β2-Adrenergic Receptor Down-regulation
1999; Elsevier BV; Volume: 274; Issue: 41 Linguagem: Inglês
10.1074/jbc.274.41.28900
ISSN1083-351X
AutoresRalf Jockers, Stéphane Angers, Angelo Da Silva, Philippe Benaroch, A. Donny Strosberg, Michel Bouvier, Stéfano Marullo,
Tópico(s)Pancreatic function and diabetes
ResumoSustained activation of most G protein-coupled receptors causes a time-dependent reduction of receptor density in intact cells. This phenomenon, known as down-regulation, is believed to depend on a ligand-promoted change of receptor sorting from the default endosome-plasma membrane recycling pathway to the endosome-lysosome 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 β2-adrenergic receptors (β2ARs), sustained agonist treatment caused a time-dependant decrease in both β2AR binding sites and immuno-detectable receptor. Blocking β2AR 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 proteasome-associated, were ineffective in preventing β2AR down-regulation. Further evidence for an endocytosis-independent pathway of β2AR down-regulation was provided by studies in A431 cells, a cell line expressing both endogenous β2AR and EGF receptors. In these cells, inhibition of endocytosis and inactivation of the lysosomal degradation pathway did not block β2AR 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 β2AR down-regulation and that the inactivation of β2ARs, leading to a reduction in binding sites, may occur at the plasma membrane. Sustained activation of most G protein-coupled receptors causes a time-dependent reduction of receptor density in intact cells. This phenomenon, known as down-regulation, is believed to depend on a ligand-promoted change of receptor sorting from the default endosome-plasma membrane recycling pathway to the endosome-lysosome 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 β2-adrenergic receptors (β2ARs), sustained agonist treatment caused a time-dependant decrease in both β2AR binding sites and immuno-detectable receptor. Blocking β2AR 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 proteasome-associated, were ineffective in preventing β2AR down-regulation. Further evidence for an endocytosis-independent pathway of β2AR down-regulation was provided by studies in A431 cells, a cell line expressing both endogenous β2AR and EGF receptors. In these cells, inhibition of endocytosis and inactivation of the lysosomal degradation pathway did not block β2AR 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 β2AR down-regulation and that the inactivation of β2ARs, leading to a reduction in binding sites, may occur at the plasma membrane. β2-adrenergic receptor human embryonic kidney cells epidermal growth factor Dulbecco's modified Eagle's medium phosphate-buffered saline green fluorescent protein polyacrylamide gel electrophoresis cycloheximide hemagglutinin fluorescence-assisted cell sorting N-acetyl-Leu-Leu-norleucinal or calpain inhibitor I N-acetyl-Leu-Leu-methioninal or calpain inhibitor II carbobenzoxyl-l-leucyl-l-leucyl-l-leucinal, a cell-permeable proteasome inhibitor 125I-iodocyanopindolol, a β-adrenergic antagonist 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 stimulus. 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 (1Benovic J.L. Bouvier M. Caron M. Lefkowitz R.J. Annu. Rev. Cell Biol. 1988; 4: 405-427Crossref PubMed Scopus (328) Google Scholar). 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 (2Yang J. Williams J.A. Yule D.I. Logsdon C.D. Mol. Pharmacol. 1995; 48: 477-485PubMed Google Scholar). 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 (3Chen J. Ishii M. Wang L. Ishii K. Coughlin S.R. J. Biol. Chem. 1994; 269: 16041-16045Abstract Full Text PDF PubMed Google Scholar); this peptide activates the receptor irreversibly, promoting its endocytosis and its subsequent degradation in lysosomes (4Hein L. Ishii K. Coughlin S.R. Kobilka B.K. J. Biol. Chem. 1994; 269: 27719-27726Abstract Full Text PDF PubMed Google Scholar). Desensitization of β2-adrenergic receptors (β2ARs)1 is mostly dependent on rapid phosphorylation by the cAMP-dependent protein kinase and G protein-coupled receptor kinases (5Benovic J.L. Pike L.J. Cerione R.A. Staniszewski C. Yoshimasa T. Codina J. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1985; 260: 7094-7101Abstract Full Text PDF PubMed Google Scholar, 6Benovic J.L. Major Jr., F. Somers R. Caron M.G. Lefkowitz R.J. Nature. 1986; 321: 869-872Crossref PubMed Scopus (139) Google Scholar, 7Bouvier M. Hausdorff P. De Blasi A. O'Dowd B.F. Kobilka B.K. Caron M.G. Lefkowitz R.J. Nature. 