibodies and a secondary antibody coupled to horseradish peroxidase, tetramethylbenzidine-based color development was determined using a microplate reader. Cells incubated in the absence of GABA were taken as 100% controls. The data represent the mean ± S.D. of four independent experiments done in quadruplicate. Stimulation of cells with GABA did not result in a loss of cell surface receptors.

 
Most G protein-coupled receptors displaying agonist-induced internalization recruit an arrestin protein, which serves as a signal for endocytosis (24). 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 (25). 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).


Figure 2
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  		FIGURE 2.
Internalization of GABAB receptors is not induced by antibodies. A, immunofluorescence assay revealed inhibition of GABAB receptor internalization at high antibody concentrations. Living HEK 293 cells transiently expressing GABAB1b and GABAB2 were labeled for 30 min at 4 °C with GABAB2(N) antiserum at the indicated dilutions and tested for internalization (120 min at 37 °C). At high antibody concentration (dilution 1:250), only a few small clusters of internalized receptors were observed, whereas at low antibody concentration (dilutions 1:1000 and 1:5000), large clusters of internalized receptors abundantly accumulated within the cells. Bar, 10 µm. B, quantification of inhibitory effect of antibodies by cell surface ELISA. Cell surface ELISA on HEK 293 cells transiently expressing GABAB1b and GABAB2 revealed an inhibition of GABAB receptor internalization at high antibody concentration (dilution 1:250). The data represent the mean ± S.D. of five independent experiments done in quadruplicate.

 
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 (26, 27) 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 (28, 29), did not affect internalization of GABAB receptors (Fig. 4A).


Figure 3
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  		FIGURE 3.
Demonstration of GABAB receptor internalization using a cell surface biotinylation assay. A, surface proteins of HEK 293 cells expressing GABAB1b and GABAB2 were biotinylated for 15 min at 4 °C and incubated thereafter for 120 min at 37 °C in the presence or absence of 100 µM GABA or 100 µM GABA + 10 µM CGP 55999A. Subsequently, biotin on cell surface proteins was cleaved off with glutathione, and biotinylated internalized proteins were purified with streptavidin-Sepharose. The presence and abundance of GABAB receptors in the streptavidin-Sepharose eluate was determined by Western blotting with GABAB2 antibodies. Each lane represents the signal from one individual culture plate. Total surface receptors, cells were left on ice to prevent internalization and were not subjected to cleavage with glutathione; nonspecific, cells were left on ice to prevent internalization and were subjected to cleavage with glutathione; internalized receptors, cells were incubated for 120 min at 37 °C to allow endocytosis, followed by stripping off cell surface biotin with glutathione. Robust constitutive but no agonist-induced internalization of GABAB receptors was observed. B, quantification of the Western blots shown in A. Chemoluminescence signals captured using a Fuji LAS-1000 plus imaging system were quantified with the AIDA software. Signals of "total surface receptors" (control) were set to 100%. Signals for "nonspecific" (i.e. cell surface biotin that was not cleaved off by glutathione) were subtracted from signals of "internalized receptors." The data represent the mean ± S.D. of four independent experiments performed in duplicate.

 
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 (30). The antibody used recognized the beta2-adaptin subunit of the AP2 complex located predominantly at the plasma membrane and in addition also the beta1-adaptin subunit of the AP1 complex, which is restricted to clathrin-coated membranes of the trans-Golgi network (30). In order to detect co-localization of GABAB receptors specifically with beta2-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 beta-adaptin antibody. This experimental set-up prevented the co-detection of a potential co-localization with beta1-adaptin of intracellular GABAB receptors in the exocytotic pathway. Under these experimental conditions, we observed a frequent co-localization of beta2-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 beta-adaptin by immunoprecipitation using GABAB1a,b or GABAB2(C) antibodies coupled to protein A-agarose. As expected, beta-adaptin immunoreactivity was detected in both GABAB receptor immunoprecipitates (Fig. 4C). Although we cannot exclude a possible association also with beta1-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.


