COMMUNICATION
Feedback Regulation of
-Arrestin1 Function by
Extracellular Signal-regulated Kinases*
Fang-Tsyr
Lin,
William E.
Miller,
Louis M.
Luttrell, and
Robert
J.
Lefkowitz
From the Howard Hughes Medical Institute, Departments of Medicine
and Biochemistry, Duke University Medical Center,
Durham, North Carolina 27710
 |
ABSTRACT |
The functions of
-arrestin1 to facilitate
clathrin-mediated endocytosis of the
2-adrenergic
receptor and to promote agonist-induced activation of extracellular
signal-regulated kinases (ERK) are regulated by its
phosphorylation/dephosphorylation at Ser-412. Cytoplasmic
-arrestin1
is almost stoichiometrically phosphorylated at Ser-412.
Dephosphorylation of
-arrestin1 at the plasma membrane is required
for targeting a signaling complex that includes the agonist-occupied
receptors to the clathrin-coated pits. Here we demonstrate that
-arrestin1 phosphorylation and function are modulated by an
ERK-dependent negative feedback mechanism. ERK1 and ERK2
phosphorylate
-arrestin1 at Ser-412 in vitro. Inhibition of ERK activity by a dominant-negative MEK1 mutant significantly attenuates
-arrestin1 phosphorylation, thereby increasing the concentration of dephosphorylated
-arrestin1. Under such conditions,
-arrestin1-mediated
2-adrenergic receptor
internalization is enhanced as is its ability to bind clathrin. In
contrast, if ERK-mediated phosphorylation is increased by transfection
of a constitutively active MEK1 mutant, receptor internalization is
inhibited. Our results suggest that dephosphorylated
-arrestin1
mediates endocytosis-dependent ERK activation. Following
activation, ERKs phosphorylate
-arrestin1, thereby exerting an
inhibitory feedback control of its function.
 |
INTRODUCTION |
The life cycle of G protein-coupled receptors
(GPCRs)1 includes receptor
activation, desensitization, sequestration, and either resensitization
(recycling) or degradation (1).
-Arrestins were initially discovered
as molecules that bind to agonist-occupied receptors following receptor
phosphorylation by G protein-coupled receptor kinases, thereby
interdicting signal transduction to G proteins and causing receptor
desensitization (2, 3). More recently, however,
-arrestins have been
shown to be involved in the internalization and signaling of GPCRs (4).
For example, they serve as clathrin adaptors, which help to target
agonist-occupied GPCRs to clathrin-coated pits for internalization (5).
This function is regulated by phosphorylation/dephosphorylation of
-arrestin1 at a carboxyl-terminal serine, Ser-412 (6). Cytosolic
-arrestin1 is constitutively phosphorylated by heretofore
unidentified kinase(s) and is rapidly dephosphorylated when it is
recruited to the plasma membrane in response to agonist stimulation.
Dephosphorylation of
-arrestin1 at the plasma membrane is not
required for receptor binding and receptor desensitization but is
required for its clathrin binding and receptor internalization. The
S412A mutant of
-arrestin1, which mimics the dephosphorylated form,
has been shown to be more active than wild-type
-arrestin1 in
promoting clathrin-mediated endocytosis of the
2-adrenergic receptor. In contrast, the S412D mutant,
which simulates the phosphorylated form of
-arrestin1, acts as a
dominant-negative inhibitor of receptor endocytosis (6). Moreover, in
addition to regulating the internalization of classical GPCRs, such as
the
2-adrenergic receptor,
-arrestin1 has been shown
to bind to the tyrosine kinase insulin-like growth factor I receptor
and mediate its endocytosis in an analogous fashion (7).
