From the Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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Previous studies have demonstrated that
non-visual arrestins function as adaptors in clathrin-mediated
endocytosis to promote agonist-induced internalization of the
2-adrenergic receptor (
2AR). Here,
we characterized the effects of arrestins and other modulators of
clathrin-mediated endocytosis on down-regulation of the
2AR. In COS-1 and HeLa cells, non-visual arrestins
promote agonist-induced internalization and down-regulation of the
2AR, whereas dynamin-K44A, a dominant-negative mutant of
dynamin that inhibits clathrin-mediated endocytosis, attenuates
2AR internalization and down-regulation. In HEK293
cells, dynamin-K44A profoundly inhibits agonist-induced internalization
and down-regulation of the
2AR, suggesting that receptor
internalization is critical for down-regulation in these cells.
Moreover, a dominant-negative mutant of
-arrestin,
-arrestin-(319-418), also inhibits both agonist-induced receptor
internalization and down-regulation. Immunofluorescence microscopy
analysis reveals that the
2AR is trafficked to lysosomes
in HEK293 cells, where presumably degradation of the receptor occurs.
These studies demonstrate that down-regulation of the
2AR is in part due to trafficking of the
2AR via the clathrin-coated pit endosomal pathway to
lysosomes.
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INTRODUCTION |
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Stimulation of G protein-coupled receptors (GPRs)1 leads to adaptive changes that serve to modulate the responsiveness of a cell to further stimulation (1). Three temporally distinct mechanisms of agonist-promoted regulation of GPRs have been identified: desensitization, internalization, and down-regulation. Desensitization often occurs within seconds of stimulation and for many GPRs is mediated by phosphorylation of the receptor by a specific G protein-coupled receptor kinase. This phosphorylation promotes the binding of an arrestin to the receptor, which sterically prevents subsequent coupling to G proteins. Agonist stimulation also promotes rapid (minutes) internalization of many GPRs into an intracellular endosomal pool, thereby effectively removing the receptor from the cell surface. Finally, prolonged receptor stimulation (hours) results in receptor down-regulation, defined by an overall decrease in receptor number.
The 2-adrenergic receptor (
2AR) has
served as an important model for elucidating the molecular mechanisms
involved in GPR regulation. The
2AR is rapidly
desensitized after agonist stimulation, in part a consequence of
phosphorylation of carboxyl-terminal serines and threonines by G
protein-coupled receptor kinase 2 (2). This phosphorylation promotes
the binding of an arrestin (either
-arrestin or arrestin 3), which
directly inhibits the ability of the receptor to couple to
Gs (3-5). Recent studies have suggested that
-arrestin
and arrestin 3 are not only involved in the initial stages of
desensitization of the
2AR but that they are important
components in receptor internalization (6, 7). Since both
-arrestin
and arrestin 3 can specifically bind to clathrin, the major structural
protein of clathrin-coated pits, arrestins can function as adaptors to
recruit G protein-coupled receptor kinase-phosphorylated receptors into
coated pits where they can be internalized (7). Recent studies have
demonstrated that
2AR internalization plays an important
role in receptor resensitization, a process that involves
dephosphorylation of the receptor and subsequent recycling back to the
cell surface (8-10).
It has been hypothesized that trafficking of GPRs from early endosomes
to lysosomes might be a mechanism for agonist-induced down-regulation.
Indeed several GPRs, including the thrombin (11), thyrotropin (12), and
cholecystokinin (13) receptors, have been shown to be sorted to
lysosomes in an agonist-dependent manner. We have also
recently shown that a 2AR tagged with the green fluorescent protein (GFP) can also slowly accumulate in lysosomes after
agonist activation (14). However, whether internalization of the
2AR via clathrin-coated pits directly contributes to its down-regulation remains speculative. Previous radioligand binding studies utilizing mutants of the
2AR have failed to link
internalization and down-regulation and, in fact, suggest that the two
processes may be distinct events (15-19). Moreover, in addition to
protein degradation, prolonged agonist stimulation also leads to
decreased steady state levels of
2AR mRNA in some
cells (20-22). Thus, down-regulation of the
2AR is
composed of multiple mechanisms that function to both destroy existing
receptor as well as decrease the synthesis of new receptors.
