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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We investigated the role of arrestins in the
trafficking of human Three human Arrestins2 have been shown to
mediate the desensitization and internalization of G protein-coupled
receptors (reviewed in Refs. 2 and 3). This is accomplished by binding
of arrestins to agonist-activated GPCRs after phosphorylation of
receptors by GRKs. Arrestins are recruited to at least 15 different
GPCRs after agonist activation, highlighting the critical role of
arrestins in receptor turn-off (4). Binding of arrestin results in the physical uncoupling of GPCRs from heterotrimeric G proteins, thus terminating agonist-mediated signaling (5, 6). The In several different cell types, the In the present study, we investigated the role of arrestins and GRKs in
the agonist-mediated trafficking of the different Materials--
LPA, isoproterenol, epinephrine, EGF, and
pertussis toxin were purchased from Sigma. UK 14,304 was purchased from
Research Biochemicals International. Unless otherwise noted, the
standard concentrations of these compounds used in this study were: 10 µM UK 14,304, 100 µM epinephrine, 10 µM isoproterenol, 10 ng/ml EGF, and 25 µM
LPA.
Plasmid Construction--
Cell Culture and Transfection--
COS-1 cells were maintained
in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.)
supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin,
and 100 units/ml penicillin. COS-1 cells were grown in 60-mm or 100-mm
dishes (Falcon) at 37 °C in a humidified atmosphere containing 95%
air and 5% CO2. Cells were grown to 80-90% confluence
prior to transfection. Cells were transfected using FuGENE-6 reagent
(Boehringer Mannheim) according to the manufacturer's protocol. For
transfection of 60-mm dishes, 10 µl of FuGENE-6 was used, and 30 µl
of FuGENE-6 was used to transfect 100-mm dishes. Cells were incubated
with FuGENE-DNA mixture for 5 h, after which the cells were split
into 12-well dishes (for p42/p44 MAP kinase assays) or 24-well dishes
(for ELISA). All assays were performed 24 h after transfection.
Internalization of Receptors--
Internalization of
Immunofluorescence Microscopy--
COS-1 cells in 60-mm dishes
were transfected as described above with 3 µg of the different
Flag-tagged Analysis of p42/p44 MAP Kinase Activation--
COS-1 cells in
100-mm dishes were transfected as described above, then split into
12-well dishes and serum-starved overnight in DMEM containing 0.5%
fetal bovine serum. After agonist stimulation, cells were rapidly
rinsed once with phosphate-buffered saline and lysed by addition of 100 µl of SDS sample buffer. Samples were heated to 95 °C for 10 min,
and proteins (20 µl) were separated on 10% SDS-polyacrylamide gels.
The gels were transferred to nitrocellulose and blocked for 30 min in
blocking buffer consisting of 20 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20 (TBST) containing 5% nonfat dry
milk. To detect p42/p44 MAP kinase activation, blots were incubated in
blocking buffer overnight at 4 °C with a polyclonal rabbit antibody
(New England Biolabs) that specifically recognizes the amino acids
(Thr-202/Tyr-204) that are phosphorylated upon activation of p42/p44
MAP kinase (diluted 1:1000). The following day, blots were washed three
times with TBST, then incubated with goat-anti rabbit horseradish
peroxidase secondary antibody (Bio-Rad) diluted 1:2000 in blocking
buffer for 1 h. Blots were then washed three times in TBST and
developed by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).
Immunoprecipitation and Analysis of HA-tagged ERK-2
(p42)--
COS-1 cells were transfected with HA-ERK-2 and the
indicated Effect of Arrestin and GRK Expression on
We next compared the ability of arrestin-2 and arrestin-3 to promote
To further characterize the arrestin-mediated internalization of
Immunofluorescence Analysis of Role of Endocytosis in
Since the
In the experiments shown in Fig. 6 (A and B),
both Role of Endocytosis in Activation of p42/p44 MAP Kinase by
Endogenous The role of arrestins in the trafficking and signaling of the
We observed significant differences between the Interestingly, internalization of the Several observations support our conclusion that endocytosis of
Our results contrast with those of Daaka et al. (15), who
reported that activation of p42/p44 MAP kinase by endogenous
2-adrenergic receptors
(
2-ARs) and the effect of receptor trafficking on
p42/p44 MAP kinase activation.
2-ARs expressed in COS-1
cells demonstrated a modest level of agonist-mediated internalization,
with
2c >
2b >
2a.
However, upon coexpression of arrestin-2 (
-arrestin-1) or arrestin-3
(
-arrestin-2), internalization of the
2b AR was
dramatically enhanced and redistribution of receptors to clathrin
coated vesicles and endosomes was observed. Internalization of the
2c AR was selectively promoted by coexpression of
arrestin-3, while
2a AR internalization was only
slightly stimulated by coexpression of either arrestin. Coexpression of GRK2 had no effect on the internalization of any
2-AR
subtype, either in the presence or absence of arrestins.
