b2-Adrenergic Receptor Down-regulation EVIDENCE FOR A PATHWAY THAT DOES NOT REQUIRE ENDOCYTOSIS*

Sustained activation of most G protein-coupled receptors causes a time-dependent reduction of receptor density in intact cells. This phenomenon, known as downregulation, is believed to depend on a ligand-promoted change of receptor sorting from the default endosomeplasma membrane recycling pathway to the endosomelysosome degradation pathway. This model is based on previous studies of epidermal growth factor (EGF) receptor degradation and implies that receptors need to be endocytosed to be down-regulated. In stable clones of L cells expressing b2-adrenergic receptors (b2ARs), sustained agonist treatment caused a time-dependant decrease in both b2AR binding sites and immuno-detectable receptor. Blocking b2AR endocytosis with chemical treatments or by expressing a dominant negative mutant of dynamin could not prevent this phenomenon. Specific blockers of the two main intracellular degradation pathways, lysosomal and proteasomeassociated, were ineffective in preventing b2AR downregulation. Further evidence for an endocytosis-independent pathway of b2AR down-regulation was provided by studies in A431 cells, a cell line expressing both endogenous b2AR and EGF receptors. In these cells, inhibition of endocytosis and inactivation of the lysosomal degradation pathway did not block b2AR down-regulation, whereas EGF degradation was inhibited. These data indicate that, contrary to what is currently postulated, receptor endocytosis is not a necessary prerequisite for b2AR down-regulation and that the inactivation of b2ARs, leading to a reduction in binding sites, may occur at the plasma membrane.

A recurrent theme in G protein-coupled receptor physiology is that the intensity of the functional response to hormones wanes over time despite the continuous presence of the stimu-lus. This phenomenon of hormonal tolerance, also known as desensitization, reflects multiple molecular mechanisms of receptor regulation. For most receptors, the predominant mechanism of desensitization is the phosphorylation-dependent uncoupling from G proteins (1). For some receptors, such as the m3-muscarinic acetylcholine receptor, desensitization is the consequence of receptor endocytosis, which decreases the number of surface receptors that may be activated by the hormone (2). Desensitization may also be caused by down-regulation, the ligand-dependent reduction of total receptor number, as in the case of thrombin receptors. Thrombin proteolytic activity cleaves the amino terminus of the receptor, unmasking a new amino-terminal peptide (3); this peptide activates the receptor irreversibly, promoting its endocytosis and its subsequent degradation in lysosomes (4).
Desensitization of ␤ 2 -adrenergic receptors (␤ 2 ARs) 1 is mostly dependent on rapid phosphorylation by the cAMP-dependent protein kinase and G protein-coupled receptor kinases (5)(6)(7). However, although the ␤ 2 AR is less rapidly affected by downregulation than the thrombin receptor, long term agonist-promoted ␤ 2 AR down-regulation significantly contributes to the desensitization and is additive to rapid inactivation resulting from receptor uncoupling (8). Supporting the physio-pathological significance of ␤ 2 AR down-regulation are studies demonstrating that the development of heart failure may be associated with ␤ 2 AR down-regulation (9). In addition, studies on ␤ 2 AR polymorphism showed that alleles, which display accelerated ligand-dependent down-regulation in vitro (10), are associated with altered desensitization to ␤-adrenergic bronchodilators in asthmatic patients (11). ␤ 2 AR down-regulation involves at least two pathways. The first is the reduction in receptor mRNA steady-state level resulting from destabilization of the transcript (12)(13)(14). The consequences of such a phenomenon, however, become apparent only after many hours of sustained activation once the number of pre-existing ␤ 2 ARs has decreased. The second pathway of ␤ 2 AR down-regulation is detectable as early as 1 h following receptor activation (15); it consists in the loss of pre-existing ligand binding sites. Based on the observation that, upon removal of agonist, the recovery of ␤ 2 ARs to control levels requires neosynthesis, it was postu-lated that the loss of binding sites was the consequence of receptor degradation (16 -18). However, there are no reports in the literature showing that the loss of binding sites is associated with receptor proteolysis. In addition, the processes leading to the accelerated rate of receptor degradation and its topology within the cell have not been established unambiguously. The currently accepted model postulates that ␤ 2 ARs are degraded following the same mechanism described for EGF and its receptor (19 -21). Upon activation by the agonist, the ␤ 2 AR cycles between the plasma membrane and endosomes, where receptors are dephosphorylated (22)(23)(24). In the case of sustained stimulation by the agonist, ␤ 2 ARs would not be recycled to the plasma membrane but sorted instead to lysosomes and degraded by lysosomal proteases (25). A key feature of this model is that receptor endocytosis would necessarily constitute an early step in the down-regulation pathway. Consistent with this paradigm, is the observation by Gagnon et al. (26) that endocytosis and down-regulation of ␤ 2 ARs may both be inhibited in HEK293 cells by the K44A dominant negative mutant of dynamin, a mutant known to block the "pinching off" of endocytic vesicles. However, the inhibitory effect of dynamin K44A on ␤ 2 AR down-regulation was not evident in other cell lines (26). In addition, the observation that a cluster of point mutations in the carboxyl-terminal tail of the ␤ 2 AR almost completely blocks receptor endocytosis without impeding downregulation challenges the model of receptor down-regulation described above (27).
