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J Biol Chem, Vol. 274, Issue 41, 28900-28908, October 8, 1999
From the 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
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 (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 In the present study we have investigated mechanisms involved in the
loss of Materials--
125I-CYP was from Amersham Pharmacia
Biotech. ( Plasmid Constructions, Transfections, and Cell Culture--
To
obtain clonal cell lines expressing the human
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.
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 × 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.
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 × 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 µM 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 Inhibition of Endocytosis--
Chemical inhibition of
endocytosis in stable clones expressing Fluorescence Microscopy--
Three days after transfection with
the Analysis of Antibody-accessible Cell Surface Receptor by Flow
Cytometry--
Agonist-induced redistribution of HA-epitope-tagged
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),
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).
SDS-PAGE/Immunoblotting--
Membranes prepared from cells
expressing the HA- 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 125I-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 degraded
125I-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).
Ligand-dependent Inactivation of
Although indirect evidence that the decay of
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. 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 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 Down-regulation of
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
The results described above indicate that in L cells,
Down-regulation of Down-regulation of the Comparative Study of About one decade ago, a model of agonist-promoted
So far, only a limited number of studies have specifically addressed
the question of the relationship between endocytosis and
down-regulation pathways of The model supported by our study, in which Our demonstration that down-regulation of the The fact that a lysosomal function inhibition sufficient to prevent EGF
degradation did not affect Another potential site of membrane protein degradation is the plasma
membrane itself, which is known to contain multiple proteases. The
possible involvement of this compartment in the In conclusion, we have provided evidence for an alternative pathway of
*
This work was supported by the NATO and the INSERM-FRSQ
Collaborative Research Grants Programs and by grants from the CNRS, INSERM, the Université de Paris VII, the Fondation pour la
Recherche Médicale (FRM 2000417-01), the Agence Nationale pour la
Recherche sur le SIDA (ANRS N°97084), the Association pour la
Recherche sur le Cancer (ARC N°9010), the Medical Research Council of
Canada, and the Canadian Health and Stroke Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported by the Société de Secours des Amis des
Sciences and the Fondation pour la Recherche Médicale.
The abbreviations used are:
2-Adrenergic Receptor Down-regulation
EVIDENCE FOR A PATHWAY THAT DOES NOT REQUIRE ENDOCYTOSIS*
§,,,
, and
Immuno-Pharmacologie Moléculaire, UPR
415 of CNRS and University of Paris VII, ICGM, 75014 Paris, France,
** Pharmacologie Cellulaire et Moléculaire, UPRES-A 8068 of CNRS
and University of Paris V, ICGM, Pavillon Gustave Roussy, 75679 Paris
CEDEX 14, France, and ¶ Département de Biochimie,
Université de Montréal,
Montréal H3C 3J7, Canada
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptors (2ARs),
sustained agonist treatment caused a time-dependant decrease in both
2AR binding sites and immuno-detectable receptor. Blocking 2AR 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 2AR down-regulation. Further evidence for an
endocytosis-independent pathway of 2AR
down-regulation was provided by studies in A431 cells, a cell line
expressing both endogenous 2AR and EGF receptors. In
these cells, inhibition of endocytosis and inactivation of the
lysosomal degradation pathway did not block 2AR
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 2AR
down-regulation and that the inactivation of 2ARs,
leading to a reduction in binding sites, may occur at the plasma membrane.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptors
(2ARs)1 is
mostly dependent on rapid phosphorylation by the
cAMP-dependent protein kinase and G protein-coupled
receptor kinases (5-7). However, although the 2AR is
less rapidly affected by down-regulation than the thrombin receptor,
long term agonist-promoted 2AR down-regulation significantly contributes to the desensitization and is additive to
rapid inactivation resulting from receptor uncoupling (8). Supporting
the physio-pathological significance of 2AR
down-regulation are studies demonstrating that the development of heart
failure may be associated with 2AR down-regulation (9).
In addition, studies on 2AR 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). 2AR down-regulation involves at least two
pathways. The first is the reduction in receptor mRNA steady-state
level resulting from destabilization of the transcript (12-14). The
consequences of such a phenomenon, however, become apparent only after
many hours of sustained activation once the number of pre-existing
2ARs has decreased. The second pathway of
2AR 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 2ARs to control
levels requires neosynthesis, it was postulated 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
2ARs are degraded following the same mechanism described
for EGF and its receptor (19-21). Upon activation by the agonist, the
2AR cycles between the plasma membrane and endosomes,
where receptors are dephosphorylated (22-24). In the case of sustained
stimulation by the agonist, 2ARs 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 2ARs 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 2AR 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
2AR almost completely blocks receptor endocytosis
without impeding down-regulation challenges the model of receptor
down-regulation described above (27).
2AR 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.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)-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.