Recently, several studies have shown that clathrin-mediated
internalization is required for mitogenic signaling by various GPCRs
and tyrosine kinase growth factor receptors (7-12, 15). Thus,
inhibition of clathrin-mediated internalization reduces agonist-induced
activation of ERK1 and 2. The Ras-dependent activation of
ERKs by GPCRs also requires c-Src (13, 14). Very recently, it has been
shown that
-arrestins serve to recruit the activated c-Src to the
agonist-occupied
2-adrenergic receptors as well as to
target this signaling complex to the clathrin-coated pits for
internalization and activation of the ERK cascade (15). Like clathrin
targeting, the recruitment and activation of c-Src kinase is modulated
by phosphorylation/dephosphorylation of
-arrestin1 (15). The S412D
-arrestin1 mutant, defective in both binding to Src and targeting
the receptors to clathrin-coated pits, acts as a dominant-negative
inhibitor of agonist-induced ERK activation. In contrast, the S412A
-arrestin1 mutant, which binds to Src as well as the wild-type
-arrestin1, is active in promoting agonist-induced ERK phosphorylation.
ERK activity appears to be tightly regulated by an
activation/inactivation cycle. GPCR-mediated activation of ERKs
involves the sequential involvement of components of a Ras activation
complex, including c-Src, Shc, Grb2, Gab1, and Sos1, followed by
activation of Raf-1 kinase and MEK1 (13, 14). It has been shown that the inactivation of this cascade is associated with the induction of
mitogen-activated protein kinase phosphatase (MKP-1) by agonist stimulation (16). Previous studies also suggest that it may involve the
negative feedback phosphorylation of upstream activators, including
Sos1, Raf-1 kinase, and MEK1, by the activated ERK (17-21). Recently
ERK has been reported to phosphorylate IRS-1 and reduce its function,
thereby inhibiting further insulin signaling (22). These findings
underscore the requirement for stringent control of cellular ERK
activity by feedback regulatory mechanisms. Here we demonstrate a novel
form of feedback regulation controlling GPCR-mediated activation of
ERKs. Once stimulated, the ERKs phosphorylate
-arrestin1 at Ser-412,
thereby reducing its endocytic functions and thus ultimately reducing
ERK activation.
 |
EXPERIMENTAL PROCEDURES |
Expression of
-Arrestin1 in Escherichia coli and
Phosphorylation in Vitro--
A 1.26-kilobase
KpnI/HindIII fragment encoding
(S412D)
arr1-His6 was removed from
pBS/(S412D)
arr1-His6 (6) and subcloned into pKK223-3
vector (Amersham Pharmacia Biotech). After transfection of
pKK/
arr1-His6 or pKK/(S412D)
arr1-His6
plasmid into E. coli, expression of the proteins was induced
by isopropyl-1-thio-
-D-galactopyranoside.
-Arrestin1
was purified by nickel affinity chromatography followed by
Heparin-Sepharose chromatography as described before (6). 20 pmol of
wild-type or S412D
-arrestin1 were incubated with either 0.9 µg of
GST-ERK1 (Upstate Biotechnologies Inc.), 0.15 µg of ERK2 (Upstate
Biotechnologies Inc.), or 10 units of GSK-3 (New England Biolabs) in
the presence of 20 mM Tris, pH 7.4, 2 mM EDTA,
10 mM MgCl2, 1 mM dithiothreitol,
100 µM ATP, and 1 µCi of [
-32P]ATP at
30 °C for 30 min. The phosphoproteins were fractionated by SDS-PAGE.
The gel was dried and developed by autoradiography.
Two-dimensional Tryptic Phosphopeptide Mapping--
Purified
phospho-
-arrestin1 (wild-type or S412D) was resolved by SDS-PAGE and
transferred to polyvinylidene difluoride membranes (Immobilon,
Millipore). The phospho-
-arrestin1 band was cut out, digested with
trypsin in situ, and oxidized in performic acid (23).
Lyophilized peptides were resolved by electrophoresis at pH 3.5 in the
first dimension and ascending chromatography in the second dimension as
described (24). Phosphopeptides were detected by autoradiography.
Metabolic Labeling--
The His-tagged
-arrestin1 expression
vector was transfected alone or with the dominant-negative
MEK1(K97A) plasmid (25) into HEK 293 cells. Cells were labeled with
[32P]orthophosphate for 1 h and then harvested for
-arrestin1 purification as described (6).