In the present study we have investigated mechanisms involved in the loss of ␤ 2 AR binding sites upon sustained activation with the agonist. We show that receptor down-regulation is fully maintained when endocytosis is impeded or when lysosomal or proteasomal functions are blocked. A novel model of receptor down-regulation emerges where the primary inactivation step may occur at the plasma membrane.
Plasmid Constructions, Transfections, and Cell Culture-To obtain clonal cell lines expressing the human ␤ 2 AR, the pBC-HA␤2 plasmid, encoding the hemagglutinin antigen-tagged human ␤ 2 AR (22), or the pBC-␤ 2 AR, encoding the wild type receptor, were transfected in murine L cells as described previously (28). Neomycin-resistant cells were selected in DMEM supplemented with 10% (v/v) fetal bovine serum, 4.5 g/liter glucose, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 1 mM glutamine and geneticin at a concentration of 400 mg/liter. Individual clones were screened for ␤ 2 AR expression by radioligand binding assay using 125 I-CYP as the ligand. A fusion ␤ 2 AR-green fluorescent protein (GFP) cDNA was also constructed by subcloning the ␤ 2 AR-coding region within the multicloning site located 5Ј to the GFP-coding region in the Cytogem Topaze (pGFPtpz-N1) vector (Packard, Meriden, CT). Wild type and K44A dynamin cDNAs subcloned into the eukaryotic expression vector pCDNA3 were generously provided by Dr. van der Bliek (San Diego, CA).
Transient transfections were performed using the DEAE-dextran method. Transfection efficiency was 30 -40% as monitored by co-transfecting a GFP-encoding plasmid. For fluorescence studies (see below), L cells were seeded in a 6-well dish containing glass covers 12 h before transfection. A431 cells were grown in RPMI supplemented with 10% (v/v) fetal bovine serum, 1 mM glutamine in a 10% CO 2 atmosphere.
Crude Membrane Preparation-Cells were placed on ice, washed twice with ice-cold PBS, and detached mechanically in ice-cold buffer 1 (5 mM Tris, 2 mM EDTA, pH 7.4, 5 mg/liter soybean trypsin inhibitor, 5 mg/liter leupeptin, and 10 mg/liter benzamidine). Cell suspensions were homogenized with a Polytron homogenizer (Janke & Kunkel Ultra-Turrax T25) three times for 5 s at the maximal setting. The lysate was centrifuged at 450 ϫ g for 5 min at 4°C, and the supernatant was centrifuged at 43000 ϫ g for 30 min at 4°C. The final pellet was washed twice in buffer 1 and resuspended in 75 mM Tris (pH 7.4), 12.5 mM MgCl 2 , 5 mM EDTA with protease inhibitors (as above) and immediately used for radioligand binding experiments or submitted to SDS-PAGE. Protein concentrations were determined by the method of Bradford with the Bio-Rad protein assay system using bovine serum albumin as standard.
Whole Cell Radioligand Binding Assay-Nearly confluent cells grown as monolayers were washed with PBS, incubated for 5 min with 2% trypsin, EDTA at 37°C, and resuspended in DMEM supplemented with 10% (v/v) fetal bovine serum. The cells were then centrifuged at 450 ϫ g for 5 min at 4°C and washed twice with ice-cold PBS. Binding assays were carried out using 0.1 ml of cell suspension in PBS. 125 I-CYP at 200 pM was used as the radioligand. Specific binding was defined as binding displaced by 10 M D/L-propranolol. Assays were carried out for 90 min at 25°C and terminated by rapid filtration through Whatman GF/C glass fiber filters previously soaked in PBS containing 0.3% polyethyleneimine (to reduce nonspecific binding). Protein concentrations were determined on broken cell preparations as above.
Endocytosis Assay-Endocytosis was determined as reported previously (29, 30) by differential centrifugation and separation of a light vesicle fraction from plasma membranes using a 35% sucrose cushion. A recent study has confirmed that endocytotic vesicle containing internalized ␤ 2 ARs can efficiently be separated from the plasma membrane fraction using this approach (31). Indeed, the endosomal compartment was found to sediment at around 26% sucrose, whereas plasma membrane was found at 35-40% sucrose. The amount of receptor present in each membrane fraction was determined by radioligand binding assay using 125 I-CYP as the radioligand. The assay was as described above but using membrane preparations instead of cell suspensions.