The present studies were focused on addressing whether agonist-promoted
internalization of the 2AR contributes to its
down-regulation. To accomplish this, we expressed the
2AR in a variety of mammalian cell lines and examined
the effect of coexpression of various modulators of clathrin-mediated
endocytosis on agonist-induced down-regulation. The results of these
studies indicate that increases or decreases in agonist-mediated
internalization of the
2AR directly correlate with
concomitant changes in the extent of receptor down-regulation. Moreover, in HEK293 cells,
2AR down-regulation appears
very dependent upon internalization of the receptor and subsequent
trafficking to lysosomes.
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EXPERIMENTAL PROCEDURES |
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Materials--
A BamHI fragment of dynamin-K44A was
subcloned into BamHI-digested pcDNA3, whereas
Flag-tagged 2AR was subcloned into pcDNA3 as
described (14). Poly-L-lysine was obtained from Sigma, and tissue culture dishes were treated according to manufacturer's instructions. The construct pcDNA3-
2AR-GFP, encoding
a
2AR fusion protein with GFP at its carboxyl terminus,
was created as described (14). Flag-
2AR and arrestin
constructs in the vector pBC12BI have been described (7).
Cell Culture and Transient Transfection--
COS-1, HEK293, and
HeLa cells were maintained in Dulbecco's modified Eagle's medium
(Life Technologies, Inc.) supplemented with 10% fetal bovine serum,
100 units/ml penicillin G, and 100 µg/ml streptomycin sulfate
(complete media) at 37 °C in a humidified atmosphere of 95% air,
5% CO2. Cells grown to 80-90% confluency in T75 flasks
were transfected with 65 µl (COS-1 and HeLa) or 90 µl (HEK293) of
LipofectAMINE (Life Technologies) according to the manufacturer's
instructions. For transfection of COS-1 cells, 10 µg of
pBC-Flag-2AR was transfected alone or co-transfected with 3 µg of pBC-
-arrestin, pBC-arrestin 3, or pBC-arrestin. HeLa
cells and HEK293 cells were transfected with 5-10 µg of
pcDNA3-Flag-
2AR or cotransfected with 3 µg (HeLa)
or 5 µg (HEK293) of pcDNA3-arrestin 3, pcDNA3-
-arrestin,
or pcDNA3dynamin-K44A. For microscopy studies, HEK293 cells were
transfected as above with 10 µg of
pcDNA3-
2AR-GFP.
Sequestration Assays--
For COS-1 and HeLa cells, receptor
internalization studies were performed as described (7). Briefly, cells
were harvested 48 h after transfection by trypsinization, washed
extensively, resuspended, and split into 0.5 ml aliquots in
phosphate-buffered saline (PBS) containing 0.1 mM
ascorbate. Cells were incubated with or without 1 µM
()-isoproterenol at 37 °C for the indicated time, washed with
ice-cold PBS, and resuspended in PBS for binding studies. Cell surface
receptors were measured by incubating with 10 nM
[3H]CGP-12177 for 3 h at 14 °C with or without 10 µM (
)-alprenolol. Protein concentrations were
determined by Bio-Rad assay using bovine serum albumin as a
standard.
Down-regulation Assays--
Cells were harvested by
trypsinization 24 h after transfection and plated onto 60-mm
dishes (poly-L-lysine treated for HEK293). The next day,
complete media containing 0.1 mM ascorbate was added, and
the cells were then treated with or without 10 µM ()
isoproterenol for 1 to 24 h. Cells were washed four times with 4 ml of room temperature PBS and then lysed in 1-4 ml of 20 mM Tris, pH 8, 2 mM EGTA, 5 mM
EDTA, 5 µg/ml leupeptin, 0.2 mg/ml benzamidine by homogenization on
ice using a polytron (25,000 rpm, 2× 15 s). Receptor expression
was measured in the cell lysate with 1 nM [125I]iodopindolol (NEN Life Science Products) at room
temperature for 1 h with or without 10 µM
(
)-alprenolol. Binding reactions were terminated by the addition of
5× 4 ml of ice-cold 25 mM Tris, pH 7.5, 2 mM
MgCl2 followed by rapid filtration through Whatman GF/C
filters using a Brandel Cell Harvester.