1988; 333: 370-373Crossref PubMed Scopus (339) Google Scholar). However, although the β2AR is less rapidly affected by down-regulation than the thrombin receptor, long term agonist-promoted β2AR down-regulation significantly contributes to the desensitization and is additive to rapid inactivation resulting from receptor uncoupling (8Nantel F. Bouvier M. Strosberg A.D. Marullo S. Br. J. Pharmacol. 1995; 114: 1045-1051Crossref PubMed Scopus (32) Google Scholar). Supporting the physio-pathological significance of β2AR down-regulation are studies demonstrating that the development of heart failure may be associated with β2AR down-regulation (9Ungerer M. Bohm M. Elce J.S. Erdmann E. Lohse M.J. Circulation. 1993; 87: 454-463Crossref PubMed Scopus (763) Google Scholar). In addition, studies on β2AR polymorphism showed that alleles, which display accelerated ligand-dependent down-regulation in vitro (10Green S.A. Turki J. Innis M. Liggett S.B. Biochemistry. 1994; 33: 9414-9419Crossref PubMed Scopus (711) Google Scholar), are associated with altered desensitization to β-adrenergic bronchodilators in asthmatic patients (11Tan S. Hall I.P. Dewar J. Dow E. Lipworth B. Lancet. 1997; 350: 995-999Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). β2AR down-regulation involves at least two pathways. The first is the reduction in receptor mRNA steady-state level resulting from destabilization of the transcript (12Hadcock J.R. Malbon C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5021-5025Crossref PubMed Scopus (236) Google Scholar, 13Bouvier M. Collins S. O'Dowd B.F. Campbell P.T. de Biasi A. Kobilka B.K. MacGregor C. Irons G.P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1989; 264: 16786-16792Abstract Full Text PDF PubMed Google Scholar, 14Danner S. Frank M. Lohse M.J. J. Biol. Chem. 1998; 273: 3223-3229Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The consequences of such a phenomenon, however, become apparent only after many hours of sustained activation once the number of pre-existing β2ARs has decreased. The second pathway of β2AR down-regulation is detectable as early as 1 h following receptor activation (15Nantel F. Marullo S. Krief S. Strosberg A.D. Bouvier M. J. Biol. Chem. 1994; 269: 13148-13155Abstract Full Text PDF PubMed Google Scholar); it consists in the loss of pre-existing ligand binding sites. Based on the observation that, upon removal of agonist, the recovery of β2ARs to control levels requires neosynthesis, it was postulated that the loss of binding sites was the consequence of receptor degradation (16Doss R.C. Perkins J.P. Harden T.K. J. Biol. Chem. 1981; 256: 12281-12286Abstract Full Text PDF PubMed Google Scholar, 17Morishima I. Thompson W.S. Robison G.A. Strada S.J. Mol. Pharmacol. 1980; 18: 370-378PubMed Google Scholar, 18Waldo G.L. Doss R.C. Perkins J.P. Harden T.K. Mol. Pharmacol. 1984; 26: 424-429PubMed Google Scholar). 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 β2ARs are degraded following the same mechanism described for EGF and its receptor (19Dunn W.A. Hubbard A.L. J. Cell Biol. 1984; 98: 2148-2159Crossref PubMed Scopus (185) Google Scholar, 20Renfrew C.A. Hubbard A.L. J. Biol. Chem. 1991; 266: 21265-21273Abstract Full Text PDF PubMed Google Scholar, 21Kornilova E. Sorkina T. Beguinot L. Sorkin A. J. Biol. Chem. 1996; 271: 30340-30346Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Upon activation by the agonist, the β2AR cycles between the plasma membrane and endosomes, where receptors are dephosphorylated (22von Zastrow M. Kobilka B.K. J. Biol. Chem. 1992; 267: 3530-3538Abstract Full Text PDF PubMed Google Scholar, 23Yu S.S. Lefkowitz R.J. Hausdorff W.P. J. Biol. Chem. 1993; 268: 337-341Abstract Full Text PDF PubMed Google Scholar, 24Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar). In the case of sustained stimulation by the agonist, β2ARs would not be recycled to the plasma membrane but sorted instead to lysosomes and degraded by lysosomal proteases (25Perkins J.P. Hausdorff W.P. Lefkowitz R.J. Perkins J.P. The β-Adrenergic Receptor. Humana Press Inc., Clifton, NJ1991: 73-124Google Scholar). 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. (26Gagnon A.W. Kallal L. Benovic J.L. J. Biol. Chem. 1998; 273: 6976-6981Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar) that endocytosis and down-regulation of β2ARs 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 β2AR down-regulation was not evident in other cell lines (26Gagnon A.W. Kallal L. Benovic J.L. J. Biol. Chem. 1998; 273: 6976-6981Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). In addition, the observation that a cluster of point mutations in the carboxyl-terminal tail of the β2AR almost completely blocks receptor endocytosis without impeding down-regulation challenges the model of receptor down-regulation described above (27Hausdorff W.P. Campbell P.T. Ostrowski J., Yu, S.S. Caron M.G. Lefkowitz R.J. Proc. Natl. Acad. Sci U. S. A. 1991; 88: 2979-2983Crossref PubMed Scopus (139) Google Scholar). In the present study we have investigated mechanisms involved in the loss of β2AR 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. 