Internalization of the
2b and
2c ARs was
inhibited by coexpression of dominant negative dynamin-K44A. However,
2-AR-mediated activation of either endogenous or
cotransfected p42/p44 mitogen-activated protein (MAP) kinase was not
affected by either dynamin-K44A or arrestin-3. Moreover, activation of
p42/p44 MAP kinase by endogenous epidermal growth factor,
lysophosphatidic acid, and
2-adrenergic receptors was
also unaltered by dynamin-K44A. In summary, our data suggest that
internalization of the
2b,
2c, and to a
lesser extent
2a ARs, is both arrestin- and
dynamin-dependent. However, endocytosis does not appear to
be required for
2-adrenergic, epidermal growth factor,
lysophosphatidic acid, or
2-adrenergic receptor-mediated p42/p44 MAP kinase activation in COS-1 cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptor
(
2-AR)1
subtypes have been identified, known as
2a,
2b, and
2c, which belong to the G
protein-coupled receptor (GPCR) superfamily. A fourth subtype,
2d, has been identified in rat, mouse, and bovine
tissues and is considered to be the ortholog of
2a (1).
Although the subtype-specific pharmacological differences, G protein
coupling, and desensitization of the
2-ARs have been
extensively investigated, there is little information regarding their
trafficking after agonist stimulation, in particular the role of
arrestins and G protein-coupled receptor kinases (GRKs).
2a AR has been shown to be phosphorylated by GRK2 and GRK3, but not GRK5 or
GRK6, whereas the
2c AR does not appear to be a
substrate for GRK phosphorylation (7). The
2b AR also
undergoes agonist-promoted phosphorylation (8, 9). Similarly, the
2a and
2b ARs undergo rapid
agonist-mediated desensitization, whereas the
2c AR does not (7-9).
2a and
2b ARs have been shown to localize initially to the
plasma membrane (10, 11). However, a significant proportion of the
2c AR appears to be intracellular, accumulating in both
the Golgi apparatus and endoplasmic reticulum (10, 12). In HEK-293
cells, the
2b AR has been shown to traffic to endosomes
upon agonist stimulation, similar to the classic pattern of
internalization observed with the
2-adrenergic receptor
(
2-AR) (12). In contrast, the
2a AR
appears to undergo little or no agonist-mediated redistribution (12).
However, in Chinese hamster ovary cells, internalization of the
2a AR was significantly greater (13), which may be
attributed to cell type-specific differences in regulatory proteins or
in differences between the methods used to quantitate internalization.
Recently, in vitro arrestin binding to the third
intracellular loop of the
2a AR has been reported,
suggesting that arrestins have the ability to interact with the
2a AR (14). Analysis of
2c AR trafficking is complicated by the sizable pool of intracellular receptors. However,
using multiple epitope tags and labeling of cell surface receptors,
Daunt et al. observed agonist-promoted internalization of
the plasma membrane-localized
2c AR in HEK-293 cells,
but did not detect redistribution of the intracellular pool of
2c AR (12).
2-AR
subtypes. Since the different
2-ARs display clear
differences with respect to their intracellular localization and
trafficking, they constitute an interesting receptor family in which to
investigate the role of GRKs and arrestins in these processes.
Furthermore, endocytosis has recently been suggested to be important in
the GPCR-mediated activation of p42/p44 MAP kinase (15). Therefore, the
2-AR family should also be useful to further explore a
potential link between endocytosis and MAP kinase activation. In the
present work, we used COS-1 cells to study the role of GRKs and
arrestins in
2 AR trafficking, since COS cells have been
shown to contain low endogenous levels of GRKs and arrestins (16). We
show that expression of arrestins, but not GRKs, differentially
mediates the trafficking of the different
2-AR subtypes.
However, expression of wild-type arrestins or dominant negative
dynamin-K44A had no effect on activation of p42/p44 MAP kinase by
either
2-ARs or endogenous EGF, lysophosphatidic acid
(LPA), or
2-adrenergic receptors in COS-1 cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2a,
2b,
and
2c AR cDNAs in pBC12BI were kind gifts from John
Regan (University of Arizona) (17-19). A Flag epitope was created by
PCR amplification of a HindIII/NcoI fragment from
pcDNA3-Flag-
2AR. This fragment encodes a 23-amino
acid amino-terminal peptide, which permits membrane targeting and
processing to expose an epitope that is recognized by both the M1 and
M2 Flag antibodies. The amino acid sequence of this region is:
MKTIIASYIFCLVFADYKDDDDA. In order to introduce epitope tags at the
amino terminus of each of the
2-ARs, the full-length
2a and
2c ARs were digested with NcoI and SalI and ligated with the PCR-amplified
HindIII/NcoI fragment (containing the Flag
epitope) and pBC (from pBC-Flag-
2AR) that was digested
with HindIII/SalI. The 5' region that resulted from the PCR reaction was confirmed by dideoxy sequencing. The Flag-tagged
2c and
2a ARs in pBC were
digested with SalI, blunted with Klenow, then digested with
HindIII to release the insert. This insert was then ligated
with pcDNA3 that had been digested with HindIII and
EcoRV. To construct
pcDNA3-Flag-
2bAR, the pBC-based
2b AR, which has three internal NcoI sites,
was restricted with SfiI, which cleaves at an internal 5'
SfiI site, and HindIII. A 40-base pair
double-stranded overlapping oligonucleotide was then designed to
reconstruct the region from the NcoI site at the initiating
methionine to the SfiI site. The
HindIII/NcoI Flag fragment, the 40-base pair
NcoI/SfiI fragment, and the
SfiI/HindIII
2b AR fragment were
then ligated together with HindIII-digested pBluescript KS
(Stratagene). The Flag-tagged
2b AR was then digested with HindIII to liberate the insert, blunted with Klenow,
and subcloned into pcDNA3 that was digested with EcoRV.