Immunofluorescence Microscopy--
HEK293 cells were transiently
transfected with pcDNA3-2AR-GFP using LipofectAMINE
according to the manufacturer's instructions. The cells were
trypsinized and plated onto poly-L-lysine-coated coverslips
24 h after transfection. After the cells had adhered, the media
was aspirated and replaced with 1 ml of complete media plus 0.1 mM ascorbate. For labeling of lysosomes, 100 µl of 10 mg/ml rhodamine-labeled dextran (Molecular Probes) was added to each
well and allowed to incubate at 37 °C overnight. The dextran was
washed out 1.5 h before the end of the incubation period and replaced with fresh media. Isoproterenol (10 µM final)
was added to the media or rhodamine-dextran mix at the appropriate
time. At the end of the incubation, cells were washed briefly three times in PBS, fixed for 10 min in 3.7% formaldehyde, and mounted on
coverslips using SlowFade mounting medium (Molecular Probes). Confocal
microscopy analysis was performed on a Bio-Rad MRC-Zeiss Axiovert 100 confocal microscope (Hemmelholsteadt) using a Zeiss Plan-Apo 63× 1.4 NA oil immersion objective.
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RESULTS AND DISCUSSION |
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Since mutational analysis has not provided a clear picture of the
link between agonist-stimulated internalization and down-regulation of
the 2AR, we undertook studies examining the effect of
modulators of endocytosis on agonist-mediated down-regulation of the
wild-type
2AR. Presumably if internalized receptors are
targeted for down-regulation, increasing or decreasing the proportion
of these receptors should have a concomitant effect on down-regulation.
Previously we demonstrated that non-visual arrestins increase
agonist-induced internalization of the
2AR in COS-1
cells (7). Thus, COS-1 cells provide a convenient model for
establishing a potential link between agonist-mediated internalization
and down-regulation of the
2AR. COS-1 cells were transiently transfected with
2AR or cotransfected with
2AR plus either
-arrestin, arrestin 3, or visual
arrestin and assayed for both agonist-induced internalization and
down-regulation. Both
-arrestin and arrestin 3, but not visual
arrestin, promoted an ~3-fold increase in agonist-induced
internalization of the
2AR after 30 min of isoproterenol
treatment (Fig. 1A). Previous studies have established that this effect is likely mediated via a
carboxyl-terminal clathrin binding domain in
-arrestin and arrestin
3 (23) that functions to recruit the receptor complex to
clathrin-coated pits, resulting in receptor internalization (7).
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We next determined the effect of arrestins on 2AR
down-regulation in COS-1 cells. Similar to the internalization results, expression of either
-arrestin or arrestin 3, but not visual arrestin, significantly increased isoproterenol-stimulated
down-regulation of the
2AR after a 24-h incubation with
agonist (Fig. 1B). However, although the effect of
-arrestin and arrestin 3 appears specific, these proteins did not
increase agonist-promoted down-regulation as effectively as they
increase
2AR internalization. Thus, in an effort to
increase the observed effect, we expressed both arrestin 3 and G
protein-coupled receptor kinase 2 with the
2AR. G
protein-coupled receptor kinase 2 expression augments the effect of
arrestins on agonist-stimulated
2AR sequestration in
COS-1 cells (Ref. 24 and data not shown), presumably by promoting
receptor phosphorylation and thereby increasing arrestin binding to the
receptor. Expression of G protein-coupled receptor kinase 2 and
arrestin 3 in COS-1 cells increased
2AR down-regulation
from 32 ± 5 to 40 ± 1%. These results suggest that some
fraction of the receptors that are internalized in response to
isoproterenol are targeted for down-regulation.
We also wanted to assess whether inhibitory modulators of
clathrin-mediated endocytosis would affect down-regulation of the 2AR. For these studies we chose to examine the effect of
dynamin-K44A on agonist-stimulated down-regulation. Dynamin is a
GTP-binding protein that plays a critical role in clathrin-coated
vesicle formation (25). Previous studies have demonstrated that
dynamin-K44A is deficient in GTP binding and functions as an effective
dominant negative protein to potently block clathrin-mediated
endocytosis (26). Indeed, dynamin-K44A overexpression in several cell
lines has been demonstrated to significantly attenuate agonist-induced internalization of the
2AR (27, 28). Since COS-1 cells
have a relatively low intrinsic level of
2AR
down-regulation, we tested the effect of dynamin-K44A on arrestin
3-promoted down-regulation in these cells. Overexpression of
dynamin-K44A inhibited arrestin 3-promoted
2AR
internalization ~72% (Fig. 1A), whereas an ~53% reduction in down-regulation was observed (Fig. 1B). This
suggests that both
2AR internalization and
down-regulation are primarily mediated via clathrin-coated pits in
these cells.
