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Journal of Cell Science 112, 329-338 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS7309
Agonist-induced sorting of human β2-adrenergic receptors to lysosomes
during downregulation
Robert H. Moore1,2, Amjad Tuffaha1, Ellen E. Millman1, Wenping Dai2, Hassan S. Hall2, Burton F. Dickey2,3,4,5
and Brian J. Knoll2,3,4,*
Departments of 1Pediatrics (Pulmonary), 2Molecular Physiology & Biophysics, 3Medicine (Pulmonary) and 4Cell Biology, Baylor
College of Medicine, Houston VA Medical Center5, Houston, TX 77030, USA
*Author for correspondence
Accepted 13 November 1998; published on WWW 13 January 1999
SUMMARY
During prolonged exposure to agonist, β2-adrenergic
receptors undergo downregulation, defined by the loss of
radioligand binding sites. To determine the cellular basis
for β2-adrenergic receptor downregulation, we examined
HEK293 cells stably expressing β2-adrenergic receptors
with an N-terminal epitope tag. Downregulation was
blocked by leupeptin, a cysteine protease inhibitor, but not
by pepstatin, an inhibitor of aspartate proteases.
Immunofluorescence microscopy of cells treated with
agonist for 3-6 hours in the presence of leupeptin showed
β2-adrenergic receptors, but not transferrin receptors,
localizing with the lysosomal protease cathepsin D, and
with lysosomes labeled by uptake of a fluorescent fluidphase marker. No localization of β2-adrenergic receptors
with lysosomal markers was observed in the absence of
leupeptin, most likely due to proteolysis of the epitope. The
proton pump inhibitor, bafilomycin A1, significantly
inhibited this agonist-induced redistribution of β2adrenergic receptors into lysosomes, causing receptors to
accumulate in the rab11-positive perinuclear recycling
INTRODUCTION
G-protein coupled receptors (GPCRs) comprise an extended
family of cell surface proteins that transduce signals from diverse
kinds of extracellular stimuli to intracellular effector systems. A
particularly well-studied GPCR is the β2-adrenergic receptor
(β2AR), which is an important target for catecholamine
hormones in physiologic actions such as the relaxation of
vascular and airway smooth muscle. Agonist-bound β2ARs
work principally by activating adenylyl cyclase through a
coupled stimulatory G-protein (Gs), causing a rise in the
intracellular levels of cAMP, a second messenger that eventually
mediates most downstream physiological effects (Liggett, 1997).
The signaling capacity of β2ARs is attenuated by
desensitization processes involving several distinct, but
overlapping mechanisms. Within seconds of agonist binding,
β2ARs are rapidly phosphorylated by G-protein coupled
receptor kinases that recognize only agonist-bound receptors
compartment and slowing the rate of β2-adrenergic
receptor recycling. Control experiments showed that
leupeptin had no nonspecific effects on the cellular
trafficking of either β2-adrenergic receptors or transferrin
receptors. Although cAMP alone caused a small decline in
receptor levels without redistributing β2-adrenergic
receptors from the plasma membrane, this effect was
additive to that seen with agonist alone, suggesting that
agonist-induced β2-adrenergic receptor downregulation
resulted largely from cAMP-independent mechanisms.
These results indicate that during agonist-induced
downregulation, a significant fraction of β2-adrenergic
receptors are specifically sorted to lysosomes via the
endosomal pathway, where receptor degradation by
cysteine proteases occurs. These results provide a cellular
explanation for the loss of radioligand binding sites that
occurs during prolonged exposure to agonist.
Key words: β2-adrenergic receptor, Lysosome, Downregulation,
Laser confocal microscopy, rab11, Bafilomycin A1
(Ferguson et al., 1997), and also by protein kinases A and C
(Yuan et al., 1994), resulting in a functional uncoupling of
receptors from Gs. A second rapidly occurring event, receptor
endocytosis into endosomes, physically uncouples β2ARs from
Gs (Ferguson et al., 1997; Morrison et al., 1996) and transports
receptors to an early endosome compartment containing both
transferrin receptors (von Zastrow and Kobilka, 1992) and the
ras-related GTPase rab5 (Moore et al., 1995). β2ARs may be
dephosphorylated at some point during their intracellular
trafficking by a membrane-associated protein phosphatase 2Alike enzyme, following which receptors reappear on the cell
surface in a fully sensitized state (Krueger et al., 1997; Yu et
al., 1993). The kinetics of receptor internalization are relatively
rapid (tg < 3 minutes), as is recycling back to the cell surface
(tg < 8 minutes) (Morrison et al., 1996).
A third desensitization mechanism, downregulation, occurs
during prolonged agonist treatment (at least several hours) and
results in the loss of radioligand binding sites. In contrast to
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R. H. Moore and others
rapid desensitization events (Ferguson et al., 1997), the
mechanism of downregulation is poorly understood. At steadystate, the total number of β2ARs should be a function of the
rates of receptor synthesis and loss of radioligand binding sites,
either of which in principal could be influenced by agonist.