The plasmids pcDNA3-arrestin-2, pcDNA3-arrestin-3,
pcDNA3-GRK2-CT (containing residues 495-689 of GRK2), and
pcDNA3-dynamin-K44A have been described previously (20-23).
pcDNA-HA-p42 was a gift from J. Silvio Gutkind (National Institutes
of Health), and RasN17 was a gift from Jonathan Chernoff (Fox Chase
Cancer Center).
2-ARs was assessed by ELISA essentially as described by
Daunt et al. (12). Briefly, transfected cells in either
60-mm or 100-mm dishes were split into 24-well dishes coated with 0.1 mg/ml poly-L-lysine (Sigma). 24 h after transfection, cells were treated with or without 100 µM
(
)-epinephrine in DMEM containing 0.3 mM ascorbate, then
fixed for 5 min with 3.7% formaldehyde. The primary anti-Flag antibody
M1 (Sigma or IBI) and the secondary antibody, goat anti-mouse
conjugated with alkaline phosphatase (Bio-Rad), were used at a 1:1000
dilution. Visualization of antibody binding was performed using an
alkaline phosphatase substrate kit (Bio-Rad). Plates were read at 405 nm in a microplate reader driven by Microplate Manager software
(Bio-Rad).
2-ARs and 1 µg of the indicated arrestin-3
construct. Following transfection, cells were split and allowed to
adhere overnight on glass coverslips grown in 24-well dishes. To
visualize cell surface receptors, cells were incubated with M1 antibody
diluted 1:500 for 1 h at 4 °C in DMEM supplemented with 0.1%
bovine serum albumin. Cells were then treated with 100 µM
(
)-epinephrine for 10 min, washed, fixed with 3.7% formaldehyde,
permeabilized with 0.05% Triton X-100 for 10 min, and incubated with
goat anti-mouse fluorescein isothiocyanate-conjugated secondary
antibody (1:200 dilution). Coverslips were mounted using Slow-Fade
mounting medium (Molecular Probes) and examined by microscopy on a
Nikon Eclipse E800 fluorescence microscope using a Plan Fluor 40×
objective. Cells expressing the lowest levels of transiently expressed
proteins, but clearly above those of nonexpressing cells, were chosen
for view. Images were collected using QED Camera software and processed
with Adobe Photoshop version 3.0.
2-AR and either dynamin-K44A or pcDNA3.
Cells were serum-starved in DMEM containing 0.5% fetal bovine serum
overnight. Cells were stimulated with specific agonists as indicated in
the figure legends, washed once with phosphate-buffered saline and
lysed in 1 ml of ice-cold lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,
1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM sodium vanadate, 1 mM sodium fluoride, 1 µg/ml leupeptin). Cells were scraped off the plates and sonicated
twice using a Fisher model 550 sonic dismembrator set at 2 for 15 s each. All subsequent manipulations were performed on ice or at
4 °C with constant rocking on a platform rocker. Cell debris was
removed by centrifugation for 10 min at 13,000 rpm in an Eppendorf
microcentrifuge. The supernatant was precleared for 1 h to reduce
nonspecific binding by addition of 25 µl of protein G-agarose
(Boehringer Mannheim). Samples were then centrifuged in an Eppendorf
microcentrifuge at 3000 rpm for 2 min and 3 µl of monoclonal anti-HA
antibody (unpurified mouse ascites, Babco) was added to the supernatant for 3 h. 50 µl of protein G-agarose was then added, and the
samples were incubated overnight. The next day, protein
G-antibody-antigen complexes were collected by centrifugation and
washed three times with cold lysis buffer. The final pellet was
resuspended in 45 µl of SDS sample buffer and boiled for 10 min at
95 °C. 20 µl was electrophoresed on a 10% SDS-polyacrylamide gel,
and the gel was transferred to nitrocellulose. Activated HA-p42 was
detected using the phosphorylation state-specific p42/p44 antibodies as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-AR
Internalization--
To investigate the role of arrestins and GRKs in
trafficking of
2-ARs, we initially assessed the
internalization of the three human
2-AR subtypes in COS
cells. COS cells contain relatively low amounts of arrestins and GRKs
in comparison to other cell types such as HEK-293 (16), thus enabling
assessment of the relative contributions of GRKs and arrestins to
receptor trafficking. COS-1 cells were transfected with plasmids
expressing Flag epitope-tagged
2a,
2b,
and
2c ARs in the presence or absence of cotransfected arrestin-3, and receptor internalization was assayed by ELISA. In order
to achieve equivalent expression of all three
2-AR
subtypes (4-5 pmol/mg), it was necessary to transfect 10-fold lower
amounts of the
2a AR construct (data not shown). In the
absence of coexpressed arrestin-3, the
2b AR displayed a
slow rate of internalization, which reached 15% after 30 min (Fig.
1A). Internalization of the
2c AR occurred at a faster rate, reaching 20% after 20 min (Fig. 1B). In contrast, there was no significant
internalization of the
2a AR (Fig. 1C).