Figure 4
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  		FIGURE 4.
GABAB receptors are internalized by the dynamin and clathrin-dependent pathway. A, HEK 293 cells expressing GABAB1b and GABAB2 were subjected to the immunofluorescence-based internalization assay using GABAB2(N) antibodies for 120 min at 37 °C in the absence (control) or presence of a dominant-negative mutant of dynamin (dynamin K44A), 450 mM sucrose, 100 µg/ml chlorpromazine, 50 µg/ml nystatin, or 5 µg/ml filipin. Although nystatin and filipin, inhibitors of the caveolin-dependent pathway, did not affect internalization of GABAB receptors, dynamin K44A, sucrose, and chlorpromazine completely blocked endocytosis, indicating that receptors were internalized via the classical dynamin and clathrin-dependent pathway. For the sake of clarity, expression of dynamin K44A is not depicted in the cells shown. Bar, 10 µm. B, GABAB receptors co-localize with beta-adaptin of the AP2 complex. Cell surface GABAB receptors were labeled with GABAB2(N) antibodies for 30 min at 4 °C and allowed to endocytose for 120 min at 37 °C. Subsequently, cells were fixed, permeabilized, and double-stained for GABAB2 (red, cell surface and internalized receptors) and beta-adaptin (green), caveolin 1 (green), or caveolin 2 (green). beta-Adaptin frequently co-localized with cell surface and internalized GABAB receptors, whereas caveolin 1 and caveoline 2 displayed only rare co-localization with GABAB receptors. Bar, 10 µm. C, GABAB receptors directly associate with beta-adaptins. HEK 293 cells expressing GABAB1b and GABAB2 were harvested, solubilized with 0.5% deoxycholate, and subjected to immunoprecipitation using GABAB1a,b, GABAB2(C), or nonimmune antibodies (Control) coupled to protein A-agarose. The immunoprecipitates were analyzed by Western blotting for the presence of GABAB receptors and beta-adaptin. In GABAB1 and GABAB2 immunoprecipitates, strong beta-adaptin signals were observed, indicating the association of GABAB receptors with beta-adaptin. Faint signals for GABAB2 and beta-adaptin were also detected in the control precipitation using nonimmune antibodies and thus represent background staining due to technical reasons. Control, precipitate using nonimmune antibodies; Ab, antibody; IP, immunoprecipitate.

 


Figure 5
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  		FIGURE 5.
Fate of endocytosed GABAB receptors. A, internalized GABAB receptors are not recycled back to the cell surface. Cell surface GABAB receptors expressed on HEK 293 cells were labeled with GABAB2(N) antibodies for 90 min at 37 °C, a condition that permits endocytosis of receptors during the labeling period. Thereafter, antibodies bound to cell surface GABAB receptors were removed with two washes of 0.2 M glycine, pH 2.5, 0.5 M NaCl for 3 min, followed by further incubation of the cells at 37 °C for 10–30 min. Potential cell surface receptors (recycled) were stained at 4 °C with a green fluorescent secondary antibody, followed by fixation, permeabilization, and staining of internalized receptors with a red fluorescent secondary antibody. No recycling of GABAB receptors (green) was detected. Bar, 10 µm. B, GABAB receptors are targeted to lysosomes for degradation. Cell surface receptors on HEK 293 cells were labeled with GABAB2(N) antibodies for 30 min at 4 °C and allowed to endocytose for 60 min at 37 °C in the presence or absence of 100 µM chloroquine (a blocker of lysosomal function). Cell surface receptors were then stained with a green fluorescent secondary antibody for 60 min at 4 °C, and cells were incubated again at 37 °C for 30–120 min (chase). Subsequently, the cells were fixed, permeabilized, and stained for internalized receptors with a red fluorescent secondary antibody. Cell surface receptors (chase: 0 min, green) were progressively internalized at 30, 60, and 120 min of incubation at 37 °C and were found to be largely co-localized (yellow) with formerly internalized receptors (red). In the absence of chloroquine, GABAB receptor staining strongly decreased after 60 and 120 min, indicating degradation of GABAB receptors. The presence of chloroquine prevented the loss of GABAB receptor staining and resulted in a pronounced intracellular accumulation of the receptors. Bar, 10 µm. C, inhibition of lysosomal proteases but not of proteasomal activity leads to an increased intracellular accumulation of endocytosed GABAB receptors. HEK 293 cells expressing GABAB1b and GABAB2 were subjected to the internalization assay for 2 h in the absence (control) or presence of inhibitors of lysosomal function (100 µM chloroquine, 100 µM leupeptin, or 1 µM pepstatin A) or inhibitors of proteasomal activity (10 µM MG132 or 50 µM lactacystin). Cell surface receptors were stained with a green fluorescent secondary antibody, and internalized receptors were stained with a red fluorescent secondary antibody. Bar, 10 µm. D, internalized GABAB receptors do not accumulate in lysosomes. HEK 293 cells expressing GABAB1b and GABAB2 were subjected to the immunofluorescence internalization assay for 120 min at 37 °C using GABAB2(N) antibodies in the absence or presence of 100 µM chloroquine (to inhibit lysosomal activity). Cell surface receptors were stained with a blue fluorescent secondary antibody, internalized receptors were stained with a red fluorescent secondary antibody, and lysosomes were labeled using a monoclonal antibody directed against Lamp 1 (green). No co-localization (yellow) of internalized receptors (red) with the lysosomal marker Lamp 1 (green) was observed, irrespective of blocking lysosomal activity with chloroquine. Thus, internalized GABAB receptors appear to accumulate in an endosomal compartment.