Inhibition of Endocytosis-Chemical inhibition of endocytosis in stable clones expressing ␤ 2 ARs was performed by potassium depletion (32), by incubating the cells in hypertonic medium (33), by acidification of the cytosol (34), or by incubating cells with concanavalin A (35). Cells were grown in 75-cm 2 flasks to 90% confluence. In all cases, protein synthesis was inhibited by the addition of cycloheximide (CHX) to eliminate the contribution of ␤ 2 AR mRNA regulation to the down-regulation phenomenon. For potassium depletion, cells were washed once with depletion buffer (20 mM Hepes, pH 7.4, 0.14 M NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , and 4.5 g/liter D-glucose). Subsequently, cells were incubated for 5 min in depletion buffer/H 2 O (1:1). Next, cells were incubated for 150 min in depletion buffer supplemented with CHX (5 g/ml). During the last 120 min isoproterenol (10 M final concentration) was added or not. Control cells were incubated under the same conditions but with 10 mM KCl added to the buffer. Inhibition of endocytosis by hypertonic shock was performed in maintaining cells in hypertonic medium (DMEM, 4.5 g/liter D-glucose, 10% fetal calf serum, and 0.5 M sucrose). Cells were washed once in hypertonic medium and incubated for 150 min in this medium supplemented with CHX (5 g/ml). During the last 120 min, isoproterenol (10 M final concentration) was added or not. For inhibition of endocytosis by cytosol acidification, cells were incubated for 150 min in DMEM, pH 5.0, 4.5 g/liter D-glucose, 10% fetal calf serum, 10 mM acetic acid, and CHX (5 g/ml). During the last 120 min isoproterenol (10 M final concentration) was added or not. Control cells were incubated under the same conditions but without acetic acid. Incubation in the presence of concanavalin A (0.25 mg/ml) was carried out for 150 min in DMEM, pH 5.0, 4.5 g/liter D-glucose, 10% fetal calf serum, and CHX (5 g/ml). During the last 120 min isoproterenol (10 M final concentration) was added or not. Inhibition of endocytosis was also measured in cells transiently co-transfected with plasmids encoding HA␤ 2 AR and K44A dynamin 48 -72 h after transfection. Wild type dynamin was used instead of the K44A mutant in control experiments.
Fluorescence Microscopy-Three days after transfection with the ␤ 2 AR-GFP construct, cells were subjected to various treatments aimed to inhibit receptor internalization. Inhibition of endocytosis by hypertonic shock, by potassium depletion, or by the incubation with concanavalin A was carried out as described above. After treatment, cells were washed in ice-cold PBS and fixed for 20 min at room temperature in a fresh solution of 4% paraformaldehyde in PBS. Coverslips were then mounted on microscope slide. Fluorescence microscopy was performed using a Zeiss Axioskop equipped with a mercury 100-watt lamp (AttoArc HBO 100). Pictures were taken using a CCD camera (Zeiss).
Analysis of Antibody-accessible Cell Surface Receptor by Flow Cytometry-Agonist-induced redistribution of HA-epitope-tagged ␤ 2 ARs (HA-␤ 2 AR) was determined using fluorescence-assisted cell sorting (FACS). Briefly, L cells were seeded in a 6-well plate the day before the experiment. After appropriate treatments, plates were kept on ice and washed twice with PBS. Cells were then incubated with a 1/100 dilution of the anti-HA 3F10 antibody (Roche Molecular Biochemicals) for 45 min, followed by an incubation with an anti-rat IgG coupled to Oregon green (dilution 1/500, Molecular Probes). Cells were detached with 5 mM EDTA, fixed with paraformaldehyde, and analyzed by FACS.
Inactivation of Lysosomal and Proteasome Pathways-Various compounds interfering with the lysosomal pathway and/or the proteasome degradation pathway were added to the cell culture medium 1 h before the incubation with isoproterenol and maintained in the medium throughout the experiment: concanamycin B (100 nM), NH 4  SDS-PAGE/Immunoblotting-Membranes prepared from cells expressing the HA-␤ 2 AR were denatured in 62.5 mM Tris/HCl (pH 6.8), 5% SDS, 3% 2-mercaptoethanol, 10% glycerol, 0.05% bromphenol blue for 3 h at 37°C. Seventy g of proteins were separated by 12% SDS-PAGE and transferred to nitrocellulose. Immunoblot analysis was carried out with the monoclonal HA-specific 3F10 antibody (Roche Molecular Biochemicals, 250 ng/ml). As a control for the inhibition of the proteasomedependent degradation pathway (36), ␤-catenin immunoreactivity was measured on whole cell extract using rabbit polyclonal antibodies to ␤-catenin (Sigma) after SDS-PAGE and transfer to nitrocellulose. Immunoreactivity was revealed using appropriate secondary antibodies coupled to horseradish peroxidase and the ECL chemiluminescent reagent (Amersham Pharmacia Biotech). Autoradiograms were digitalized using a CCD camera, and the densitometric analysis of the images were carried out with the NIH Image 1.6 software.
EGF Degradation Assay in A431 Cells-The EGF degradation assay was performed as described previously (37) with minor modifications. A431 cells were plated in 12-well culture dishes and serum-starved in RPMI supplemented with 1% bovine serum albumin 24 before the experiments. Cells were then incubated with 0.5 nM 125 I-EGF for 1 h at 15°C in RPMI containing 0.2% bovine serum albumin with or without the appropriate inhibitors. Leupeptin was added 24 h before the experiments. For K ϩ -depletion experiments, the depletion buffer described above replaced RPMI. After the incubation, plates were chilled on ice, and cells were washed three times with ice-cold buffer. Plates were then shifted to 37°C for various periods of time. Supernatants were collected, mixed with the same volume of 20% trichloroacetic acid in RPMI, and incubated on ice for 2 h. Cells were lysed by a 2-h incubation in 1 M NaOH. After centrifugation of trichloroacetic acid precipitates, pellets, supernatants, and cell lysates were counted separately. The fraction of degraded 125 I-EGF was determined by calculating the ratio between the radioactivity remaining in the supernatant after trichloroacetic acid precipitation and total radioactive load (sum of radioactivity values in supernatant, pellet, and cell lysate).