Internalization of both the
2b and
2c ARs
was significantly promoted upon coexpression of arrestin-3 (Fig. 1,
A and B). Both the rate and total amount of
receptor internalization was increased in the presence of arrestin-3.
In contrast, internalization of the
2a AR was only
modestly promoted by arrestin-3 coexpression (Fig.
1C).
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Fig. 1.
Internalization of
2-ARs in the presence or absence of
arrestin-3. COS-1 cells in 100-mm dishes were transfected with 5 µg of pcDNA-Flag-
2bAR (A), 5 µg of
pcDNA-Flag-
2cAR (B), or 0.3 µg of
pcDNA-Flag-
2aAR (C) and 5 µg of
pcDNA-arrestin-3 or vector. In the case of the
2a
AR, an additional 4.7 µg of vector was cotransfected such that all
dishes were transfected with a total of 10 µg of DNA. 5 h after
transfection, cells were split to 24-well dishes. 24 h after
transfection, cells were exposed to 100 µM epinephrine
for the indicated times, fixed, and internalization of receptors was
quantitated by ELISA as described under "Experimental Procedures."
Values shown are the mean ± S.E. of at least four independent
experiments performed in triplicate.
2-AR internalization. We have previously shown that transfection of arrestin-2 or arrestin-3 results in approximately 20-30-fold overexpression compared with endogenous arrestin levels (21, 22). Both arrestin-2 and arrestin-3 promoted internalization of
the
2b AR, with arrestin-3 promoting a significantly
higher level (Fig. 2A).
Coexpression of GRK2, in either the presence or absence of arrestins,
had no effect on
2b AR internalization, suggesting that
GRK2 is not limiting for
2b AR internalization in COS-1
cells (Fig. 2A). Expression of GRK2 also did not promote internalization of either the
2a or
2c
ARs even at times earlier than 30 min (data not shown). Interestingly,
2c AR internalization was significantly promoted by
arrestin-3, but not by arrestin-2 (Fig. 2B) even though
expression of arrestin-2 was more efficient. These data suggest that
either arrestin-2 or arrestin-3 can promote
2b AR
internalization, but that the
2c AR may preferentially respond to arrestin-3. In contrast, arrestin-2 or arrestin-3 (Fig. 2C) only modestly promoted
2a AR
internalization.
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Fig. 2.
Effects of arrestins and GRKs on
2-AR internalization. COS-1 cells
in 60-mm dishes were transfected with 3 µg of
pcDNA-Flag-
2bAR (A), 3 µg of
pcDNA-Flag-
2cAR (B), or 0.1 µg of
pcDNA-Flag-
2aAR (C) and 1 µg of
pcDNA-arrestin-2, pcDNA3arrestin-3, pcDNA-GRK2, or vector.
In the case of
2a, an additional 0.7-1.7 µg of vector
was cotransfected such that all dishes were transfected with a total of
4 µg (arrestins alone) or 5 µg (arrestins + GRK2) of DNA. 5 h
after transfection, cells were split to 24-well dishes. 24 h after
transfection, cells were exposed to 100 µM epinephrine
for 30 min, and internalization of receptors was quantitated by ELISA.
Values shown are the mean ± S.E. of at least four independent
experiments performed in triplicate. Asterisks (*) indicate
the result of statistical analysis by paired t test with
p < 0.05 sufficient to reject the null hypothesis.
Insets, immunoblot analysis of arrestin expression from a
representative experiment in cells cotransfected with vector alone
(left), arrestin-2 (middle), or arrestin-3
(right).
2-ARs, we transfected cells with either the
2b or
2c AR and arrestin-3, with or
without dynamin-K44A, a dominant negative mutant of dynamin-I which
does not bind GTP (24). Arrestin-mediated internalization of the
2b and
2c ARs was completely inhibited by
coexpression of dynamin-K44A (Fig. 3,
A and B). Coexpression of dynamin-K44A in the
absence of arrestin-3 also revealed a modest inhibition of
internalization of both receptors. These data suggest that
agonist-induced internalization of both
2b and
2c ARs in both the presence and absence of arrestin-3 is
a dynamin-dependent process.
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Fig. 3.
Effect of dominant negative dynamin-K44A
on 2-AR internalization.
COS-1 cells in 60-mm dishes were transfected with 3 µg of
pcDNA-Flag-
2bAR (A) or 3 µg of
pcDNA-Flag-
2cAR (B), 1 µg of
pcDNA3-arrestin-3 or vector, and 1 µg of pcDNA-dynamin-K44A.
5 h after transfection, cells were split to 24-well dishes.
24 h after transfection, cells were exposed to 100 µM epinephrine for 30 min, and internalization of
receptors was quantitated by ELISA. Values shown are the mean ± S.E. of three or four independent experiments performed in
triplicate.
2-AR
Trafficking--
We next examined the redistribution of
2-ARs by immunofluorescence. In permeabilized cells, the
2a and
2b ARs were predominantly localized to the cell surface. In contrast, a significant amount of the
2c AR was localized to the Golgi apparatus and
endoplasmic reticulum (Ref. 12 and data not shown). In order to follow
the redistribution of receptors present on the cell surface, cells were
preincubated with Flag antibody at 4 °C prior to agonist exposure.