2AR, the
pBC-HA2 plasmid, encoding the hemagglutinin antigen-tagged human 2AR (22), or the pBC-2AR, 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 2AR expression by radioligand
binding assay using 125I-CYP as the ligand. A fusion
2AR-green fluorescent protein (GFP) cDNA was also
constructed by subcloning the 2AR-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).
2ARs 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 125I-CYP as the
radioligand. The assay was as described above but using membrane
preparations instead of cell suspensions.
2ARs 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-cm2 flasks to 90% confluence. In all cases, protein
synthesis was inhibited by the addition of cycloheximide (CHX) to
eliminate the contribution of 2AR 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/liter
D-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/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 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 mM
acetic 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 HA2AR and K44A
dynamin 48-72 h after transfection. Wild type dynamin was used instead of the K44A mutant in control experiments.
2AR-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).
2ARs (HA-2AR) 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.
2AR 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 (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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2ARs in
L Cells--
To investigate the molecular basis of 2AR
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
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
2ARs 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 2AR decay curve, consistent with
previous studies (15) and with the model in which sustained agonist
treatment increases the 2AR degradation rate.
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Fig. 1.
Down-regulation profile of wild type 2ARs in L cells. L cells
expressing 2ARs at approximately 200 fmol/mg of protein
were incubated with 5 µg/ml CHX in the presence (
) or absence
(
) of 10 µM isoproterenol for 0-20 h at 37 °C. The
number of 2ARs in intact cells was determined by
radioligand binding assay using 125I-CYP as ligand. The
number of receptors is expressed as the percent of 125I-CYP
binding sites in untreated cells. Data are the means ± S.E. of
three independent experiments performed in duplicate.
2AR 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-2AR antibodies were not available. We have used
anti-HA epitope antibodies to quantify the HA-epitope-tagged receptor (HA-2AR) 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.
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Fig. 2.
Isoproterenol-induced decay of
immunoreactive 2ARs.
Panel A, L cells expressing the wild type 2AR
or HA-2AR were stimulated (+) or not () for 4 h
with 10 µM isoproterenol in the presence of 5 µg/ml CHX
at 37 °C. Crude membranes were prepared, and aliquots of 50 µg of
protein were submitted to SDS-PAGE and immunoblot analysis with the
anti-HA-epitope 3F10 monoclonal antibody. Panel B, L cells
expressing HA-2AR were incubated with isoproterenol and
CHX for the indicated time, and crude membranes (50 µg/lane) were analyzed by immunoblot analysis as in
panel A. Panel C, the decay of
125I-CYP binding sites was compared with that of
immunoreactive material in crude membrane preparations from
isoproterenol-treated L cells expressing HA-2AR; the
amount of immunoreactive material was measured by densitometry; data
are the means ± S.E. of four independent experiments. Panel
D, plasma and light membrane fractions were prepared from
untreated L cells expressing HA-2AR by centrifugation on
a sucrose cushion (see "Experimental Procedures"). Western blots
were carried on aliquots of 25 µg of protein, and the amount of
immunoreactive material was assessed by densitometry. The density of
125I-CYP binding sites was measured in the same fractions.
Histograms represent the ratio between 125I-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.
2AR down-regulation is
accompanied by a loss of receptor protein resulting from a proteolytic
degradation of the receptor.
2ARs 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 2ARs. 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 2AR endocytosis, probably by
interfering with the formation of clathrin-coated vesicles (39).
Incubation with lectins that bind to the sugar moiety of the
2AR, such as concanavalin A, also block receptor
internalization (24). To investigate whether blocking endocytosis would
also affect 2AR 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 2AR 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 ~25% 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 contrast, 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).
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Fig. 3.
Inhibition of 2AR endocytosis does not abolish
receptor down-regulation. Endocytosis of the wild type
2AR expressed in L cells was inhibited by hypertonic
sucrose (Suc), concanavalin A (ConA), potassium
depletion (KCl) or cytosolic acidification (Acid)
as described under "Experimental Procedures." Cells were incubated
with or without CHX (5 µg/ml) in the presence or absence of 10 µM isoproterenol (ISO) for 2 h at
37 °C. Cells were collected, plasma membranes and light vesicle
fractions were prepared by centrifugation on a sucrose cushion, and the
number of 2ARs were determined in each fraction by a
radioligand binding assay using 125I-CYP as ligand.
Endocytosis is expressed as the isoproterenol-induced change in the
proportion of receptors associated with the light membrane fraction
(panel A). Receptor down-regulation was assessed by
measuring the total 125I-CYP binding sites (in both plasma
and light membrane fractions) still present after isoproterenol
treatment. Down-regulation was expressed as % of receptor number
measured under basal conditions (panel B). Data are the
means ± S.E. of at least three independent experiments carried
out in triplicate.