Significant levels of receptor downregulation can be observed
in cells treated with cell permeable analogs of cAMP or by
activators of adenylyl cyclase, largely as a result of increased
mRNA degradation (Bouvier et al., 1989). β2AR mRNA levels
also are reduced following agonist treatment in some systems
(Collins et al., 1992; Hadcock and Malbon, 1988), though not
in others (Kelsen et al., 1997), suggesting that agonist may
reduce the synthesis of β2ARs. However, this effect would be
significant only if the half-lives of β2ARs themselves were
relatively short, and available evidence indicates in fact that
receptors are long-lived. Estimates of β2AR half-life,
determined by measuring the rate of receptor restoration after
blockade by an irreversible antagonist, suggests a basal halflife of up to 30 hours (Mahan and Insel, 1986), while the half
time for downregulation is considerably shorter (3-10 hours)
(Pittman et al., 1984). The implication is that while the rate of
β2AR synthesis may be affected by agonist, there must also be
significant receptor degradation.
For some signal-transducing receptors, the sequence of
events during downregulation is understood in considerable
detail. For example, ligand-bound epidermal growth factor
receptors internalize into early endosomes before rapidly
trafficking to lysosomes (within about 30 minutes), as assessed
by detailed microscopic examination and subcellular
fractionation (Chang et al., 1993). Also, the ligand-induced
degradation of these receptors has been directly measured by
biochemical procedures, and correlates temporally with
lysosomal trafficking (Beguinot et al., 1984; Stoscheck and
Carpenter, 1984). In contrast, there is little information about
the intracellular fates of β2ARs during chronic agonist
exposure. In recent reports (Gagnon et al., 1998; Kallal et al.,
1998), during agonist-induced downregulation, β2AR-green
fluorescent protein chimeras (β2AR-GFP) were shown to
localize in compartments that accumulated fluorescentlylabeled dextran. However, the exact nature of these
compartments was not defined and the intracellular itineraries
of β2ARs during downregulation were not fully examined. In
the present study, we determine the intracellular events leading
to agonist-induced β2AR downregulation and specifically
demonstrate the sorting of receptors to lysosomes during
prolonged exposures to agonist.
MATERIALS AND METHODS
Materials
12β6 cells (a gift of B. Kobilka, Stanford, CA) were cultured in
Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine
serum and 200 µg/ml Geneticin (Life Technologies, Gaithersburg,
MD). This cell line was derived by stable transfection of HEK293
cells, and expresses human β2ARs with an N-terminal epitope tag
derived from influenza hemagglutinin (HA-tag) at a level of
300,000/cell (Moore et al., 1995; von Zastrow and Kobilka, 1992).
Mouse monoclonal antibodies against the HA-tag were 12CA5
(obtained from Boehringer-Mannheim, Indianapolis, IN) or mHA.11
(from Berkeley Antibody Co., Berkeley, CA). A mouse monoclonal
antibody against human transferrin receptors was purchased from
Pharmingen (San Diego, CA), and rabbit polyclonal antiserum against
human cathepsin D and rab11 were obtained from Calbiochem (La
Jolla, CA) and Zymed Laboratories (South San Franscisco, CA),
respectively. Species-appropriate goat secondary antibodies were
purchased from Molecular Probes (Eugene, OR). [3H]CGP12177 (44
Ci/mmol) was obtained from Dupont-New England Nuclear (Boston,
MA). Texas Red conjugated bovine serum albumin (BSA-Texas Red)
was purchased from Molecular Probes, and digitonin from
Boehringer-Mannheim. All other reagents were obtained from Sigma
Chemical Co. (St Louis, MO) unless otherwise noted.
Measurement of receptor downregulation
12β6 cells were grown in poly-L-lysine coated 12-well clusters to 5070% confluence, then exposed to 5 µM isoproterenol (ISO) in
triplicate wells at 37°C for 24 hours or left untreated. Some wells were
treated with 8-bromo-cAMP (1 mM) alone or in addition to ISO. In
the protease inhibitor experiments, cells were treated similarly with
ISO except that leupeptin (100 µM) and/or pepstatin (100 µM) were
added one hour prior to and 12 hours after the addition of ISO. Control
cells received the same concentrations of vehicle alone (0.1%
dimethyl sulfoxide, Me2SO). Following agonist treatment, the cells
were washed four times with ice-cold phosphate buffered saline
(PBS), then incubated with 6 nM [3H]CGP12177 in DMEM with 20
mM Hepes, pH 7.4 (DMEM-H) containing 0.05% digitonin at 4°C for
90 minutes. The cells were washed twice with DMEM-H, then lysed
in the wells with 0.1% Triton X-100, and the lysates counted by
scintillation spectroscopy. Nonspecific binding of radioligand was
determined by parallel incubations in the presence of propranolol (3
µM), and was less than 5% of total. Statistical significance was
determined using a one-way ANOVA and defined by P<0.05.
Previous control experiments to evaluate the need for inhibitors of
oxidation demonstrated that the extent of ISO-induced β2AR
downregulation was not altered upon the addition of ascorbic acid (0.1
mM) and thiourea (1 mM). Also, following a 24 hour incubation with
12β6 cells, medium containing ISO alone was able to fully stimulate
receptor internalization.