In this way, only the trafficking of receptors initially present on the
cell surface would be detected. Cells were transfected with
2-ARs in the absence or presence of arrestin-3. In the
absence of agonist and arrestin, each
2-AR subtype was
clearly detected on the cell surface (Fig.
4, left panels).
When cells were stimulated with agonist for 10 min in the absence of
coexpressed arrestin-3, little or no redistribution of
2-ARs was detected (Fig. 4, middle panels). In the presence of coexpressed arrestin-3, the
2a,
2b, and
2c ARs
demonstrated agonist-dependent redistribution to
clathrin-coated pits and endosomes (Fig. 4, right
panels). No redistribution of receptors was observed in
arrestin-3-expressing cells in the absence of agonist and
internalization was significantly inhibited by coexpression of
dynamin-K44A (data not shown). These results suggest that each
2-AR subtype internalizes in an
arrestin-dependent manner. Taken together with the results
of the ELISA, however, internalization of the
2a AR
appears to be significantly less efficient than either the
2b or
2c ARs.
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Fig. 4.
Immunofluorescence analysis of
2-AR internalization. COS-1 cells
in 60-mm dishes were transfected with 3 µg of
pcDNA-Flag-
2bAR, 3 µg of
pcDNA-Flag-
2cAR, or 0.1 µg of
pcDNA-Flag-
2aAR and 1 µg of pcDNA-arrestin-3
or vector. In the case of
2a AR, an additional 0.7 µg
of vector was cotransfected such that all dishes were transfected with
a total of 4 µg of DNA. 5 h after transfection, cells were split
onto glass coverslips in 24-well dishes. 24 h after transfection,
cells were incubated for 1 h at 4 °C with M1 anti-Flag antibody
to label cell surface receptors, then washed to remove unbound antibody
and warmed to 37 °C. Where indicated, cells were exposed to 100 µM epinephrine for 10 min. Cells were then fixed,
incubated with goat anti-mouse fluorescein isothiocyanate-conjugated
secondary antibody, and imaged as described under "Experimental
Procedures."
2-AR-mediated Activation of
p42/p44 MAP Kinase--
Recently, it was reported that
arrestin-mediated internalization of GPCRs in HEK-293 cells plays a
critical role in their ability to activate p42/p44 MAP kinase (15).
Therefore, we examined the possibility that internalization may be
required for
2-AR-mediated MAP kinase activation.
Moreover, the differences between the
2-AR subtypes with
respect to their internalization suggested that they may constitute an
ideal model for studying the role of endocytosis in p42/p44 MAP kinase
activation. Initially, we examined the ability of each
2-AR to activate p42/p44 MAP kinase in the absence of arrestin overexpression. Although little internalization of the
2-ARs was observed in the absence of arrestins (Figs. 1
and 4), each
2-AR subtype rapidly activated p42/p44 MAP
kinase to a similar extent after agonist addition, whereas no
activation of p42/p44 was observed in untransfected cells (Fig.
5A).
2-AR-mediated p42/p44 MAP kinase activation was maximal
between 5 and 30 min after agonist addition and slowly declined
thereafter. The activation of p42/p44 MAP kinase by
2-ARs was inhibited by both dominant negative Ras
(RasN17, Fig. 5B) and pertussis toxin treatment (Fig. 5C). Thus, activation of p42/p44 MAP kinase by all three
2-AR subtypes appears to proceed via activation of
Gi/o and Ras, in agreement with previous studies on the
2a AR (25, 26).
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Fig. 5.
Activation of p42/p44 MAP kinase by
2-ARs. A, COS-1 cells
in 100-mm dishes were transfected with 5 µg of pcDNA3Flag-
2bAR, 5 µg of
pcDNA3-Flag-
2cAR, or 0.3 µg of pcDNA3Flag-
2aAR. Activation of endogenous p42/p44 was measured
at the indicated times after addition of 10 µM UK 14,304 by immunoblot using a polyclonal rabbit antibody specific to the
phosphorylated form of p42/p44. The data shown are representative of
three independent experiments. B, COS-1 cells were
transfected with
2-ARs as described in A
together with 5 µg of pcDNA (vector), or 5 µg
pcDNA-RasN17 (RasN17), and stimulated with 10 µM UK 14,304. Endogenous p42/p44 MAP kinase activation
was determined as described in A. C, Activation
of endogenous p42/p44 was determined in COS-1 cells transfected with
2-ARs as described in A. Where indicated,
cells were treated with 10 µM UK 14,304 for 15 min in the
presence or absence of 100 ng/ml pertussis toxin added to cells 16 h prior to stimulation and present for the duration of the
experiment.
2-ARs exhibited minimal agonist-mediated
internalization in the absence of coexpressed arrestins, we next
examined the effect of expressing either wild-type arrestin-3 or
dominant negative dynamin-K44A on
2-AR-mediated p42/p44
MAP kinase activation.
2-AR-mediated activation of
p42/p44 was not significantly altered by coexpression of either
arrestin-3 or dynamin-K44A (Fig. 6, A and B). We also attempted to assess the effect
of receptor internalization on p42/p44 MAP kinase activation by
treating cells with concanavalin A, hypotonic sucrose, or
monodansylcadaverine, which each inhibit receptor internalization
albeit by different mechanisms (27). Monodansylcadaverine, which
inhibited
2-AR internalization as assessed by ELISA, had
no effect on
2-AR-stimulated p42/p44 activation (data
not shown), supporting our dynamin-K44A results. In contrast, we
observed that concanavalin A and sucrose directly activated p42/p44 in
COS cells (i.e. in the absence of receptor and agonist) and
were therefore not suited for this analysis.