 
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 stripping procedure did not interfere with recycling of receptors, a second series of experiments was done, where the stripping step was omitted and bound cell surface antibodies were instead saturated with secondary antibody. Again, no recycling of receptors back to the cell surface was observed (not shown).

Since internalized GABAB receptors appear not to recycle back to the cell surface, they are expected to be targeted to lysosomes and eventually be degraded. To test for lysosomal degradation, cell surface receptors were labeled with antibodies and allowed to internalize for 60 min at 37 °C. After staining of the remaining cell surface receptors with a green fluorescent secondary antibody, cells were incubated again at 37 °C for 30–120 min and were subsequently fixed, permeabilized, and stained for internalized receptors with a red fluorescent secondary antibody. Under these conditions, cell surface labeled receptors were progressively endocytosed with time and co-localized with formerly internalized receptors (Fig. 5B, top). In addition, staining of cell surface and internalized receptors strongly diminished after 60 and 120 min, indicating degradation of the receptors (Fig. 5B, upper panel). Pretreatment of cells with the lysosomotropic drug chloroquine, which increases endosomal and lysosomal pH and impairs lysosomal function (31, 32), completely prevented the loss of staining and led to the co-localization of signals in large intracellular clusters (Fig. 5B, bottom).

Furthermore, blocking directly lysosomal proteases with either 100 µM leupeptin or 1 µM pepstatin A likewise resulted in an increased intracellular accumulation of internalized GABAB receptors, supporting the view that endocytosed GABAB receptors are degraded in lysosomes (Fig. 5C). In contrast, blocking proteasomal activity with 10 µM MG 132 or 50 µM lactacystin did not affect intracellular accumulation of GABAB receptors (Fig. 5C). These experiments indicate that internalized GABAB receptors are predominantly degraded by lysosomes with no appreciable contribution of proteasomal activity.

Since inhibition of lysosomal activity resulted in a pronounced formation of intracellular clusters, we expected that the receptors accumulate in lysosomes. Surprisingly, co-localization experiments of internalized GABAB receptors with the lysosomal marker Lamp 1 did not reveal any overlap in localization, even after blocking lysosomal activity with chloroquine (Fig. 5D). Therefore, internalized GABAB receptors appear to accumulate in an endosomal compartment before entering lysosomes for degradation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Endocytosis of G protein-coupled receptors involves a sequence of highly orchestrated molecular events. It serves as a mechanism for regulating cell surface levels of receptors and thereby contributes to signaling strength. Currently, the process of GABAB receptor endocytosis is poorly understood. The results of the present study suggest that heterologously expressed GABAB receptors are constitutively endocytosed predominantly via the dynamin- and clathrin-dependent pathway and are finally sorted to lysosomes for degradation.