125I-CYP was from Amersham Pharmacia Biotech. (−)-Isoproterenol, (−)-alprenolol,dl-propranolol, bovine serum albumin, E-64, ALLN, ALLM, cycloheximide, concanavalin A, leupeptin, soybean trypsin inhibitor and benzamidine were from Sigma. MG132, concanamycin B, and lactacystin were obtained from Calbiochem. DMEM, RPMI, fetal bovine serum, PBS, trypsin-EDTA, Geneticin (G418), penicillin, and streptomycin were from Life Technologies, Inc. To obtain clonal cell lines expressing the human β2AR, the pBC-HAβ2 plasmid, encoding the hemagglutinin antigen-tagged human β2AR (22von Zastrow M. Kobilka B.K. J. Biol. Chem. 1992; 267: 3530-3538Abstract Full Text PDF PubMed Google Scholar), or the pBC-β2AR, encoding the wild type receptor, were transfected in murine L cells as described previously (28Nantel F. Bonin H. Emorine L.J. Zilberfarb V. Strosberg A.D. Bouvier M. Marullo S. Mol. Pharmacol. 1993; 43: 548-555PubMed Google Scholar). 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 mmglutamine and geneticin at a concentration of 400 mg/liter. Individual clones were screened for β2AR expression by radioligand binding assay using 125I-CYP as the ligand. A fusion β2AR-green fluorescent protein (GFP) cDNA was also constructed by subcloning the β2AR-coding region within the multicloning site located 5′ 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% CO2 atmosphere. 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 ×g for 5 min at 4 °C, and the supernatant was centrifuged at 43000 × 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 MgCl2, 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. 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 × 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. 125I-CYP at 200 pm was used as the radioligand. Specific binding was defined as binding displaced by 10 μmd/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 was determined as reported previously (29Lohse M.J. Benovic J.L. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1990; 265: 3202-3209Abstract Full Text PDF PubMed Google Scholar, 30Jockers R. Da Silva A. Strosberg A.D. Bouvier M. Marullo S. J. Biol. Chem. 1996; 271: 9355-9362Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) 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 β2ARs can efficiently be separated from the plasma membrane fraction using this approach (31Marullo S. Faundez V. Kelly R.B. Recept. Channels. 1999; 6: 255-269PubMed Google Scholar). 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 125I-CYP as the radioligand. The assay was as described above but using membrane preparations instead of cell suspensions. Chemical inhibition of endocytosis in stable clones expressing β2ARs was performed by potassium depletion (32Larkin J.M. Brown M.S. Goldstein J.L. Anderson R.G. Cell. 1983; 33: 273-285Abstract Full Text PDF PubMed Scopus (342) Google Scholar), by incubating the cells in hypertonic medium (33Daukas G. Zigmond S.H. J. Cell Biol. 1985; 101: 1673-1679Crossref PubMed Scopus (172) Google Scholar), by acidification of the cytosol (34Sandvig K. Olsnes S. Petersen O.W. van Deurs B. J. Cell Biol. 1987; 105: 679-689Crossref PubMed Scopus (252) Google Scholar), or by incubating cells with concanavalin A (35Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (919) Google Scholar). Cells were grown in 75-cm2 flasks to 90% confluence. In all cases, protein synthesis was inhibited by the addition of cycloheximide (CHX) to eliminate the contribution of β2AR 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 CaCl2, 1 mm MgCl2, and 4.5 g/literd-glucose). Subsequently, cells were incubated for 5 min in depletion buffer/H2O (1:1). Next, cells were incubated for 150 min in depletion buffer supplemented with CHX (5 μg/ml). During the last 120 min isoproterenol (10 μm 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/literd-glucose, 10% fetal calf serum, and 0.5 msucrose). Cells were washed once in hypertonic medium and incubated for 150 min in this medium supplemented with CHX (5 μg/ml). During the last 120 min, isoproterenol (10 μm 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 mmacetic acid, and CHX (5 μg/ml). During the last 120 min isoproterenol (10 μm 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 μg/ml). During the last 120 min isoproterenol (10 μm final concentration) was added or not. Inhibition of endocytosis was also measured in cells transiently co-transfected with plasmids encoding HAβ2AR and K44A dynamin 48–72 h after transfection. Wild type dynamin was used instead of the K44A mutant in control experiments. Three days after transfection with the β2AR-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). Agonist-induced redistribution of HA-epitope-tagged β2ARs (HA-β2AR) 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. 