Co-immunoprecipitation and Immunoblotting--
The FLAG-tagged
-arrestin1 expression vector was transfected alone or with the
MEK1(K97A) plasmid into HEK 293 cells. Two days after transfection,
cells were harvested and lysed for co-immunoprecipitation as described
(6). The FLAG-tagged
-Arrestin1 (15) was immunoprecipitated with a
polyclonal antibody directed against the FLAG epitope (Santa Cruz
Inc.). After SDS-PAGE, the immunoblot was probed with a monoclonal antibody specific to clathrin heavy chain (Transduction Laboratories) and was visualized by enhanced chemiluminescence assay (ECL, Amersham Pharmacia Biotech). The expression levels of phospho-ERKs, total cellular ERKs, and MEK1 mutants (K97A and S218D/S222D) (25, 26) in
whole cell extracts were determined by probing the immunoblots separately with the antibodies specific to phospho-ERK (Promega), cellular ERK2 (Transduction Laboratories), or MEK1 (Transduction Laboratories).
Agonist-promoted Sequestration of the
2-Adrenergic
Receptors--
HEK 293 cells were transiently transfected with the
plasmid encoding FLAG-tagged
2-adrenergic receptors with
or without the expression vectors of
-arrestin and a MEK1 mutant.
Two days after transfection, cells were incubated with 10 µM (
)-isoproterenol in 0.1 mM ascorbic acid
for 30 min before harvesting. The agonist-promoted sequestration of
2-adrenergic receptors was determined by
immunofluorescence flow cytometry as described previously (27).
 |
RESULTS AND DISCUSSION |
ERKs Phosphorylate
-Arrestin1 at Ser-412 in
Vitro--
Previously we have shown that cytosolic
-arrestin1 is
highly phosphorylated and is dephosphorylated only when it is recruited to the plasma membrane in response to agonist stimulation (6). The
major phosphorylation site is located at the carboxyl-terminal Ser-412,
which accounts for 90% of
-arrestin1 phosphorylation. To identify
the candidate kinase(s) that phosphorylate
-arrestin1 at Ser-412, we
tested the ability of several kinases to phosphorylate
-arrestin1
in vitro. Because Ser-412 is followed by a proline residue,
a consensus phosphorylation sequence recognized by members of the
mitogen-activated protein kinase family as well as by glycogen synthase
kinase-3 (GSK-3), we speculated that these kinases might be potential
candidates for mediating Ser-412 phosphorylation. Therefore, equal
amounts of wild-type and S412D
-arrestin1 purified from E. coli were subjected to phosphorylation by ERK1, ERK2, or GSK-3
in vitro. As shown in Fig. 1,
wild-type
-arrestin1 was highly phosphorylated by either ERK1 or
ERK2. The stoichiometry was ~0.8 mol of Pi/mol of
protein. Mutation of Ser-412 to Asp markedly reduced ERK-mediated
-arrestin1 phosphorylation. Both wild-type and S412D
-arrestin1
were equally but weakly phosphorylated by GSK-3, indicating that GSK-3
is not the kinase responsible for Ser-412 phosphorylation.

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Fig. 1.
Phosphorylation of
-arrestin1 by ERK1, ERK2, and GSK-3 in
vitro. 20 pmol of His-tagged wild-type (WT)
or S412D -arrestin1 purified from E. coli were
phosphorylated in vitro by ERK1, ERK2, or GSK-3 in the
presence of [ -32P]ATP as described under
"Experimental Procedures." The phosphoproteins were fractionated by
SDS-PAGE. The gel was dried and developed by autoradiography.
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Next, we compared the two-dimensional tryptic phosphopeptide map of
cellular
-arrestin1 with those of wild-type and S412D
-arrestin1
phosphorylated by ERK2 in vitro. As shown in Fig. 2A, the two-dimensional
phosphopeptide mapping of cellular
-arrestin1 purified from HEK 293 cells indicates that it contains three phosphopeptides: a1, a2, and b.
The major phosphopeptides, a1 and a2, are partial digestion
products (amino acids 401-418 and 398-418) containing Ser-412 (Fig.