Measurement of β2AR internalization and recycling
kinetics
12β6 cells were grown to 50-70% confluence in poly-L-lysine-coated
24-well clusters and pretreated for 6 hours with leupeptin (100 µM)
and pepstatin (100 µM) or left untreated as a control. Cells were
exposed to ISO (5 µM) in triplicate wells for varying times up to 20
minutes, washed with ice-cold DMEM-H, and incubated with 6 nM
[3H]CGP12177 in DMEM-H at 4°C for 90 minutes in the absence of
digitonin to measure surface receptors only. The cells were washed,
lysed and counted as described above. Nonspecific binding of
radioligand was determined by parallel incubations in the presence of
3 µM propranolol, and was less than 5% of total. The fraction of
receptors left on the cell surface was plotted versus time of agonist
exposure, and the curves fitted by nonlinear regression using the
computer program GraphPad Prism (version 2; San Diego, CA). The
rate of approach to a steady-state between surface and internal
receptors is determined by first-order rate constants for receptor
endocytosis (ke) and recycling (kr). Unique values for these rate
constants were estimated by curve-fitting to equation 4 of Morrison
et al. (1996), and the level of statistical significance was determined
using a paired Student’s t-test and defined by P<0.05. Rate constants
are presented as the mean ± s.e.m. of five independent experiments.
In separate experiments, 12β6 cells were pretreated with leupeptin
(100 µM) in the presence and absence of bafilomycin A1 (1 µM) for
30 minutes, then exposed to ISO (5 µM) for 6 hours. Cells were
harvested by tituration in ice-cold DMEM-H and washed by
centrifugation. To allow receptor recycling, cells were suspended in
DMEM-H prewarmed to 37°C and containing 10% normal goat serum
and 6 nM [3H]CGP12177. Samples containing 75 µg of protein were
removed at varying times up to 60 minutes, rapidly diluted into ice-
β2-adrenergic receptors and lysosomes
331
cold DMEM-H containing 6 nM [3H]CGP12177, and left on ice for
60 minutes for binding to equilibrate. The cells were collected by
rapid filtration and the bound tritium quantified by scintillation
spectroscopy. Nonspecific binding of radioligand was determined by
parallel incubations with 3 µM propranolol and was less than 5% of
total binding. The fraction of receptors on the cell surface was plotted
versus time of recycling, and the curves fitted by nonlinear regression
as described by Morrison et al. (1996). Recycling rate constants are
presented as the mean ± s.e.m. of three separate experiments.
Immunofluorescence laser confocal microscopy
12β6 cells were grown on poly-L-lysine-coated 22 mm glass coverslips
to 50-70% confluence in 6-well clusters and treated as indicated in the
figure legends. In some experiments, prior to ISO treatment, the cells
were fed BSA-Texas Red (5 mg/ml) in DMEM-H for one hour at 37°C,
washed twice with DMEM-H, and chased for one hour at 37°C with
DMEM-H to label lysosomes. In separate experiments, cells were
pretreated with 1 µM bafilomycin A1 in Me2SO (final concentration,
0.1%) for 30 minutes prior to the addition of agonist. In control
experiments, cells were pretreated with 0.1% Me2SO alone. Following
treatment, the cells were washed with PBS containing 1.2% sucrose
(PBSS), fixed with 4% paraformaldehyde in PBSS at 4°C for 10
minutes, then washed again with PBSS. The following steps were done
at room temperature, with PBSS used for washes. The fixed cells were
incubated in 0.34% L-lysine, 0.05% Na-m-periodate for 20 minutes,
then washed and permeabilized with 0.2% Triton X-100. After further
wash, the cells were blocked with 10% heat-inactivated goat serum
(HIGS) for 15 minutes. Primary antibodies were diluted in PBSS with
0.2% HIGS and 0.05% Triton X-100, then added to the cells and left
for 1-12 hours. The cells were washed four times before labeling with
secondary antibodies using the same procedure as for the primary
antibodies. We used the following concentrations of antibodies:
12CA5, 1 µg/100 µl; mHA.11, 0.5 µg/100 µl; monoclonal antitransferrin receptor, 2 µg/100 µl; anti-cathepsin D, 2 µl/100 µl; antirab11, 1 µg/100 µl; FITC-anti-mouse IgG, 1:100 dilution; Texas Red
anti-rabbit IgG, 1:200 dilution. The coverslips were mounted in
Mowiol and viewed using a Molecular Dynamics Multiprobe 2001
laser confocal scanning microscope. Optical sections through cells
were collected at a thickness of 0.49 µm. The digitalized images were
transferred to Adobe Photoshop for processing, and printed using a
Codonics 1600 dye sublimation printer.