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Fig. 6.
Effect of dynamin-K44A and wild-type
arrestin-3 on 2-AR-mediated
p42/p44 MAP kinase activation. A, COS-1 cells were
transfected with 5 µg of pcDNA-Flag-
2bAR, 5 µg
of pcDNA-Flag-
2cAR, or 0.3 µg of
pcDNA-Flag-
2aAR together with 5 µg of vector,
dynamin-K44A, or wild-type arrestin-3. Cells were stimulated with 10 µM UK 14,304 for the indicated times, and p42/p44
activation was determined as described in Fig. 5. In all experiments,
overexpression of dynamin K44A or arrestin-3 was confirmed by
immunoblot. B, the experiment described in A was
repeated and p42/p44 activation was quantitated by densitometric
scanning of immunoblots. Values shown are the mean ± S.E. of six
independent experiments. C, COS-1 cells were transfected
with 5 µg of pcDNA3-Flag-
2bAR together with 5 µg
of vector, dynamin-K44A, wild-type arrestin-3, or RasN17 and 5 µg of
HA-p42. The total amount of DNA transfected was held constant at 15 µg. Cells were stimulated with 10 µM UK 14,304 for the
indicated times and lysed, and HA-p42 was immunoprecipitated with a
mouse monoclonal antibody directed against the HA epitope. Activation
of HA-p42 was determined by immunoblot with a polyclonal rabbit
antibody specific to phosphorylated p42/p44. Immunoprecipitation of
HA-p42 was assessed in parallel immunoblots using a phosphorylation
state-independent antibody directed against p42. This experiment was
repeated twice with similar results.
2-ARs and either arrestin-3 or dynamin-K44A were
cotransfected. Thus, it is unlikely that the expression of arrestin-3
or dynamin-K44A would be limiting with respect to their ability to
modulate the activation of endogenous p42/p44. Moreover, cotransfection
of dominant negative Ras significantly inhibited activation of
endogenous p42/p44 MAP kinase (Fig. 5B). However, to address
the possible limitations of transient transfection on p42/p44
activation, we cotransfected HA-tagged p42 MAP kinase together with the
2b AR and either dynamin-K44A, arrestin-3 or RasN17. The
2b AR was chosen since its internalization was the most
responsive to arrestin-3 and was completely inhibited by dynamin-K44A
(Figs. 2 and 3). The coexpression of either wild-type arrestin-3 or
dynamin-K44A did not affect
2b AR-mediated activation of
transfected HA-p42 MAP kinase (Fig. 6C). In contrast,
cotransfection of RasN17 resulted in almost complete inhibition of
2b AR-mediated HA-p42 activation. Taken together, these
results strongly suggest that internalization of the
2-ARs is not required for activation of p42/p44 MAP kinase.
2-Adrenergic, LPA, and EGF Receptors--
We
next addressed the question of whether the lack of effect of arrestin
or dynamin-K44A on
2-AR-mediated p42/p44 MAP kinase activation was specific to the
2-ARs or a general
property of receptor function in COS-1 cells. Therefore, we examined
the effect of dynamin-K44A expression on activation of p42/44 by
endogenous LPA,
2-adrenergic, and EGF receptors in COS-1
cells. The dynamin-dependent internalization of the
2-AR has been well documented, and internalization of
the EGF receptor has also been reported to be mediated by
clathrin-coated pit endocytosis (28, 29). The role of arrestin or
dynamin in the trafficking of the LPA receptor has not been reported, although it has been observed that dynamin-K44A and a dominant negative
arrestin-2 mutant inhibit LPA-mediated activation of p42/p44 in HEK-293
cells (15). We found that overexpression of dynamin-K44A had no
significant effect on the ability of isoproterenol, EGF, or LPA to
activate p42/p44 (Fig. 7A).
2-AR-mediated activation was specifically inhibited by
the
-antagonist alprenolol, and LPA-mediated p42/p44 activation was
sensitive to overexpression of GRK2-CT, which sequesters G
subunits (data not shown). However, it was possible that transient
transfection of dynamin-K44A would be insufficient to detect an effect
on activation of both endogenous p42/p44 MAP kinase and receptors
present in all cells. Therefore, this experiment was repeated by
cotransfecting both dynamin-K44A and HA-tagged p42 MAP kinase. HA-p42
was immunoprecipitated, and its activation was assessed by immunoblot
using a phosphorylation state-specific p42/p44 antibody. Activation of
HA-p42 MAP kinase by EGF, LPA, and isoproterenol was unaltered by
overexpression of dynamin-K44A (Fig. 7B). Taken together,
these data suggest that endocytosis is not required for
receptor-mediated activation of p42/p44 MAP kinase in COS-1 cells.
View larger version (39K):
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Fig. 7.