2B) as confirmed by amino acid sequencing (6). The
two-dimensional phosphopeptide map of
-arrestin1 phosphorylated by
ERK2 in vitro (Fig. 2C) was identical with the
pattern derived from cellular phospho-
-arrestin1. This was further
confirmed by the identical map derived from a mixture of equal amounts
of cellular phospho-
-arrestin1 and ERK2-phosphorylated
-arrestin1 (data not shown). These two phosphopeptides, a1 and a2, were missing in
the map of S412D
-arrestin1 phosphorylated by ERK2 (Fig.
2D). Taken together, our results indicate that ERK is
capable of phosphorylating Ser-412 of
-arrestin1. Although
-arrestin1 could be phosphorylated by protein kinases A and C, GRK2,
and casein kinase I and II in vitro, in no case did the
two-dimensional tryptic phosphopeptide maps match those of
-arrestin1 purified from cells (data not shown). Moreover, mutation
of Ser-412 to Asp did not reduce in vitro phosphorylation of
-arrestin1 by these kinases, indicating that these kinases are not
responsible for Ser-412 phosphorylation.

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Fig. 2.
Two-dimensional tryptic phosphopeptide
mapping of cellular phospho- -arrestin1 and
ERK2-phosphorylated -arrestin1.
A, 32P-labeled -arrestin1
( arr1) was purified from HEK 293 cells overexpressing
His-tagged -arrestin1, resolved by SDS-PAGE, and transferred to
Immobilon polyvinylidene difluoride membranes. The
32P-labeled -arrestin1 was cut out, digested with
trypsin, resolved in two dimensions by electrophoresis and
chromatography, and detected by autoradiography. × is the
origin of sample loading. The arrows indicate the major
partially digested phosphopeptides a1 and a2 and the minor
phosphopeptide b. B, tryptic phosphopeptides as described
above were purified by reverse-phase high pressure liquid
chromatography. Two major phosphopeptides identified as amino acids
401-418 and 398-418 (6) were mixed and subjected to two-dimensional
phosphopeptide mapping analysis. C and D,
ERK-phosphorylated wild-type (C) and S412D (D)
-arrestin1 as described in Fig. 1 were digested with trypsin and
subjected to two-dimensional phosphopeptide mapping analysis as
described.
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|
Inhibition of
-Arrestin1 Phosphorylation in HEK 293 Cells
by a Dominant-negative MEK1 Inhibitor--
To investigate whether ERK1
and ERK2 mediate
-arrestin1 phosphorylation in cells, we employed a
dominant-negative MEK1(K97A) inhibitor (25) to determine whether
inhibition of ERK activity might affect
-arrestin1 phosphorylation.
Overexpression of the MEK1(K97A) mutant in HEK 293 cells significantly
reduced ERK phosphorylation (Fig. 3,
lower panel). This was associated with ~70% reduction of
-arrestin1 phosphorylation (Fig. 3, upper panel).
Increasing the level of activated ERKs with a constitutively active
S218D/S222D mutant of MEK1 (26) did not significantly elevate
-arrestin1 phosphorylation (data not shown), consistent with the
high stoichiometry of cellular phosphorylation of
-arrestin1 at
Ser-412 (0.85 mol Pi/mol protein) (6). These results
demonstrate that inhibition of ERK activation blocks
-arrestin1
phosphorylation in HEK 293 cells, thus further implicating ERKs as the
kinases responsible for phosphorylating
-arrestin1 in cells.

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Fig. 3.
Effect of the dominant-negative MEK1(K97A)
mutant on phosphorylation of -arrestin1 in HEK
293 cells. HEK 293 cells, transiently expressing His-tagged
-arrestin1 alone or with MEK1(K97A), were split onto two
plates each. Upper panel, one plate was metabolically
labeled with [32P]orthophosphate for 1 h.