Fig. 1. Agonist-induced β2AR downregulation in 12β6 cells. Each
bar indicates the total remaining radioligand binding sites following
24 hours of the indicated treatment. The data are expressed as the
mean ± s.e.m. of at least four separate experiments. Control cells had
a mean specific binding of 12,700±1,000 cpm/well. Background
binding determined in the presence of propranolol was always less
than 500 cpm/well. Control, vehicles alone; ISO, 5 µM ISO; cAMP,
1 mM 8-bromo-cAMP; ISO + cAMP, 5 µM ISO plus 1 mM 8bromo-cAMP. *P<0.05 of control group versus ISO and cAMP
groups. **P<0.05 of ISO versus ISO + cAMP group.
suggesting different mechanisms for cAMP- and agonistinduced β2AR downregulation. To determine if lysosomal
proteases contributed to the observed agonist-induced loss of
receptors, we measured agonist-induced downregulation in
cells pretreated with the protease inhibitors leupeptin and
pepstatin, which have been shown previously to block ligandactivated degradation of epidermal growth factor receptors in
rat hepatocytes (Renfrew and Hubbard, 1991). In 12β6 cells,
pretreatment with these protease inhibitors almost completely
blocked agonist-induced β2AR downregulation (Fig. 2).
RESULTS
We determined the extent of β2AR downregulation in HEK293
cells stably expressing a human β2AR with an N-terminal HAepitope tag (12β6 cells) (von Zastrow and Kobilka, 1992).
Total binding sites (surface and internal) for the βARantagonist [3H]CGP12177 were measured in digitoninpermeabilized cells before and after chronic agonist exposure
(Fig. 1). Using this method, we found that approximately 30%
of total cellular receptors were no longer detectable (i.e.
downregulated) following 24 hours of treatment with 5 µM
ISO. There were no changes in cell numbers, total protein, or
cell viability (Trypan Blue exclusion) after a 24 hour ISO
treatment compared with untreated control cells, excluding
altered growth or viability as a cause of agonist-induced loss
of radioligand binding sites (data not shown). Since cAMPdependent mechanisms also have been shown to induce β2AR
loss in some cell systems, we determined the extent of receptor
downregulation in 12β6 cells treated with 1 mM 8-bromo
cAMP in the absence and presence of agonist. While cAMP
alone significantly reduced receptor levels by approximately
13%, this effect was additive to that seen with agonist,
Fig. 2. Effect of protease inhibitors on agonist-induced β2AR
downregulation in 12β6 cells. Each bar indicates the total remaining
radioligand binding sites following 24 hours of the indicated
treatment. The data are expressed as the mean ± s.e.m. of at least
four separate experiments. Control, vehicles alone; ISO, 5 µM ISO
plus vehicles; PI (protease inhibitor) control, 100 µM each of
pepstatin and leupeptin; PI + ISO, 5 µM ISO plus 100 µM each of
pepstatin and leupeptin; Leu + ISO, 5 µM ISO plus 100 µM
leupeptin; ISO + Pep, 5 µM ISO plus 100 µM pepstatin. *P<0.05 of
control group versus ISO and pepstatin + ISO groups. **P<0.05 of
ISO group versus protease inhibitors + ISO and leupeptin + ISO
groups (one-way ANOVA).
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R. H. Moore and others
Fig. 3. Time- and protease inhibitordependent localization of β2ARs with
lysosomal markers during agonistinduced downregulation. 12β6 cells
were exposed to 5 µM ISO for 5
minutes or 6 hours in the presence of
100 µM each of leupeptin and
pepstatin, then fixed and stained with
antibodies as described in Materials
and Methods. (A-F) The cells were
stained simultaneously with an
antibody against the HA epitope and an
antibody against cathepsin D. (G-I) the
cells were exposed to BSA-Texas Red
for one hour, then washed and chased
with medium without BSA-Texas Red
for one hour before adding ISO. The
cells were then stained with an
antibody against the HA-epitope. In
merged images (C,F,I), localization of
the markers at 6 hours is seen as
yellow. Bar, 10 µm.
Since inhibiting lysosomal proteases significantly blocked
β2AR downregulation, it seemed likely that downregulation
might require the trafficking of receptors to lysosomes during
prolonged agonist exposure. To determine whether this might
be the case, we tracked receptors using immunofluorescence
laser confocal microscopy. To label lysosomes, we used two
markers: antibodies against cathepsin D, an aspartic lysosomal
protease (Tang and Wong, 1987) which also localizes with the
lysosomal integral membrane protein LAMP-1 in 12β6 cells
(data not shown); and BSA-Texas Red, a fluorescent fluidphase marker given to cells in a pulse-chase manner to label
lysosomes (Racoosin and Swanson, 1994). We treated 12β6
cells with ISO in the presence and absence of leupeptin and
pepstatin for varying times up to 6 hours and examined
specimens for the localization of HA-tagged β2ARs with either
cathepsin D or BSA-Texas Red. In protease inhibitor-treated
cells, there was minimal localization of β2ARs with cathepsin
D (Fig. 3A-C) or BSA-Texas Red (data not shown) following
a 5 minute exposure to agonist. Very little colocalization also
was observed after treatment periods up to 60 minutes (data
not shown). However, after 6 hours of agonist treatment,
increased localization of β2ARs with both cathepsin D (Fig.
3D-F) and BSA-Texas Red (Fig. 3G-I) was observed.
Significant localization also was observed after 3 hours of
agonist treatment (data not shown). Some intracellular β2ARs
do not localize with cathepsin D but substantially localize with
transferrin receptors (data not shown), and most likely
represent β2ARs that have not yet reached lysosomes or have
been sorted from endosomes for transit to the plasma
membrane. No internalized receptors were seen following 24
hours of exposure to 8-bromo cAMP alone (data not shown).