Effect of dynamin-K44A on p42/p44 activation
by EGF, 2-adrenergic, and LPA
receptors. A, COS-1 cells were transfected with 5 µg
of vector or dynamin-K44A and stimulated for the indicated times with
10 µM (
)-isoproterenol, 25 µM
lysophosphatidic acid, or 10 ng/ml EGF. Activation of p42/p44 was
determined as described in Fig. 5A. B, COS-1
cells were transfected with 5 µg of vector or dynamin-K44A and 5 µg
of HA-p42. Cells were stimulated as described in A and then
lysed, and HA-p42 was immunoprecipitated with a mouse monoclonal
antibody directed against the HA epitope. Activation of HA-p42 was
determined by immunoblot using a polyclonal rabbit antibody specific to
phosphorylated p42/p44. For each experiment, overexpression of HA
epitope-tagged dynamin-K44A was determined by stripping and reprobing
of blots with an antibody directed against the HA tag and
immunoprecipitation of HA-p42 was monitored in parallel immunoblots
using a phosphorylation state-independent antibody directed against
p42. These experiments were repeated twice for each receptor with
similar results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptors has not been investigated. In
this study, we show that arrestins significantly promote
internalization of the human
2b and
2c
ARs. Internalization of the
2c AR was promoted to a
significantly greater extent by arrestin-3 compared with arrestin-2. In
contrast, internalization of the
2a AR was only modestly
promoted by coexpression of arrestins. However, endocytosis of
2-ARs does not appear to be required for their ability
to activate p42/p44 MAP kinase. Furthermore, EGF, LPA, and
2-adrenergic receptor-mediated activation of p42/p44 MAP
kinase was also insensitive to overexpression of dynamin-K44A, a GTP
binding-deficient mutant that has been shown to inhibit both clathrin
coated vesicle- and caveolae-mediated receptor internalization (24,
30).
2-AR
subtypes with respect to their cellular localization and their ability to internalize in response to arrestins. Similar to previous reports using different cell types (10, 12), both the
2a and the
2b ARs were initially localized to the cell surface. In
contrast, a significant proportion of the
2cAR was
localized to the Golgi apparatus and endoplasmic reticulum. The
2bAR internalized to a greater extent in the presence of
either arrestin-2 or arrestin-3 compared with the
2a or
the
2c ARs and arrestin-3 promoted
2bAR internalization more effectively than arrestin-2. Internalization of
the
2aAR was only slightly promoted by either arrestin-2
or arrestin-3. However, the proportion of the
2a AR that
did internalize was localized to clathrin-coated pits and endosomes.
The agonist-mediated internalization of all three
2-AR
subtypes in the absence of arrestin overexpression, although modest,
was only slightly inhibited by expression of dynamin-K44A. Thus, it is
possible that internalization of receptor under these conditions may be
at least partially dynamin-independent, similar to that previously
observed with the angiotensin 1A receptor (31). Although
agonist-activated
2a and
2b ARs have been
reported to be substrates for GRK phosphorylation (7, 8),
cotransfection of GRK2 did not promote internalization of the
2-ARs or enhance arrestin-promoted internalization.
However, internalization of both the
2b and
2c ARs in HEK-293 cells has been shown to occur in the
absence of cotransfected GRKs and arrestins (12), and HEK-293 cells
possess higher endogenous levels of both GRKs and arrestins (16). In
COS cells, internalization of the
2-AR was shown to be
promoted by expression of arrestins, but not by GRK2 (16). Thus, it is
likely that trafficking of
2-ARs, as well as other
GPCRs, will be highly dependent upon the complement of GRKs and
arrestins in a given cell type, as well as the affinity of these
proteins for a given GPCR.
2c AR was promoted
to a much larger extent by arrestin-3 compared with arrestin-2. To our
knowledge, this is the first reported observation of significant preference of a GPCR for either arrestin-2 or arrestin-3. Since both
the ELISA and the immunofluorescence studies exclusively measured
surface
2c AR, the intracellular pool of receptor did not confound our results. Studies performed in HEK-293 cells also showed that cell surface
2c ARs can internalize in
response to agonist, whereas no redistribution of the intracellular
pool of receptor was observed (12). However,
2c ARs do
not appear to be a substrate for GRK phosphorylation (7), and we
observed that coexpression of GRK2 had no effect on
2cAR
internalization. It is possible that internalization of the
2cAR receptor may occur in a phosphorylation-independent
manner. Indeed, internalization of
2-AR mutants that are
deficient in agonist-mediated phosphorylation can be rescued by
overexpression of arrestins (32). More recently, phosphorylation-independent but arrestin- or
dynamin-dependent internalization has been described for
both the follitropin and
opioid receptors (33, 34). The exquisite
specificity of visual arrestin for rhodopsin has been well
characterized (35). However, the precise determinants that account for
this specificity, either within the receptor binding domain of arrestin
or the cytoplasmic loops of rhodopsin or other GPCRs are not known. The
subtype-specific differences noted here between the
2-ARs may provide a suitable model to address the
question of specificity of GPCR-arrestin interaction.