RESULTS
Ligand-dependent Inactivation of ␤ 2 ARs in L Cells-To investigate the molecular basis of ␤ 2 AR down-regulation, stable clones of L cells expressing physiological levels (100 -200 fmol) of receptor were studied in the presence of the protein synthesis inhibitor CHX. The decay of receptor number was assessed in cells treated or not with the ␤-adrenergic agonist isoproterenol. Because new protein synthesis is inhibited in both control and isoproterenol-treated cells, any effect of agonist treatment on whole cell receptor density has to be attributed to downregulation of pre-existing receptor and not to mRNA regulation. Treatment of cells with CHX induced a time-dependent decrease in the number of ␤ 2 ARs detected in whole cell binding assay using the membrane-permeable radioligand 125 I-CYP (Fig. 1). This decay was biphasic with a rapid component between 0 and 6 h and a much slower component between 6 and 24 h. The occurrence of the second slow phase might result from the progressive disappearance of a short-lived process implicated in the degradation of the receptor as a result of protein synthesis inhibition. Treatment with isoproterenol considerably steepened the first component of the ␤ 2 AR decay curve, consistent with previous studies (15) and with the model in which sustained agonist treatment increases the ␤ 2 AR degradation rate.
Although indirect evidence that the decay of ␤ 2 AR number upon sustained agonist activation is the consequence of receptor degradation exists (16 -18), this phenomenon has not been directly documented so far, mostly because high affinity and low background anti-␤ 2 AR antibodies were not available. We have used anti-HA epitope antibodies to quantify the HAepitope-tagged receptor (HA-␤ 2 AR) expressed in L cells by immunoblot on membranes prepared from cells incubated for various periods of time with isoproterenol (Fig. 2). The 3F10 anti-HA-epitope monoclonal antibody recognized tagged receptors specifically, as shown by the absence of background on membranes prepared from L cells expressing nontagged wild type receptors ( Fig. 2A). A decrease of immunoreactive material was evident in cells treated for various periods of time with isoproterenol ( Fig. 2B), consistent with agonist-induced receptor proteolysis. We could not visualize any low molecular weight fragment of the receptor containing the HA epitope that could correspond to a specific proteolytic fragment. Possible explanations are that the amino-terminal portion of the receptor was cleaved and released from the rest of the molecule or that the complete proteolysis of the receptor is achieved with a very fast kinetics.
Despite a general parallelism between the loss of binding sites and of receptor immunoreactivity, the decay of immunoreactive material appeared to be slightly slower than the loss of binding sites. We thus analyzed this decay in more detail during the first 6 h of exposure to isoproterenol and measured the loss of 125 I-CYP binding sites in the same preparations (Fig.  2C). These experiments confirmed that the loss of binding sites was faster than that of immunoreactive material during the first 2 h of agonist stimulation. A plausible explanation for this discrepancy could be that some immunoreactive receptors do not bind to the radioligand, thus slowing down the detection of the receptor protein decay. To confirm this hypothesis, total membranes were prepared from unstimulated cells and separated in plasma and light membrane fractions by centrifugation on a sucrose cushion (Fig. 2D). For each fraction, the ratio between bound 125 I-CYP and immunoreactivity was calculated. This ratio was four times higher in the plasma membrane fraction than in the light membrane fraction, indicating the existence of a pool of apparently mature and glycosylated intracellular receptors unable to bind the ligand. This pool of receptors may be responsible for the apparent discrepancy between the loss of binding and that of immunoreactivity. The observation that the proportions of down-regulated receptors measured with the 2 approaches coincided after 4 h of isoproterenol stimulation suggests that these intracellular receptors may be exported to the plasma membrane and become compe- tent for ligand binding. Taken together, our results are consistent with the current hypothesis that ␤ 2 AR down-regulation is accompanied by a loss of receptor protein resulting from a proteolytic degradation of the receptor.
Down-regulation of ␤ 2 ARs Is Unaffected by Blockers of Receptor Endocytosis-According to the current model of receptor regulation, endocytosis is viewed as an early and necessary step in the down-regulation of ␤ 2 ARs. If this model is true, one would expect that down-regulation does not occur when receptor endocytosis is blocked. Various treatments such as potassium depletion, cytosol acidification, and incubation with high sucrose concentrations (24,38) are known to block ␤ 2 AR endocytosis, probably by interfering with the formation of clathrincoated vesicles (39). Incubation with lectins that bind to the sugar moiety of the ␤ 2 AR, such as concanavalin A, also block receptor internalization (24). To investigate whether blocking endocytosis would also affect ␤ 2 AR down-regulation, both phenomena were studied in untreated control cells and cells treated with endocytosis blockers mentioned above. Endocytosis was determined by measuring the proportion of receptors translocated from the plasma membrane to light density endocytic compartment (see "Experimental Procedures"), whereas the decay of total 125 I-CYP binding sites was used to monitor ␤ 2 AR down-regulation. All experiments were carried out in the presence of CHX, which did not affect receptor endocytosis (Fig.  3). A 2-h treatment with isoproterenol promoted endocytosis of ϳ25% of the total receptor sites. Incubation with 0.5 M sucrose, 0.25 mg/ml concanavalin A, or acetic acid as well as potassium depletion all blocked endocytosis by more than 80%. In con-trast, none of these treatments inhibited the loss of receptor binding sites promoted by isoproterenol (Fig. 3B), indicating that endocytosis is not a prerequisite for down-regulation. The slight effect of sucrose on the extent of down-regulation most likely reflects pleiotropic actions of this compound. For example, sucrose by itself caused a 40% decrease in the number of binding sites in the absence of isoproterenol in L cells (data not shown).