2AR agonist-dependent
redistribution. Previous experiments have shown that the fusion of GFP
to the carboxyl-terminal extremity of the 2AR does not
modify ligand-dependent receptor endocytosis (40, 41). L
cells were transiently transfected with a plasmid carrying the
2AR-GFP fusion cDNA and depleted or not of
intracellular K+ 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,
2AR-GFP was internalized in punctiform structures
probably corresponding to endosomes (panel B). In cells
depleted of K+ (panel C) or preincubated with
high sucrose or concanavalin A or with a cytosol-acidifying medium (not
shown), no isoproterenol-dependent accumulation of
2AR-GFP could be visualized in endosomes. FACS analysis
of HA-2AR 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-2AR. 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+ (not shown).
FACS analysis could not be used to study the effect of concanavalin A,
since this treatment causes cell aggregation.
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Fig. 4.
Agonist-promoted redistribution of GFP- and
HA-tagged 2ARs in the presence and
absence of endocytosis inhibitors. L cells expressing either
GFP-2AR or HA-tagged 2AR were
preincubated or not for 1 h with the indicated treatments
inhibiting endocytosis and then stimulated or not for 1 h with 10 µM isoproterenol. Panels A-C,
GFP-2AR redistribution was studied by fluorescence
microscopy under basal conditions (A) following
isoproterenol stimulation (B) or following isoproterenol
stimulation in a K+-depleted medium (C). Data
shown are representative of 3 independent experiments. Panels
D-E, antibody-accessible HA-tagged 2AR 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 (+ 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.
2AR 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 2AR 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
down-regulation. Indeed, incubation with isoproterenol caused a similar
decrease of 2AR 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 2AR endocytosis
and down-regulation can be functionally dissociated in L cells and that
the latter is likely to occur at the plasma membrane.
View larger version (22K):
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Fig. 5.
Expression of the dominant negative K44A
dynamin mutant in L cells blocks 2AR endocytosis but not
down-regulation. L cells were co-transfected with a plasmid coding
for either wild type (wt) or dominant negative K44A dynamin
(K44A) and a plasmid encoding HA-2AR. Forty-eight h
after transfection cells were incubated for 6 h with CHX (5 µg/ml) in the absence or presence of 10 µM
isoproterenol. Plasma and light membrane fractions were prepared as
described under "Experimental Procedures." Panel A, the
endocytosis assay was conducted as in Fig. 3. Panel B,
Western blot showing the decay of immunoreactive receptor in plasma
membrane fractions of cells transfected as above and treated or not
with isoproterenol. Panel C, 125I-CYP binding
sites and densitometric analysis of immunoreactive material measured on
plasma membrane fractions. Data are the means ± S.E. of three
independent experiments.
2ARs Is Maintained upon
Inactivation of the Lysosomal Pathway--
Since the results described
above argue against a role for endocytosis in the 2AR
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 NH4Cl, and concanamycin B, a highly specific
inhibitor of vacuolar H+-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
2AR 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 2AR does not occur in the lysosomal compartment.
Consistent with this idea is the observation that alkalinization with
NH4Cl did not block agonist-promoted receptor loss of
immunoreactivity (data not shown).
View larger version (20K):
[in a new window]
Fig. 6.
Inactivation of lysosomal pathway does not
abolish 2AR down-regulation.
L cells expressing 2AR were preincubated for 1 h
with various compounds interfering with the lysosome function in the
presence (panel A) or absence (panel B) of CHX (5 µg/ml) at 37 °C. Isoproterenol (ISO, 10 µM) was then added for 2 (panel A) or 24 additional h (panel B). Crude cell membranes were prepared,
and 2AR binding sites were determined by radioligand
binding assay. The number of receptors is expressed as the percent of
125I-CYP binding sites in untreated cells. Data are the
means ± S.E. of three independent experiments carried out in
triplicate. Chlo, chloroquine; Con B,
concanamycin B; Leu, leupeptin.
2AR Is Maintained upon
Inactivation of the Ubiquitin Proteasome Pathway--
In an attempt of
identifying the mechanism of 2AR 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
(45-47). 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 2AR 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 2AR 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 2AR in
L cells is not the consequence of ubiquitination and proteasome
degradation.
View larger version (42K):
[in a new window]
Fig. 7.
Inactivation of the proteasome does not
interfere with 2AR
down-regulation. L cells expressing 2AR were
pretreated for 1 h with various compounds interfering with the
proteasome activity in the presence (panel A) or absence
(panel B) of CHX (5 µg/ml) at 37 °C. Isoproterenol
(ISO, 10 µM) was then added for 2 (panel
A) or 24 additional h (panel B). Crude cell membranes
were prepared, and 2AR binding sites were determined by
radioligand binding assay. The number of receptors is expressed as the
percent of 125I-CYP binding sites in untreated cells. Data
are the means ± 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.