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), NH4Cl (10 mm), E-64 (1 mm), leupeptin (1 mm), chloroquine (0.1 mm), MG132 (50 μm), ALLN (100 μm), ALLM (100 μm), lactacystin (10 μm). Membranes prepared from cells expressing the HA-β2AR 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 μg 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 proteasome-dependent degradation pathway (36Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2172) Google Scholar), β-catenin immunoreactivity was measured on whole cell extract using rabbit polyclonal antibodies to β-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. The EGF degradation assay was performed as described previously (37Futter C.E. Pearse A. Hewlett L.J. Hopkins C.R. J. Cell Biol. 1996; 132: 1011-1023Crossref PubMed Scopus (439) Google Scholar) 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 nm125I-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+-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 degraded125I-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). To investigate the molecular basis of β2AR 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 β-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 down-regulation of pre-existing receptor and not to mRNA regulation. Treatment of cells with CHX induced a time-dependent decrease in the number of β2ARs detected in whole cell binding assay using the membrane-permeable radioligand 125I-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 β2AR decay curve, consistent with previous studies (15Nantel F. Marullo S. Krief S. Strosberg A.D. Bouvier M. J. Biol. Chem. 1994; 269: 13148-13155Abstract Full Text PDF PubMed Google Scholar) and with the model in which sustained agonist treatment increases the β2AR degradation rate. Although indirect evidence that the decay of β2AR number upon sustained agonist activation is the consequence of receptor degradation exists (16Doss R.C. Perkins J.P. Harden T.K. J. Biol. Chem. 1981; 256: 12281-12286Abstract Full Text PDF PubMed Google Scholar, 17Morishima I. Thompson W.S. Robison G.A. Strada S.J. Mol. Pharmacol. 1980; 18: 370-378PubMed Google Scholar, 18Waldo G.L. Doss R.C. Perkins J.P. Harden T.K. Mol. Pharmacol. 1984; 26: 424-429PubMed Google Scholar), this phenomenon has not been directly documented so far, mostly because high affinity and low background anti-β2AR antibodies were not available. We have used anti-HA epitope antibodies to quantify the HA-epitope-tagged receptor (HA-β2AR) 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.2 A). A decrease of immunoreactive material was evident in cells treated for various periods of time with isoproterenol (Fig.2 B), 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 125I-CYP binding sites in the same preparations (Fig. 2 C). 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. 2 D). For each fraction, the ratio between bound 125I-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 competent for ligand binding. Taken together, our results are consistent with the current hypothesis that β2AR down-regulation is accompanied by a loss of receptor protein resulting from a proteolytic degradation of the receptor. According to the current model of receptor regulation, endocytosis is viewed as an early and necessary step in the down-regulation of β2ARs. 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 (24Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar, 38Raposo G. Dunia I. Delavier-Klutchko C. Kaveri S. Strosberg A.D. Benedetti L. Eur J. Cell Biol. 1989; 50: 340-352PubMed Google Scholar) are known to block β2AR endocytosis, probably by interfering with the formation of clathrin-coated vesicles (39Hansen S.H. Sandvig K. van Deurs B. J. Cell Biol. 1993; 121: 61-72Crossref PubMed Scopus (297) Google Scholar). Incubation with lectins that bind to the sugar moiety of the β2AR, such as concanavalin A, also block receptor internalization (24Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar). To investigate whether blocking endocytosis would also affect β2AR 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 125I-CYP binding sites was used to monitor β2AR down-regulation. All experiments were carried out in the presence of CHX,
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