In the absence of protease inhibitors, there was little or no
localization of receptors with either cathepsin D or BSA-Texas
Red, even after 6 hours of ISO exposure (Fig. 4). Instead, these
receptors almost completely overlapped with transferrin
receptors (data not shown). Although this result was likely a
result of degradation of the β2AR N-terminal HA epitope by
lysosomal proteases, we also considered the possibility that
treatment with leupeptin and pepstatin may alter vesicular
trafficking by inducing a nonspecific sorting of receptors to
lysosomes. To test for this possibility, we first determined if
transferrin receptors localized with cathepsin D in cells
incubated with β-agonist in the presence and absence of
protease inhibitors. Normally, transferrin receptors undergo
constitutive internalization from the cell surface into early
endosomes and the perinuclear recycling compartment and
then recycle to the plasma membrane, with few receptors ever
reaching lysosomes (Daro et al., 1996; Dautry-Varsat et al.,
β2-adrenergic receptors and lysosomes
1983; Green et al., 1997). We found minimal localization of
transferrin receptor with cathepsin D in either control or in
protease inhibitor-treated cells, suggesting that leupeptin and
pepstatin did not cause a nonspecific shunting of endosomal
receptors to lysosomes (data not shown). In addition, if normal
endosomal sorting of receptors were affected by the
pretreatment of cells with leupeptin and pepstatin, we
considered it likely that the first-order rate constants of agonistinduced β2AR endocytosis (ke) and recycling (kr) would also
be altered. However, the rate constants for agonist-induced
β2AR endocytosis during a 20 minute exposure to ISO were
not significantly different comparing control cells and cells
pretreated with protease inhibitors for 6 hours (ke for control
cells = 0.171±0.014 minute−1; ke for protease inhibitor treated
cells = 0.151±0.024 minute−1). In addition, values for kr,
derived from curve-fitting as previously described (Morrison et
al., 1996), did not differ significantly in the two groups (kr for
control cells = 0.132±0.020 minute−1; kr for protease inhibitor
treated cells = 0.135±0.041 minute−1).
We next performed studies to determine which class of
lysosomal protease is responsible for β2AR downregulation.
Cells were incubated with either leupeptin (100 µM), which
inhibits cysteine proteases such as cathepsins B, H, and L, or
pepstatin (100 µM), which inhibits the activity of the aspartic
protease, cathepsin D (Erickson, 1989). As shown in Fig. 2,
most of the receptor downregulation following a 24 hour
exposure to agonist was inhibited by leupeptin, while pepstatin
had no effect. This result is supported by morphologic data
indicating that the localization of HA-β2AR with cathepsin D
is maintained in the presence of leupeptin alone (Fig. 5A-C)
and is lost when pepstatin is used alone (Fig. 5D-F). The effect
of leupeptin might be explained if it permitted the
333
accumulation of β2ARs in lysosomes as a result of normal
receptor turnover. If this were the case, we should observe
localization of β2ARs with cathepsin D during prolonged
leupeptin exposure even in the absence of agonist. To test for
this, 12β6 cells were incubated with leupeptin alone for 30
hours, the maximal estimated half-life of β2ARs in untreated
cells (Mahan and Insel, 1986), then fixed and labeled with
antibodies to localize both β2ARs and cathepsin D. In
examining 300 cells treated with leupeptin, only 6 cells
appeared to have a small amount of punctate staining for
β2ARs, a fraction similar to that observed in 12β6 cells not
treated with leupeptin, and no receptors localized with
cathepsin D (data not shown).
We and others (Moore et al., 1995; von Zastrow and Kobilka,
1992) have shown that β2ARs enter early endosomes following
brief exposures to agonist. To determine if β2ARs transit the
normal endocytic pathway en route to degradation within
lysosomes during chronic exposures to agonist or use some
alternative pathway, we studied the effects of bafilomycin A1,
a vacuolar-type H+-ATPase inhibitor that raises endosome and
lysosome pH (Yoshimori et al., 1991) and blocks the formation
of vesicles carrying endocytosed materials from early
endosomes to lysosomes (Clague et al., 1994; van Deurs et al.,
1996). In cells pretreated with bafilomycin A1 (1 µM) in the
presence of leupeptin followed by ISO for 6 hours, little or no
colocalization of β2ARs and cathepsin D was observed (Fig.
6A-C), whereas significant colocalization of the two markers
was noted in cells not pretreated with bafilomycin A1 (compare
with Fig. 5A-C). Indeed, in most cells, receptors appeared in
a dense perinuclear compartment which was completely devoid
of cathepsin D labeling. To determine the nature of this
compartment, we labeled cells treated similarly in the presence
Fig. 4. Localization of β2ARs
with lysosomal markers not
detected in the absence of
protease inhibitors. 12β6 cells
were treated with ISO for 6
hours in the absence of
leupeptin and pepstatin, then
fixed and stained as described
in Materials and Methods to
visualize HA-β2AR or
cathepsin D. Some cells were
labeled with BSA-TxR for 60
minutes followed by a 60
minute chase. (A-C)
Localization of β2AR with
cathepsin D; (D-F)
Localization of β2AR with
BSA-TxR. Bar, 10 µm.