To confirm with a different approach that the treatments used indeed inhibited endocytosis in L cells, we examined their effect on GFP-or HA epitope-tagged ␤ 2 AR agonist-dependent redistribution. Previous experiments have shown that the fusion of GFP to the carboxyl-terminal extremity of the ␤ 2 AR does not modify ligand-dependent receptor endocytosis (40,41). L cells were transiently transfected with a plasmid carrying the ␤ 2 AR-GFP fusion cDNA and depleted or not of intracellular K ϩ before isoproterenol stimulation (Fig. 4). In the absence of ligand, no detectable endocytosis was observed in control cells (panel A). In the presence of isoproterenol at 37°C, ␤ 2 AR-GFP was internalized in punctiform structures probably corresponding to endosomes (panel B). In cells depleted of K ϩ (panel C) or preincubated with high sucrose or concanavalin A or with a cytosol-acidifying medium (not shown), no isoproterenoldependent accumulation of ␤ 2 AR-GFP could be visualized in endosomes. FACS analysis of HA-␤ 2 AR redistribution with anti HA-antibodies was used in previous studies as an indirect technique to quantify receptor endocytosis (42,43). Indeed, the amount of agonist-promoted loss of surface fluorescence was found to be proportional, although not identical, to endocytosis measured with radioligand-based assays (42). In Fig. 4 (panels D-F), the isoproterenol-induced loss of surface anti-HA antibody binding sites was studied in stable clones of L cells expressing HA-␤ 2 AR. In untreated cells, the agonist caused a marked reduction of cell surface fluorescence (Fig. 4, panels D and F). In contrast, in cytosol-acidified cells, isoproterenol did not induce any significant change in cell surface fluorescence (Fig. 4, panels E and F). Similar results were obtained in cells preincubated with sucrose or depleted of their intracellular K ϩ (not shown). FACS analysis could not be used to study the effect of concanavalin A, since this treatment causes cell aggregation.
The results described above indicate that in L cells, ␤ 2 AR down-regulation does not require endocytosis and, thus, is likely to occur at the plasma membrane. To test this possibility, down-regulation was measured directly in plasma membrane preparations that were separated from the light density endocytotic compartment by ultracentrifugation on sucrose cushion (see "Experimental Procedures"). For these experiments, the endocytosis-independent down-regulation was assessed by overexpressing the K44A dominant-negative mutant of dynamin. Inhibiting the function of this GTPase, which regulates the formation and internalization of endocytic vesicles from the plasma membrane (43), represents an alternative approach to the chemical treatments to block endocytosis. Epitope-tagged ␤ 2 AR cDNA was co-transfected in L cells with either wild type or K44A dynamin. Forty-eight h after transfection, cells were stimulated with isoproterenol in the presence of CHX. As shown in Fig. 5A, co-transfection with K44A dynamin completely prevented agonist-promoted endocytosis as assessed by radio-ligand binding on the light membrane fraction. In contrast, K44A dynamin was without effect on receptor downregulation. Indeed, incubation with isoproterenol caused a similar decrease of ␤ 2 AR binding sites and of receptor immunoreactivity in the plasma membrane fraction of both wild type-and K44A-dynamin-transfected cells (Fig. 5, B and  C). Taken together these data indicate that ␤ 2 AR endocytosis FIG. 2. Isoproterenol-induced decay of immunoreactive ␤ 2 ARs. Panel A, L cells expressing the wild type ␤ 2 AR or HA-␤ 2 AR were stimulated (ϩ) or not (Ϫ) for 4 h with 10 M isoproterenol in the presence of 5 g/ml CHX at 37°C. Crude membranes were prepared, and aliquots of 50 g of protein were submitted to SDS-PAGE and immunoblot analysis with the anti-HA-epitope 3F10 monoclonal antibody. Panel B, L cells expressing HA-␤ 2 AR were incubated with isoproterenol and CHX for the indicated time, and crude membranes (50 g/lane) were analyzed by immunoblot analysis as in panel A. Panel C, the decay of 125 I-CYP binding sites was compared with that of immunoreactive material in crude membrane preparations from isoproterenol-treated L cells expressing HA-␤ 2 AR; the amount of immunoreactive material was measured by densitometry; data are the means Ϯ S.E. of four independent experiments. Panel D, plasma and light membrane fractions were prepared from untreated L cells expressing HA-␤ 2 AR by centrifugation on a sucrose cushion (see "Experimental Procedures"). Western blots were carried on aliquots of 25 g of protein, and the amount of immunoreactive material was assessed by densitometry. The density of 125 I-CYP binding sites was measured in the same fractions. Histograms represent the ratio between 125 I-CYP binding sites and densitometric measurements in the plasma membrane fraction (P) and in the light membrane fraction (L). A representative Western blot and a Ponceau red staining of blotted proteins is shown. and down-regulation can be functionally dissociated in L cells and that the latter is likely to occur at the plasma membrane.