2AR and EGF Down-regulation in
A431 Cells--
To investigate whether the endocytosis-independent
2AR 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 2AR
(53) and were previously used as a model to study 2AR
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 2AR
down-regulation, with 50-60% of the 2AR 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). 2AR down-regulation and EGF degradation
were tested in parallel after various treatments that inhibited
endocytosis or lysosomal degradation. Concanavalin A and K+
depletion blocked at least 80% of EGF degradation, whereas they had
minor or no effect on 2AR down-regulation (Fig.
8C). Sucrose totally blocked EGF degradation, compared with
a 40% inhibition of 2AR down-regulation. Treatments
blocking lysosomal function also had very different effects on the two
receptors; neither leupeptin nor NH4Cl could affect
2AR down-regulation (Fig. 8D), whereas they
inhibited 80 and 100% of EGF degradation, respectively (Fig.
8B). These results show that in A431 cells,
2AR down-regulation involves a different pathway than
that of EGF degradation.
View larger version (36K):
[in a new window]
Fig. 8.
Down-regulation of 2AR and degradation of EGF follow
distinct pathways in A431 cells. Panel A,
isoproterenol-induced down-regulation of 2AR in A431
cells. Cell were stimulated for the indicated times with 10 µM isoproterenol, crude cell membranes were prepared, and
125I-CYP binding sites were determined by radioligand
binding assay. The bars indicate S.E. of triplicates.
Panel B, time course of 125I-EGF degradation in
intact control A431 cells and A431 cells treated with the lysosome
inhibitors leupeptin and NH4Cl. 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 2AR down-regulation and
125I-EGF degradation. Cells were pretreated for 1 h
with the indicated inhibitors. Down-regulation of 2AR
was measured as in panel A, and 125I-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
2AR 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 2AR
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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2AR 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 2ARs in L cells is the first direct
indication that 2AR 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 2AR down-regulation.
Chemical treatments that block endocytosis or alter lysosomal function
are ineffective on 2AR 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 2AR endocytosis but
not down-regulation.
2ARs. The idea, supported by our data, that endocytosis and down-regulation could involve different pathways is consistent with the previous observation that the S355-364-2AR 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 2AR
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
2AR binding sites coincided with the appearance of
fluorescence in lysosomes was interpreted as evidence of the role of
this compartment in 2AR 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.
2AR
endocytosis and down-regulation are independent, challenges an
observation reported by Gagnon et al. (26). In HEK293 cells,
endocytosis and down-regulation of the 2AR 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
2AR 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
2AR 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 2AR down-regulation could be inhibited
following a chemical block of endocytosis in HEK293 cells (data not shown).
2AR 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.
2AR 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
2ARs excluding the involvement of this degradation
pathway. Interestingly, long term incubation of L cells with proteasome
inhibitors markedly increased the number of 125I-CYP
binding sites. This observation may suggest that most of the
2ARs 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 2AR
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.
2AR
down-regulation is supported by two of our observations: (i)
fluorescence microscopy studies confirmed that GFP- or HA-tagged
2ARs were not internalized under conditions where
receptor down-regulation was fully effective; (ii) a comparable
decrease of 125I-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 2AR 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 (~80%
cleavage in 5 min versus ~50% of 2AR
inactivation after 6 h). Second, the metalloprotease that cleaves
V2 receptor was blocked by 0.1 mM ZnCl2,
whereas Zn2+ ions had no effect on 2AR
inactivation (data not shown). Additional studies are required to
uncover the precise mechanism of 2AR down-regulation at
the plasma membrane.
2AR 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.
FOOTNOTES
A Medical Research Council scientist.
To whom correspondence should be addressed: Pharmacologie
Cellulaire et Moléculaire, ICGM, Pavillon Gustave Roussy, 27 rue du Faubourg St Jacques, 75679 Paris CEDEX 14 France. Tel.: 331 44 41 25 58; Fax: 331 44 41 25 57; E-mail: marullo@cochin.inserm.fr.
ABBREVIATIONS
2AR, 2-adrenergic receptor;
HEK, human embryonic kidney
cells;
EGF, epidermal growth factor;
DMEM, Dulbecco's modified
Eagle's medium;
PBS, phosphate-buffered saline;
GFP, green fluorescent
protein;
PAGE, polyacrylamide gel electrophoresis;
CHX, cycloheximide;
HA, hemagglutinin;
FACS, fluorescence-assisted cell sorting;
ALLN, N-acetyl-Leu-Leu-norleucinal or calpain inhibitor I;
ALLM, N-acetyl-Leu-Leu-methioninal or calpain inhibitor II;
MG132, carbobenzoxyl-L-leucyl-L-leucyl-L-leucinal,
a cell-permeable proteasome inhibitor;
125I-CYP, 125I-iodocyanopindolol, a -adrenergic antagonist.
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