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R. H. Moore and others
Fig. 5. Specificity of lysosomal proteases in β2AR localization with cathepsin D. Cells were treated as in Fig. 2 except that leupeptin or
pepstatin (100 µM each) were added individually. Localization of HA-β2ARs with cathepsin D is observed in leupeptin-treated (A-C) but not in
pepstatin-treated (D-F) cells. Bar, 10 µm.
and absence of bafilomycin A1 with antiserum against the
perinuclear recycling compartment marker, rab11 (Daro et al.,
1996; Green et al., 1997). In cells exposed to ISO for 6 hours
in the presence of leupeptin alone, there was only a small
degree of β2AR localization with rab11 (Fig. 6G-I), which
increased with more prolonged exposures up to 18 hours (data
not shown). However, pretreatment with bafilomycin A1
caused the marked accumulation of β2ARs in a rab11containing perinuclear compartment (Fig. 6D-F) and
significantly slowed the rate of receptor recycling to the plasma
membrane (kr following 6 hour exposure to ISO =
0.076±0.014; kr for cells pretreated with bafilomycin A1 =
0.024±0.007) without affecting the final extent of recycling at
60 minutes. Despite this striking redistribution of β2ARs,
bafilomycin A1 did not noticeably alter the distribution of
transferrin receptors (data not shown), similar to previously
published data (Johnson et al., 1993).
DISCUSSION
The data reported here provide direct evidence that the agonistdependent loss of β2ARs defined by radioligand binding is
caused by the specific trafficking of these receptors to
lysosomes for degradation. Previous studies of β2AR
downregulation have used the loss of radioligand binding as an
index of receptor degradation, an activity which in principle
may be affected by factors other than receptor degradation,
including a selective inhibition of receptor synthesis. In
addition to demonstrating an important role for proteolysis in
β2AR downregulation, we also define the cellular basis for
downregulation by showing that the mechanism of β2AR
downregulation involves the specific, agonist-induced sorting
of receptors from endosomes to lysosomes.
This interpretation of our immunofluorescence data depends
upon the identification of lysosomes in 12β6 cells, and for this
purpose, we used three criteria: (1) the accumulation of BSATexas Red with long labeling and chase times (Fig. 2); (2)
labeling with an antibody against human cathepsin D (Fig. 2);
(3) labeling with an antibody against LAMP-1, in a pattern
overlapping with that of cathepsin D (data not shown).
Although proteases such as cathepsin D can be found within
endosomes in other cell types, possibly during protease
maturation and transport to lysosomes, the abundance of such
proteases is much lower in endosomes compared with
lysosomes (Rodman et al., 1990).
The inclusion of protease inhibitors in the incubation
medium was necessary for the immunofluorescence
localization of receptors in lysosomes (Fig. 4), most likely
because the HA-epitope tag on the receptor N terminus would
otherwise be degraded in protease-containing compartments.
To exclude nonspecific effects of leupeptin, we examined
β2-adrenergic receptors and lysosomes
several parameters of intracellular receptor trafficking events.
Leupeptin had no detectable effect on the intracellular
distribution of transferrin receptors relative to lysosomes in the
presence of agonist, because under these conditions, there was
no localization of transferrin receptors with cathepsin D (data
not shown). This experiment also shows that the effect of
agonist is receptor-specific, because under the same conditions,
β2ARs were extensively localized with this lysosome marker
(Fig. 3). This result is of further significance since transferrin
receptors and β2ARs both traffic through early endosomes
(Moore et al., 1995; von Zastrow and Kobilka, 1992), and
indicates that prolonged agonist treatment results in the specific
sorting of β2ARs, but not transferrin receptors, from
endosomes to lysosomes. Protease inhibitors were not
nonspecifically affecting normal endosomal trafficking of
β2ARs, because the incubation of cells with leupeptin and
pepstatin for 6 hours had no affect upon the rates of β2AR
endocytosis and recycling over a subsequent short time course
of agonist exposure. Incubation of 12β6 cells in the presence
of leupeptin by itself did not cause localization of β2ARs with
cathepsin D (data not shown), indicating that the accumulation
of these receptors in lysosomes required prolonged treatment
with agonist. Moreover, in the absence of agonist and the
presence of leupeptin, there was no significant change in the
receptor number as assessed by radioligand binding,
suggesting that protease inhibitor was not influencing the basal
335
half-life of β2ARs (Fig. 2). These data strongly suggest that the
localization of β2ARs with lysosomal markers we observed
was not the result of receptor trafficking in association with
normal receptor turnover and was instead dependent on
agonist.
The finding that the protease inhibitor leupeptin was needed
to reveal β2AR trafficking to lysosomes and at the same time
blocked receptor downregulation is strong evidence that these
processes are mechanistically related. An apparent temporal
disconnection between the occurrence of measurable
downregulation (24 hours of agonist exposure), and trafficking
to lysosomes (3-6 hours), can be explained by the fact that
ligand binding is relatively resistant to proteases and does not
require the N-terminal domain (Rands et al., 1990). Also, since
the ligand binding pocket is sequestered within the lipid bilayer
(Liggett, 1997), radioligand-binding activity might be expected
to persist in the presence of proteases for a longer period of
time than an epitope tag exposed to the interior of lysosomes.