To gain the first insights into the mechanisms involved in endocytosis of GABAB receptors, we expressed in HEK 293 cells the GABAB1b/GABAB2 combination, which is the most abundant GABAB receptor subtype in the adult brain (10, 11). Heterologous expression of receptors was chosen, since it is a robust, easy to manipulate and to evaluate system. Using an immunofluorescence assay, robust fast constitutive internalization was detected. Already after 10 min, the first clusters of internalized receptors were observed in the proximity of the cell membrane, and a strong intracellular accumulation of receptors was found after 2 h. Interestingly, after 2 h, only 50–60% of cells expressing GABAB receptors displayed intracellular accumulation of receptors (not shown), and its extent varied among cells as well as experiments. However, after blocking lysosomal activity, we observed in almost the entire population of cells expressing GABAB receptors a pronounced accumulation of receptors. This observation indicates that in some cells, targeting of receptors to lysosomes and subsequent degradation may be very efficient, thus preventing the intracellular accumulation of a pool of endocytosed receptors.

Endocytosed GABAB receptors were found to accumulate in an intracellular compartment before being degraded. Since internalized receptors did not co-localize with the lysosomal marker protein Lamp 1, accumulation of internalized GABAB receptors did not take place in lysosomes but most likely in an endosomal sorting compartment. This observation suggests that sorting of internalized receptors to lysosomes is a relatively slow process in most cells, which leads to the accumulation of endocytosed receptors. Even after blocking lysosomal degradation with chloroquine, endocytosed GABAB receptors were not detected in lysosomes. However, chloroquine does not only inhibit proteolytic activity by enhancing the acidic pH of lysosomes; it also interferes with endosomal sorting and recycling (30, 31). Therefore, in the presence of chloroquine also sorting of GABAB receptors from an endosomal compartment to lysosomes appeared to be perturbed.

Quantification of GABAB receptor internalization by cell surface ELISA indicated that about 40–50% of cell surface receptors were endocytosed within 2 h. A similar amount of internalization was found using a cell surface biotinylation assay, where directly the amount of intracellularly accumulated receptors was determined in the presence of chloroquine to prevent lysosomal degradation. Since both the antibody-based and the biotinylation-based assay yielded a similar extent of GABAB receptor endocytosis, it is unlikely that binding of the antibody to GABAB receptors triggered endocytosis. In contrast, high concentrations of antibodies seemed to stabilize GABAB receptors at the cell surface, as indicated by the lower extent of receptor internalization observed in the immunocytochemical assay and by cell surface ELISA. It may well be that the failure to detect GABAB receptor internalization in previous studies is due to the use of high antibody concentrations that inhibited endocytosis of the receptors.

In agreement with previous studies (16, 19, 20), no agonist-induced internalization of GABAB receptors was observed upon prolonged agonist treatment (10–120 min), irrespective of the method used. Thus, unlike many other G protein-coupled receptors, GABAB receptors display robust fast constitutive but no agonist-promoted internalization. However, with the methods used, we cannot exclude the possibility of very fast agonist-induced endocytosis of GABAB receptors coupled with rapid recycling back to the cell membrane. In this respect, it is interesting to note that a recent study analyzing GABAB receptor-induced internalization of calcium channels in cultured chick sensory neurons suggests the fast endocytosis of a complex of calcium channels, arrestin, and GABAB receptor 20 s after baclofen stimulation (33). Intracellular co-localization of calcium channels and arrestin with GABAB receptors strongly diminished already after 1 min, indicating a rapid and transient phenomenon. Further experiments are required to clarify the existence of a fast and transient agonist-induced endocytosis.

Endocytosis of GABAB receptors was found to be mediated predominantly via the dynamin- and clathrin-dependent pathway, as indicated by its inhibition after overexpressing a dominant negative mutant of dynamin (dynamin K44A) that has been shown to restrain invaginated pits from pinching off (25) and hypertonic concentrations of sucrose as well as chlorpromazine, both of which inhibit the formation of clathrin-coated pits (26, 27). In addition, cell surface and internalized GABAB receptors frequently co-localized with the beta-adaptin subunit of the AP2 complex, which recruits plasma membrane proteins into clathrin-coated pits (30). This finding was further substantiated by co-immunoprecipitation experiments, which suggest a direct association of the AP2 complex with GABAB receptors.