2-ARs is not a prerequisite for their activation of
p42/p44 MAP kinase. First, agonist-induced activation of all three
2-AR subtypes resulted in activation of p42/p44 MAP
kinase under conditions in which little or no receptor endocytosis was
observed. Second, overexpression of arrestins, which significantly
promoted endocytosis, did not affect the magnitude of p42/p44
activation. Conversely, expression of dynamin-K44A, which ablated
arrestin-mediated internalization, did not inhibit activation of
p42/p44 even in the absence of coexpressed arrestin. Third, in
experiments in which receptor, dynamin-K44A, or wild-type arrestin-3
and HA-tagged p42 MAP kinase were transfected into cells, we observed
no effect of either dynamin-K44A or arrestin-3 on
2-AR-mediated HA-p42 activation. Thus, it is unlikely
that the limitations imposed by transfection efficiency account for the
lack of effects of either dynamin-K44A or arrestin-3 on activation of
endogenous p42/p44 MAP kinase. Moreover, cotransfection of dominant
negative RasN17 inhibited activation of endogenous p42/p44 MAP kinase
and transfected HA-p42 MAP kinase by all three
2-AR subtypes. Interestingly, expression of dynamin-K44A did not inhibit p42/p44 activation by the
2-adrenergic, EGF, or LPA
receptors. Dynamin-dependent endocytosis of both the
2-AR (31) and the EGF receptor (29) has been reported,
although evidence of dynamin-dependent trafficking of the
LPA receptor is lacking. Therefore, our results suggest that the lack
of effect of mediators of receptor internalization on p42/p44 MAP
kinase activation is not unique to
2-ARs, but extends to
endogenous receptors in COS-1 cells as well.
2-adrenergic and LPA receptors in HEK-293 cells is
inhibited by cotransfection of either dynamin-K44A or dominant negative
arrestin-2 (V53D). It was proposed that inhibition of p42/p44 MAP
kinase occurred at a step downstream from Raf, since second messenger
generation, Shc phosphorylation, and Raf activity were not affected by
either dominant negative dynamin or arrestin. The differences between our studies might be accounted for by the use of different cell lines
and their respective levels of arrestins and signaling intermediates or
by differences in their endocytic pathways. Other GPCRs have been
reported to activate p42/p44 MAP kinase in the absence of detectable
receptor internalization. Activation of the µ-opioid receptor by
morphine activates p42/p44 (36), but fails to induce receptor
internalization (37). Furthermore, cell-type and agonist-specific activation of
-opioid receptors also promotes p42/p44 MAP kinase activation independent of receptor internalization (38). Recently, endocytosis of the insulin-responsive glucose transporter GLUT4 has
been reported to be sensitive to expression of dynamin-K44A, whereas
insulin signaling, including MAP kinase activation, was unaffected
(39). In HeLa cells made defective for receptor-mediated endocytosis by
conditional expression of dynamin-K44A, EGF-mediated cell proliferation
was enhanced (40). In contrast, both EGF receptor and p42/p44
phosphorylation appeared to be reduced, while Shc phosphorylation was
increased. Whether reduced MAP kinase activation was a consequence of
reduced activation of the EGF receptor or other signaling intermediates
in the MAP kinase pathway or reduced EGF receptor internalization
per se remains unclear. A recent report also suggests that
activation of p42/p44 MAP kinase may be involved in the phosphorylation
and desensitization of µ-opioid receptor (36). Thus, there is likely
to be a complex interplay between signal transduction molecules and
regulators of receptor endocytosis such as GRKs and arrestins that
ultimately determines the extent and duration of responses to different
stimuli. Analysis of the interaction of molecules that facilitate
receptor desensitization and internalization with components of the
GPCR and receptor tyrosine kinase signaling pathways should greatly enhance our understanding of how cells control responses to
extracellular stimuli.
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ACKNOWLEDGEMENTS |
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We thank the members of the Sidney Kimmel Nucleic Acid facility for oligonucleotide synthesis and sequencing and members of the Benovic laboratory for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM47419 (to J. L. B.) and National Institutes of Health Training Grants 5-T32-DK07705 (to M. J. O.) and 5-T32-AI07523 (to J. L. D.).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.
Current address: Cardiovascular Drug Discovery, Rhone-Poulenc
Rorer Pharmaceuticals, Collegeville, PA 19426.
§ Established investigator of the American Heart Association. To whom correspondence should be addressed: Thomas Jefferson University, 233 S. 10th Street, Philadelphia, PA 19107. Tel.: 215-503-4607; Fax: 215-923-1098; E-mail: benovic{at}lac.jci.tju.edu.
2
Although a variety of names have been used for
the various mammalian arrestins, we propose that the following
nomenclature be used based on the order of discovery of the arrestins:
arrestin-1 (visual arrestin, S-antigen, 48-kDa protein); arrestin-2
(-arrestin,
-arrestin-1); arrestin-3 (
-arrestin-2, arrestin3,
thy-X arrestin); arrestin-4 (C-arrestin, X-arrestin).
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ABBREVIATIONS |
---|
The abbreviations used are:
2-AR,
2-adrenergic receptor;
2-AR,
2-adrenergic receptor;
EGF, epidermal growth factor;
GPCR, G protein-coupled receptor, GRK, G protein-coupled receptor
kinase;
LPA, lysophosphatidic acid;
p42/p44 MAP kinase, p42/p44
mitogen-activated protein kinase;
ELISA, enzyme-linked immunosorbent
assay;
PCR, polymerase chain reaction;
DMEM, Dulbecco's modified
Eagle's medium;
TBST, Tris-buffered saline with Tween 20;
HA, hemagglutinin.
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REFERENCES |
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