Further, although trafficking to lysosomes is observed within
3-6 hours (Figs 3 and 5A-C), the quantity of receptor providing
the immunofluorescence signal could be relatively small
compared with the total receptor population. Although
proteolysis of the HA epitope on the receptor’s N terminus is
not necessarily synonymous with degradation of its ligand
binding site, these data suggest that lysosomal cysteine
proteases are largely responsible for the downregulation of
Fig. 6. Inhibition of β2AR traffic to
lysosomes by bafilomycin A1. Cells
were pretreated with leupeptin (100
µM) for 30 minutes in the presence (AF) or absence (G-I) of bafilomycin A1
(1 µM) before adding ISO (5 µM) for
6 hours. Cells were labeled with
mHA.11 (B,E,H) and antisera against
either cathepsin D (A) or rab11 (D and
G). Merged images are shown in C, F,
and I. Minimal localization of HAβ2ARs with cathepsin D is noted in AC. There is a striking redistribution of
β2ARs to a rab11-containing
perinuclear compartment in cells
pretreated with bafilomycin A1 (D-F)
compared to those treated with ISO
and leupeptin alone (G-I). Bar, 10 µm.
336
R. H. Moore and others
HA-β2ARs. Further, although β2ARs localize with cathepsin
D, this aspartic protease appears to contribute minimally, if at
all, to its downregulation.
The finding that ISO-induced downregulation was almost
completely inhibited by the addition of protease inhibitor,
together with the small increment of 8-bromo cAMP-induced
downregulation, suggests that receptor degradation is the major
component of agonist-induced downregulation in 12β6 cells.
Further, since transcription in the 12β6 cell line is under the
control of a viral promoter element (Cullen, 1987) whose
activity is not likely to be affected by agonist treatment, our
measurements probably underestimate the true extent of
receptor degradation since there is likely to be some continued
receptor synthesis during the 24 hours of agonist treatment.
This would not confound the interpretation of our trafficking
data, since newly synthesized receptors are rapidly transported
to the cell surface, where they bind agonist and begin their
continuous rounds of endocytosis and recycling (Morrison et
al., 1996). Although the 3′ untranslated region of β2AR cDNA
expressed in this cell line has all four cAMP-dependent mRNA
destabilization motifs (Danner et al., 1998; von Zastrow and
Kobilka, 1992), the effect of 8-bromo cAMP on receptor
downregulation was small and additive to that seen with ISO
alone (Fig. 1). Further, if the small amount of 8-bromo cAMPmediated β2AR downregulation that we measured were due to
degradation in lysosomes, we would have expected there to be
localization of receptors with cathepsin D, because such
colocalization was observed in agonist treated cells at times
points (3 hours) before significant agonist-induced
downregulation was detected (data not shown). These results
suggest that the mechanisms of receptor downregulation
mediated by cAMP and by agonist are separable and that
cAMP-dependent events contributed little to agonist-induced
β2AR downregulation in 12β6 cells, similar to results from
studies using BEAS-2B cells (Kelsen et al., 1997) and signal
transduction defective S49 lymphoma (cyc- and kin-) cell lines
(Proll et al., 1992).
Other studies have examined the half-life of β2AR
radioligand binding activity in the presence and absence of
agonist. Estimates were made by the use of an irreversible
antagonist and computer modeling of receptor synthesis and
degradation (Mahan and Insel, 1986; Nantel et al., 1994; Neve
and Molinoff, 1986). All of these studies used loss of
radioligand binding activity as an index of receptor
degradation, and none were able to address the molecular or
cellular mechanisms of downregulation. Several other studies
have evaluated the trafficking of β2ARs to lysosomes. β2ARs
in A431 cells pre-bound with gold-labeled monoclonal antireceptor antibody apparently traffic to lysosomes; however,
this process is not agonist-dependent, was not reported to
correlate with downregulation, and initiates through nonclathrin coated vesicles (Raposo et al., 1992). In contrast,
agonist-induced receptor endocytosis in the 12β6 line used in
this study occurs through clathrin-coated vesicles (Moore et al.,
1995; von Zastrow and Kobilka, 1994), suggesting that the
receptor trafficking events we observed are specific and not
related to bulk turnover of membrane proteins. In other recent
reports prolonged exposures to agonist caused the apparent
trafficking of a β2AR-green fluorescent protein chimera
(β2AR-GFP) to a rhodamine-dextran-labeled compartment,
presumably lysosomes, in stably transfected HeLa cells and
transiently transfected HEK293 cells (Gagnon et al., 1998;
Kallal et al., 1998). Interpretation of these results is
complicated by the presence of a significant pool of
intracellular β2ARs-GFP in untreated cells, suggesting
abnormal processing of this protein within the endoplasmic
reticulum and constitutive trafficking of receptors to undefined
intracellular compartments. The cells used in the present study
express a β2AR with a small N-terminal epitope tag, and no
intracellular receptors can be identified in the absence of
agonists (data not shown; Moore et al., 1995; von Zastrow and
Kobilka, 1992). Agonist-dependent downregulation of these
receptors correlates with their intracellular trafficking to
protease-containing compartments, which are identified by
several criteria as lysosomes (Fig. 3). The colocalization of
β2ARs-GFP with rhodamine-dextran did not require the
presence of protease inhibitors (Gagnon et al., 1998; Kallal et
al., 1998), suggesting that either the C terminus of β2ARs-GFP
is relatively resistant to proteolysis or that these receptors
localized with rhodamine-dextran in a compartment devoid of
proteases. Our data clearly show proteolytic action on
receptors, as assessed by loss of their N-terminal HA-epitopes
and that inhibition of proteases also inhibits downregulation.