To determine whether agonist-promoted internalization via
clathrin-coated pits plays a cell-specific role in 2AR
down-regulation, we tested the effects of arrestins and dynamin-K44A in
additional cell lines. HeLa cells appear to display many of the
characteristics of agonist-induced internalization and down-regulation
of the
2AR that we observed in COS-1 cells, although
down-regulation was significantly higher after a 24-h treatment with
isoproterenol (~48%). Coexpression of arrestin 3 with the receptor
promoted agonist-mediated internalization ~2-2.5-fold, whereas
coexpression of dynamin-K44A inhibited internalization ~50% (Fig.
2A). Similarly, arrestin 3 promoted and dynamin-K44A inhibited receptor down-regulation, although
the effects of arrestin 3 and dynamin-K44A appeared to be more
pronounced at the shorter time points (1-5 h) compared with 24 h
(Fig. 2B). Notably, dynamin-K44A appeared only modestly effective at blocking agonist-induced down-regulation in these cells at
24 h. This suggests that additional pathways of intracellular protein degradation might contribute to
2AR
down-regulation in HeLa cells. Such pathways could include receptor
trafficking of the
2AR via caveolae, which appears to
occur in A431 cells (29, 30), as well as ubiquitin-targeted protein
degradation, which plays a role in rhodopsin (31) and
-mating factor
receptor degradation (32).
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We also assessed the effects of arrestins and dynamin-K44A on
2AR internalization and down-regulation in HEK293 cells.
As previously demonstrated (6, 28), overexpression of
-arrestin only
marginally promoted isoproterenol-stimulated internalization of the
2AR in HEK293 cells (Fig.
3A). Similarly,
-arrestin
overexpression also had no effect on agonist-induced down-regulation of
the
2AR in HEK293 cells after 24 h of isoproterenol
treatment (Fig. 3B). Conversely, dynamin-K44A effectively
attenuated agonist-mediated internalization of the
2AR
(Fig. 3A and Refs. 27 and 28). Strikingly, inhibition of
2AR internalization by dynamin-K44A also resulted in
virtually a complete blockade of
2AR down-regulation (Fig. 3B). Moreover,
-arrestin (319-418), a
dominant-negative mutant that contains the carboxyl-terminal clathrin
binding domain of
-arrestin (28), had an intermediate effect on both
internalization and down-regulation of the
2AR (Fig. 3).
The ability of the
-arrestin dominant-negative mutant to partially
inhibit agonist-induced internalization and down-regulation supports
the idea that arrestins are involved in
2AR
internalization, and by association, in receptor down-regulation. Taken
together, the results from COS-1, HeLa, and HEK293 cells indicate that
a proportion of
2ARs internalized via clathrin-mediated
endocytosis are targeted for down-regulation.
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Presumably, the trafficking pattern for the 2AR might be
similar to that seen for other G protein-coupled receptors in which activated receptors are sorted to lysosomes for degradation (11-13). Thus, we might expect that some proportion of the
2ARs
that are internalized might also be sorted to lysosomes for
degradation. To better visualize agonist-induced trafficking of the
2AR, we expressed a previously characterized green
fluorescent protein-conjugated
2AR
(
2AR-GFP) (14) in HEK293 cells. HEK293 cells transfected with
2AR-GFP were plated onto coverslips and initially
incubated with rhodamine-labeled dextran, which is taken up by fluid
phase endocytosis and accumulates in late endosomes and lysosomes (33). Before agonist stimulation, the fluorescence pattern of
2AR-GFP suggested localization primarily at the plasma
membrane (Fig. 4A). This
contrasts with the fluorescence pattern of rhodamine-labeled dextran,
which has a distinct intracellular punctate appearance (Fig. 4). After
a 30-min incubation with isoproterenol,
2AR-GFP was
primarily visualized as a distinct punctate pattern of fluorescence (Fig. 4B). However, this staining pattern did not
significantly colocalize with rhodamine-labeled dextran, suggesting
that the
2AR is likely in early endocytic vesicles.
Indeed previous studies have demonstrated that an epitope-tagged
2AR in HEK293 cells colocalizes with markers for early
endocytic vesicles after several minutes of isoproterenol stimulation
(34). In contrast, 3 h of stimulation with isoproterenol resulted
in significant colocalization of
2AR-GFP and
rhodamine-labeled dextran, as seen by the appearance of
yellow where the green and red
patterns are coincident (Fig. 4C). Colocalization of
2AR-GFP and dextran can be detected as early as 1 h
after agonist addition (data not shown) and increased out to 24 h
(Fig. 4D). These results are similar to those observed in
HeLa cells, where
2AR-GFP colocalizes with
rhodamine-labeled transferrin, a marker for early endosomes, after
short term agonist treatment, whereas colocalization of
2AR-GFP with dextran is observed with longer incubation
times (>1 h) (14). Thus, these results demonstrate that in both HEK293
and HeLa cells some portion of the
2AR are sorted from
early endosomes to lysosomes upon prolonged stimulation with
isoproterenol, defining a mechanism for
internalization-dependent, agonist-mediated degradation of the
2AR.