Down-regulation of ␤ 2 ARs Is Maintained upon Inactivation of the Lysosomal Pathway-Since the results described above argue against a role for endocytosis in the ␤ 2 AR down-regulation, one could predict that this process should not be affected by blockers of lysosomal degradation. To test this hypothesis, the effects of lysosomal function inhibitors were studied. Chloroquine, the weak base NH 4 Cl, and concanamycin B, a highly specific inhibitor of vacuolar H ϩ -ATPases (44) are known to inhibit lysosomal proteases by interfering with the acidic lysosomal pH. Other compounds such as leupeptin and E-64 prevent lysosomal proteolysis by directly inhibiting cysteine proteases. None of these drugs inhibited ␤ 2 AR down-regulation promoted by 2-h incubation with isoproterenol in the presence of CHX (Fig. 6A). Similar results were obtained after a 24-h incubation with the agonist in the absence of CHX (Fig. 6B). These results indicate that the down-regulation of the ␤ 2 AR does not occur in the lysosomal compartment. Consistent with this idea is the observation that alkalinization with NH 4 Cl did not block agonist-promoted receptor loss of immunoreactivity (data not shown).
Down-regulation of the ␤ 2 AR Is Maintained upon Inactivation of the Ubiquitin Proteasome Pathway-In an attempt of identifying the mechanism of ␤ 2 AR degradation at the plasma membrane, we investigated the potential involvement of the ubiquitin-proteasome pathway in this process. This pathway was shown to play a role in ligand-induced degradation of several membrane receptors (45)(46)(47). It was proposed that proteasome could recognize and degrade ubiquitinated cytoplasmic domains of plasma membrane proteins (48). The photoreceptor rhodopsin was previously found to be ubiquitinated and degraded in the rod outer segment, indicating that G protein-coupled receptors may be a substrate for ubiquitination (49). To rule out the possibility that ␤ 2 AR down-regulation could involve proteasome, we investigated the effects of various blockers of this pathway in intact cells. Peptide-aldehyde compounds, such as MG132, ALLN, and ALLM inhibit cysteine protease calpain and cathepsin. Among them, MG132 and ALLN can also inhibit the proteolytic activity of proteasomes, whereas ALLM is inactive on this pathway (50). Lactacystin, a Streptomyces metabolite, is one of the most potent and specific inhibitors of proteasome activity (51). None of these compounds blocked the reduction of ␤ 2 AR binding sites promoted by a 2-h incubation with isoproterenol in the presence of CHX (Fig. 7A). The amount of ␤-catenin, a protein undergoing a tonic degradation by the proteasome pathway (36), was then used as a control for the efficacy of proteasome inhibition. As shown in Fig. 7C, treatment with ALLN led to an increased accumulation of immunoreactive ␤-catenin that reached 170% of the control values, confirming the inhibitory effect of ALLN on proteasome proteolytic activity. After a 24-h incubation with isoproterenol in the absence of CHX, lactacystin, MG132, and ALLN also failed to block receptor down-regulation, confirming the noninvolvement of the proteasome in this process. However, the long term treatment with the inhibitors caused an unexpected and striking increase of the steady-state receptor density (Fig. 7B). Lactacystin induced a 4-fold increase in receptor number, whereas the peptide-aldehydes MG132 and ALLN caused an 8-and 15-fold increase, respectively. The control compound ALLM did not cause any change in receptor density, suggesting that this effect resulted from selective proteasome inhibition. The lower efficiency of lactacystin in promoting the elevation of binding sites as compared with the peptide-aldehydes is probably attributable to its spontaneous hydrolysis in aqueous solutions (52). In any case, the observation that down-regulation was still observed following treatment with proteasome inhibitors indicates that the inactivation of the ␤ 2 AR in L cells is not the consequence of ubiquitination and proteasome degradation.