A role for clathrin-mediated receptor endocytosis in the
process of downregulation has been suggested in a recent study
in which a dominant negative mutant of dynamin (K44A)
almost completely abolished agonist-induced β2AR
downregulation in HEK293 cells, although the effects were
less in HeLa and COS cells (Gagnon et al., 1998). However, it
was unclear if β2ARs that are destined for degradation in
lysosomes traffic through endosomes or use an alternate
pathway. In the present study, treatment with bafilomycin A1,
which prevents acidification of endosomes and lysosomes in
many cell systems, including HEK293 cells (Shrader-Fischer
and Paganetti, 1996; Yoshimori et al., 1991), significantly
inhibited the localization of β2ARs with cathepsin D (Fig. 6AC), caused the enrichment of receptors in the rab11-containing
perinuclear recycling compartment (Fig. 6D-F), and slowed the
rate of β2AR recycling following a 6 hour exposure to agonist.
Bafilomycin A1 is known to inhibit the formation of endosomal
carrier vesicles between early and late endosomes (Clague et
al., 1994), to modestly slow the externalization of transferrin
receptors from the perinuclear recycling compartment
(Johnson et al., 1993; Presley et al., 1997), and to prevent the
transport of horseradish peroxidase (van Weert et al., 1995) and
cationized gold (van Deurs et al., 1996) to lysosomes. Our data
indicate that the transport to lysosomes of β2ARs destined for
degradation also is dependent on a functional vacuolar-type
H+-ATPase and provide strong evidence that β2ARs are
specifically sorted from endosomes en route to degradation in
lysosomes by a pH-dependent mechanism. A bafilomycin A1sensitive mechanism has been described in the recycling of
transferrin receptors to the plasma membrane (Johnson et al.,
1993; Presley et al., 1997), a process which requires the
presence of a critical YTRF motif within the receptor’s
cytoplasmic tail. However, a bafilomycin A1-sensitive
mechanism previously has not been implicated in the
trafficking of any G protein-coupled receptors from endosomes
to lysosomes or to any other intracellular destination. Further,
the accumulation of β2ARs in the perinuclear recycling
compartment observed in bafilomycin A1-treated cells can
probably be explained by both a decreased efflux of receptors
β2-adrenergic receptors and lysosomes
from this compartment (Johnson et al., 1993; Presley et al.,
1997) and an increased influx due to the diversion of receptors
from lysosomes, although an additional bafilomycin A1sensitive pathway from the perinuclear recycling compartment
to lysosomes cannot be excluded.
The accumulated data thus indicate that in HEK293 cells
β2ARs are dynamically sorted from early endosomes to at least
three destinations, directly to the plasma membrane by rapid
recycling; the perinuclear recycling compartment, from which
slow recycling to the cell surface occurs; and lysosomes, where
they are degraded by cysteine proteases. Most previous studies
where the trafficking of other GPCRs to lysosomes has been
demonstrated have been restricted to receptors that recycled to
only a small extent. Activated thrombin receptors quite rapidly
localize with LAMP-1, with a limited degree of receptor
recycling (Hoxie et al., 1993). LH/hCG receptors labeled with
immunogold move rapidly into lysosomes and multivesicular
bodies (possibly late endosomes), and little or no recycling of
label or receptors is observed (Ghinea et al., 1992). Because
GPCRs predominantly targeted to lysosomes and those that are
largely recycled, such as β2ARs, traffic through similar
endocytic pathways (Hein et al., 1994), it is interesting to
speculate on the presence of specific sorting signals within
their C-terminal domains. Candidate signals include dileucine
or tyrosine-based motifs (for review see Trowbridge et al.,
1993; Bonifacino et al., 1996), which may promote receptor
interactions with adaptor proteins such as AP-3 (Dell’Angelica
et al., 1997) or sorting nexins (Kurten et al., 1996). Additional
studies are underway to define the complex relationships
among receptor endocytosis, sorting to lysosomes, and
receptor recycling during prolonged agonist exposure.
We thank Drs R. B. Clark, K. J. Morrison, and Marianne WesslingResnick for discussions and critical reading. This work was supported
by NIH grants HL50047 (B.J.K.), HL43161 (B.F.D.) and grants from
the Veteran’s Administration (B.F.D.), Glaxo (B.F.D.), the American
Heart Association, Texas Affiliate (R.H.M.), and the Baylor College
of Medicine Child Health Research Center (R.H.M.). R.H.M. is the
recipient of a Clinical Investigator Development Award from the NIH
(HL03463).
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