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The mechanisms involved in 2AR down-regulation are
diverse and include both translational (15-19) and transcriptional
(20-22) regulation. Studies utilizing carboxyl-terminal mutants of the
2AR that disrupt its ability to internalize in an
agonist-dependent manner have not demonstrated an effect on
down-regulation (15, 16). Additional mutations ablate the ability of
the
2AR to undergo agonist-mediated down-regulation,
seemingly without affecting its ability to internalize (17-19). The
assumption from these studies was that these two regulatory processes
were distinct. In contrast, our data demonstrate that
2AR internalization can directly contribute to
down-regulation. Although some of the differences between these studies
may be attributable to cell type, it also seems plausible that
wild-type and mutant
2ARs may behave differently. Thus, we have focused our studies on the ability of proteins downstream of
the receptor (arrestins and dynamin) to modulate internalization and
down-regulation of the wild-type
2AR.
The mechanisms involved in specific lysosomal targeting of G
protein-coupled receptors remain poorly defined. However, some receptors appear to contain distinct cytoplasmic domains that are
critical for differential intracellular targeting. For example, the
thrombin receptor contains distinct but overlapping regions within its
carboxyl tail that are involved in either tonic cycling or
agonist-stimulated internalization of the receptor (35). It has been
postulated that targeting of the thrombin receptor is dependent on the
binding of an as yet undefined protein to this region of the receptor
(35). For the yeast -mating factor receptor, agonist-promoted
phosphorylation and subsequent ubiquitination of the carboxyl tail
appears to function as an efficient signal for internalization and
trafficking of the receptor to lysosomes (32). Some growth factor
receptors are also efficiently trafficked to lysosomes after
activation. Perhaps the best example is the epidermal growth factor
receptor, where receptor internalization and degradation is mediated by
distinct endocytic and lysosomal targeting sequences (36). Recent
studies have identified a protein called sorting nexin-1 that binds to
the lysosomal targeting region of the epidermal growth factor receptor
and likely plays a role in lysosomal sorting of the receptor (37).
Whether lysosomal targeting of internalized
2ARs occurs
randomly or via the binding of another protein remains unknown.
However, it seems likely that other as yet unidentified factors will be
involved in determining the fate of internalized
2ARs.
In summary, agonist-induced down-regulation of the 2AR
can be mediated by trafficking of the receptor to lysosomes. We have demonstrated in three cell lines that by regulating the amount of
2AR internalized via clathrin-coated pits, we can
modulate the extent of agonist-mediated
2AR
down-regulation. This indicates that after prolonged stimulation, some
of the internalized
2ARs are sorted from early endosomes
to lysosomes for degradation. Utilization of this pathway does not
preclude simultaneous regulation of the
2AR by other
mechanisms, such as regulation of expression at the transcriptional or
translational level. Indeed, our results suggest that agonist-mediated
down-regulation in HeLa cells is likely composed of additional
mechanisms, supporting the idea that cells utilize several pathways to
regulate
2AR expression levels. Furthermore, it appears
that the amount that each regulatory mechanism contributes to
2AR down-regulation varies from cell to cell.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. Keen and members of
the Benovic lab for helpful discussions, P. Hingorani for confocal
microscopy, Dr. B. Kobilka for the Flag-tagged 2AR
construct, and Drs. H. Damke and S. Schmid for the dynamin-K44A
construct.
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FOOTNOTES |
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* This research was supported in part by National Institutes of Health Grants GM44944 and GM47417 and by National Institutes of Health Training Grant 5-T32-CA09662 (to A. W. G).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.
An established investigator of the American Heart Association. To
whom correspondence should be addressed: Thomas Jefferson University,
233 S. 10th St., Philadelphia, PA 19107. Tel.: 215-503-4607; Fax:
215-923-1098; E-mail: benovic{at}lac.jci.tju.edu.
1
The abbreviations used are: GPR, G
protein-coupled receptor; 2AR,
2-adrenergic receptor; GFP, green fluorescent protein; PBS, phosphate-buffered saline.
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REFERENCES |
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