Comparative Study of ␤ 2 AR and EGF Down-regulation in A431 Cells-To investigate whether the endocytosis-independent ␤ 2 AR down-regulation that we documented in L cells could be present in other cell systems, similar studies were conducted in A431 cells. These cells endogenously express the ␤ 2 AR (53) and were previously used as a model to study ␤ 2 AR endocytosis (24). In addition, A431 cells also express endogenous EGF receptors (54), whose regulation represent the paradigm of the endosome-and lysosome-dependent degradation pathway (55). In A431 cells, isoproterenol caused a fast ␤ 2 AR down-regulation, with 50 -60% of the ␤ 2 AR binding sites being lost within 60 min (Fig. 8A). In the same cells, the extent of EGF degradation after 60 min was similar, close to 50% (Fig. 8B). ␤ 2 AR down-regulation and EGF degradation were tested in parallel after various treatments that inhibited endocytosis or lysosomal degradation. Concanavalin A and K ϩ depletion blocked at least 80% of EGF degradation, whereas they had minor or no effect on ␤ 2 AR down-regulation (Fig. 8C). Sucrose totally blocked EGF degradation, compared with a 40% inhibition of ␤ 2 AR down-regulation. Treatments blocking lysosomal function also had very different effects on the two receptors; neither leupeptin nor NH 4 Cl could affect ␤ 2 AR down-regulation (Fig.   FIG. 4. Agonist-promoted redistribution of GFP-and HA-tagged ␤ 2 ARs in the presence and absence of endocytosis inhibitors. L cells expressing either GFP-␤ 2 AR or HA-tagged ␤ 2 AR were preincubated or not for 1 h with the indicated treatments inhibiting endocytosis and then stimulated or not for 1 h with 10 M isoproterenol. Panels A-C, GFP-␤ 2 AR redistribution was studied by fluorescence microscopy under basal conditions (A) following isoproterenol stimulation (B) or following isoproterenol stimulation in a K ϩ -depleted medium (C). Data shown are representative of 3 independent experiments. Panels D-E, antibody-accessible HA-tagged ␤ 2 AR was quantified by FACS analysis under basal conditions (control) and following isoproterenol stimulation (ISO) in untreated L cells (D) or cells preincubated in a cytosol-acidifying medium (E). Mock cells indicate background fluorescence; fluorescence intensity is shown in a logarithmic scale. Panel F, the modulation of antibody-accessible receptors was calculated from the experiments shown in panels D and E in cells preincubated (ϩ acid) or not in a cytosol-acidifying medium in the presence (ISO) or not (C) of isoproterenol. Data shown are representative of three independent experiments. 8D), whereas they inhibited 80 and 100% of EGF degradation, respectively (Fig. 8B). These results show that in A431 cells, ␤ 2 AR down-regulation involves a different pathway than that of EGF degradation.

DISCUSSION
About one decade ago, a model of agonist-promoted ␤ 2 AR down-regulation was proposed which postulates that receptors are first internalized in endosomes and then sorted to lysosomes, where degradation takes place, in a manner similar to that described for the EGF receptor (25,56). The observation that isoproterenol elicits a time-dependent decrease of immunoreactive ␤ 2 ARs in L cells is the first direct indication that ␤ 2 AR down-regulation is indeed associated with a loss of receptor protein that may result from proteolytic degradation. Although, one cannot rule out the possibility that the loss of immunoreactive receptor could result from an irreversible conformational change that would simultaneously mask the epitope and disable the binding site, the observation remains consistent with the above-mentioned model. However, other experiments reported here challenge the current model of ␤ 2 AR down-regulation. Chemical treatments that block endocytosis or alter lysosomal function are ineffective on ␤ 2 AR down-regulation in both L and A431 cells, whereas in the latter cell line these treatments almost completely inhibited EGF degradation. In addition, overexpression of K44A dynamin in L cells could block ␤ 2 AR endocytosis but not down-regulation.
So far, only a limited number of studies have specifically addressed the question of the relationship between endocytosis and down-regulation pathways of ␤ 2 ARs. The idea, supported by our data, that endocytosis and down-regulation could involve different pathways is consistent with the previous observation that the S355-364-␤ 2 AR mutant, which does not undergo significant endocytosis, displayed a normal pattern of receptor down-regulation in Chinese hamster ovary cells (27). However the possibility, raised by that study, of an alternative model for ␤ 2 AR down-regulation was dismissed, arguing that the observed phenomenon could be explained by kinetic arguments (22,27). More recently, the observation that isoproterenol-promoted loss of GFP-tagged ␤ 2 AR binding sites coincided with the appearance of fluorescence in lysosomes was interpreted as evidence of the role of this compartment in ␤ 2 AR down-regulation (41). Although this is a possible interpretation, the presence of GFP in the lysosomes does not demonstrate that lysosomal degradation is the causal event leading to down-regulation. Indeed, the tagged-receptor or fragment thereof could be targeted to this compartment following an earlier inactivation step.
The model supported by our study, in which ␤ 2 AR endocytosis and down-regulation are independent, challenges an observation reported by Gagnon et al. (26). In HEK293 cells, endocytosis and down-regulation of the ␤ 2 AR were both inhibited by the K44A dominant-negative mutant of dynamin. A possible explanation for the apparent discrepancy between that report and our results is that overexpression of K44A dynamin may also affect other pathways than endocytosis in HEK293 cells. For example, the overexpression of dominant negative dynamin is known to cause structural changes at the cell surface (57). Moreover, new evidence has emerged suggesting additional roles for dynamin, including budding regulation from the Golgi complex (58). Thus, the inhibition of ␤ 2 AR down-regulation by a dominant negative dynamin in some cell lines might not be a direct consequence of impaired endocytosis. An alternative way to interpret the distinct effects of K44A dynamin in different cell lines is that both endocytosis-dependent and -independent down-regulation are possible and that their relative contribution vary between cell types. The fact that overexpression of K44A dynamin strongly affected ␤ 2 AR down-regulation in HEK293 cells but had only little effect in HeLa cells (26) and no effect in L cells (this report) supports this hypothesis. Also consistent with this idea is the observation that ␤ 2 AR downregulation could be inhibited following a chemical block of endocytosis in HEK293 cells (data not shown).
Our demonstration that down-regulation of the ␤ 2 AR can occur in the absence of endocytosis, not only in the transfected L cells but also in A431 cells that endogenously express the receptor, supports the generality of the phenomenon. Interestingly, the observation that a mutant form of the m2-muscarinic receptor that does not undergo agonist-promoted endocytosis can be down-regulated (59) suggests that endocytosis-independent down-regulation can also be observed for other receptors.