Although GABAB receptors have been abundantly detected in lipid rafts upon expression in Chinese hamster ovary cells (34), a blocker of raft/caveolin-dependent endocytosis (nystatin and filipin (28, 29)) did not affect internalization of GABAB receptors. In addition, we found that caveolin 1 and caveolin 2 only rarely co-localized with GABAB receptors expressed in HEK 293 cells. Thus, caveolin-dependent endocytosis appears not to play a significant role in constitutive internalization of GABAB receptors expressed in HEK 293 cells. Even an abundant localization of receptors in lipid rafts does not necessarily imply their internalization via a raft/caveolin-dependent mechanism, as recently demonstrated for the apolipoprotein E receptor 2. Although apolipoprotein E receptor 2 has been shown to be associated with lipid rafts and caveolin 1, the receptor was endocytosed via the clathrin-mediated pathway (35). In line with this observation, G protein-coupled receptors have been found to predominantly use the clathrin-dependent endocytotic pathway, whereas caveolin and clathrin/caveolin-independent pathways appear to be used to a minor extent (24, 36).

Once internalized, GABAB receptors accumulate in a Lamp 1-negative endosomal sorting compartment (see above) before being degraded. Degradation of internalized GABAB receptors was detected as a strong decrease of antibody labeling 1 h after internalization, which was completely prevented by the addition of chloroquine to inhibit lysosomal degradation. The view of lysosomal degradation of internalized GABAB receptors was further supported by a direct inhibition of lysosomal proteases with either leupeptin or pepstatin A, which led to an increased intracellular accumulation of endocytosed GABAB receptors. Further experiments are required to demonstrate whether GABAB receptors indeed accumulate in lysosomes upon inhibition of lysosomal proteases. In contrast, the proteasome appears not to significantly contribute to the degradation of GABAB receptors, since inhibition of proteasomal activity with MG 132 or lactacystin did not result in an enhanced intracellular accumulation of GABAB receptors. Thus, the data suggest that GABAB receptors expressed in HEK 293 cells are endocytosed via the classical dynamin and clathrin-dependent pathway, accumulate in an endosomal sorting compartment, and are finally degraded in lysosomes.

It is currently unclear whether the mechanism of GABAB receptor endocytosis and degradation detected in the present study using heterologously expressed GABAB receptors also applies for neuronal GABAB receptors. So far, data on primary cultured neurons indicate the lack of agonist-induced internalization (16, 19), which is well in line with the findings of the present study. However, using a biotinylation assay, Fairfax et al. (19) were unable to detect the build-up of an intracellular pool of endocytosed GABAB receptors even after blocking lysosomal activity with leupeptin. Thus, they hypothesized that GABAB receptors may be targeted directly from the surface to the proteasome for degradation. In contrast, the presence of classical GABAB receptor internalization and degradation in lysosomes is well supported by recent immunohistochemical data. In neurons of the supraoptic nucleus, Richards et al. (37) detected GABAB receptors in early endosomes and in lysosomes. Therefore, it is likely that neuronal GABAB receptors follow similar mechanisms of internalization and possibly degradation, as revealed in the present study.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Institute of Microbiology, Dept. of Biology, ETH Zurich, Zurich 8093, Switzerland. Back

2 To whom correspondence should be addressed: Institute of Pharmacology and Toxicology, University Zürich, Winterthurerstrasse 190, Zürich 8057, Switzerland. Tel.: 41-44-635-5930; Fax: 41-44-635-6874; E-mail: benke{at}pharma.unizh.ch.

3 The abbreviations used are: GABAB, {gamma}-aminobutyric acid, type B; HEK 293, human embryonic kidney 293; NSF, N-ethylmaleimide-sensitive fusion protein; ELISA, enzyme-linked immunosorbent assay. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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