The fact that a lysosomal function inhibition sufficient to prevent EGF degradation did not affect ␤ 2 AR down-regulation raised the questions of the mechanism underlying this process and of its location within the cell. In an effort to address this question, we showed that proteasome inhibitors did not affect ligand-dependent down-regulation of preexisting ␤ 2 ARs excluding the involvement of this degradation pathway. Interestingly, long term incubation of L cells with proteasome inhibitors markedly increased the number of 125 I-CYP binding sites. This observation may suggest that most of the ␤ 2 ARs newly synthesized in L cells are degraded by default in the endoplasmic reticulum-associated proteasome, which controls the entry of proteins into the secretory pathway (recently reviewed in Refs. 60 and 61). A similar increase of ␤ 2 AR binding sites could not be documented in A431 or HEK293 cells because the sustained incubation of these cells with proteasome inhibitors was toxic (data not shown). Whether or not the proteasome-mediated degradation of newly synthesized receptors is restricted to transfected L cells or a more general phenomenon remains to be investigated.
Another potential site of membrane protein degradation is the plasma membrane itself, which is known to contain multi- Crude cell membranes were prepared, and ␤ 2 AR binding sites were determined by radioligand binding assay. The number of receptors is expressed as the percent of 125 I-CYP binding sites in untreated cells. Data are the means Ϯ S.E. of three independent experiments carried out in triplicate. Lacta, lactacystin. Panel C, ␤-catenin up-regulation was used as a positive control for proteasome inhibition by ALLN; after a 2-h incubation with ALLN, cells were dissolved in sample buffer, and identical amounts of total proteins were submitted to SDS-PAGE and Western blot. In the left part of panel C an autoradiogram representative of three independent experiments is shown. Autoradiograms were analyzed by densitometry (right part of panel C); the bar indicates the S.E. of three experiments. C, control cells.
FIG. 8. Down-regulation of ␤ 2 AR and degradation of EGF follow distinct pathways in A431 cells. Panel A, isoproterenol-induced down-regulation of ␤ 2 AR in A431 cells. Cell were stimulated for the indicated times with 10 M isoproterenol, crude cell membranes were prepared, and 125 I-CYP binding sites were determined by radioligand binding assay. The bars indicate S.E. of triplicates. Panel B, time course of 125 I-EGF degradation in intact control A431 cells and A431 cells treated with the lysosome inhibitors leupeptin and NH 4 Cl. The assay was conducted as described under "Experimental Procedures." S.E. on triplicate determinations are within the symbols. The data shown is representative of three independent experiments Panel C, effect of endocytosis inhibitors on ␤ 2 AR down-regulation and 125 I-EGF degradation. Cells were pretreated for 1 h with the indicated inhibitors. Downregulation of ␤ 2 AR was measured as in panel A, and 125 I-EGF degradation was measured as in panel B. Data are presented as the percentage of down-regulation/degradation blocked by endocytosis inhibitors. K-depl., potassium depletion. The bars indicate S.E. of triplicates. Panel D, effect of inhibitors of the lysosomal degradation pathway on ␤ 2 AR down-regulation in A431 cells. Cells were pretreated for 1 h with the indicated inhibitors and then incubated with isoproterenol for 1 h at 37°C in the continued presence of inhibitors. Crude cell membranes were prepared, and ␤ 2 AR binding sites were determined by radioligand binding assay. The bars indicate S.E. of triplicates. All figures are representative of two to three independent experiments giving similar results. ple proteases. The possible involvement of this compartment in the ␤ 2 AR down-regulation is supported by two of our observations: (i) fluorescence microscopy studies confirmed that GFPor HA-tagged ␤ 2 ARs were not internalized under conditions where receptor down-regulation was fully effective; (ii) a comparable decrease of 125 I-CYP binding sites and of receptor immunoreactivity induced by isoproterenol was documented in plasma membrane preparations of cells expressing the endocytosis-inhibiting mutant of dynamin. Although such a model of G protein-coupled receptor down-regulation has not been documented so far in an intact cell system, Kojro and Fahrenholz (62) report that vasopressin promotes the cleavage of the V2 receptor in membrane preparations through the action of a plasma membrane protease (62). However, if a membrane protease is indeed involved in ␤ 2 AR down-regulation in L and A431 cells, the enzyme implicated is probably different than the one described for the V2 receptor. First, the kinetics of the cleavage was much faster in the case of the vasopressin V2 receptor (ϳ80% cleavage in 5 min versus ϳ50% of ␤ 2 AR inactivation after 6 h). Second, the metalloprotease that cleaves V2 receptor was blocked by 0.1 mM ZnCl 2 , whereas Zn 2ϩ ions had no effect on ␤ 2 AR inactivation (data not shown). Additional studies are required to uncover the precise mechanism of ␤ 2 AR down-regulation at the plasma membrane.
In conclusion, we have provided evidence for an alternative pathway of ␤ 2 AR down-regulation that does not require endocytosis. This pathway seems to be predominant in several cell types compared with the lysosome-dependent degradation pathway. Such a pathway might represent a novel specific target for down-regulation inhibitors in pathological conditions such as heart disease and asthma.