-Arrestin1 was purified, resolved by SDS-PAGE, and transferred to
nitrocellulose membranes. After autoradiography, this membrane was
subjected to Western blot analysis using an antibody specific to
-arrestin1. Lower panel, equal amounts of whole cell
lysates from the other plate were fractionated by SDS-PAGE and
subjected to Western blot analysis using a specific anti-phospho-ERK
antibody. This blot was then stripped and reprobed with an ERK2
antibody to ensure equal expression of total cellular ERKs.
Overexpression of MEK1(K97A) was detected by an antibody specific to
MEK1.
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Regulation of
-Arrestin1 Function by Constitutively Active and
Dominant-negative Mutants of MEK1--
Previously we have shown that
dephosphorylation of
-arrestin1 at the plasma membrane is required
for clathrin binding and agonist-induced internalization of the
2-adrenergic receptor (6). Thus, it would be expected
that increasing the level of dephosphorylated
-arrestin1 in cells by
inhibiting ERK activation with the dominant-negative MEK1(K97A) mutant
would augment its clathrin binding ability and function in receptor
internalization. As shown in Fig.
4A, the dominant-negative
MEK1(K97A) mutant significantly enhances the co-immunoprecipitation of
wild-type
-arrestin1 with clathrin heavy chain. The S412A mutant of
-arrestin1, which mimics the dephosphorylated form of
-arrestin1,
also robustly co-immunoprecipitated clathrin (Fig. 4A). We
did not observe any effect of the constitutively active S218D/S222D
mutant of MEK1 on clathrin binding of
-arrestin1 (data not shown),
presumably because cellular
-arrestin1 is already so highly
phosphorylated that we could not detect its binding with clathrin.

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Fig. 4.
Effects of MEK1 mutants on clathrin binding
ability of -arrestin1 and
-arrestin-mediated sequestration of the
2-adrenergic receptors.
A, HEK 293 cells were transiently transfected with either an
empty vector (mock), a FLAG-tagged -arrestin1 expression vector
( arr1 or S412A) or co-transfected with the FLAG-tagged -arresitn1
and MEK1(K97A) plasmids. Equal amounts of proteins from the whole cell
lysates were immunoprecipitated (IP) with an antibody
directed at the FLAG epitope. After SDS-PAGE, the immunoblot was probed
with an antibody specific to clathrin heavy chain (HC) as
shown on the top panel. The lower four panels are
the immunoblots from 5% of the whole cell lysates probed with the
antibodies specific to either MEK1, phospho-ERKs, total cellular ERK2,
or -arrestin1. B, the 2-adrenergic
receptor ( 2-AR) expression plasmid was
transiently transfected into HEK 293 cells with a -arrestin
expression vector (mock, arr1, or
arr2) and a MEK1 mutant expression vector (empty vector,
dominant-negative K97A, or constitutively active S218D/S222D). Cells
were incubated with or without ( ) isoproterenol (ISO) for
30 min before harvesting. The assay of agonist-promoted receptor
sequestration was carried out as described. The results shown are the
means ± S.E. of three independent experiments done in
duplicate. At the right lower corner is a representative
Western blot showing the expression levels of phospho-ERKs and MEK1
mutants in the presence of MEK1(K97A) or MEK1(S218D/S222D).
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|
We further investigated the effect of dominant-negative K97A and
constitutively active S218D/S222D mutants of MEK1 on
-arrestin1-mediated sequestration of the
2-adrenergic
receptors. In the presence of the MEK1(K97A) mutant, receptor
sequestration was increased in control HEK 293 cells. It was further
promoted by overexpressing
-arrestin1 (Fig. 4B),
presumably because the level of active, dephosphorylated
-arrestin1
is highly increased by MEK1(K97A) mutant (as it is by S412A
-arrestin1 (6)). In contrast, the constitutively active S218D/S222D
mutant of MEK1 slightly reduced receptor sequestration in control
cells. This reduction was even more dramatic in cells overexpressing
-arrestin1 where receptor sequestration was now predominantly
mediated by transfected
-arrestin1 (in contrast to control cells
where both endogenous
-arrestin1 and 2 participate). In such cells,
levels of the phosphorylated
-arrestin1 are increased to such high
levels by the constitutively active MEK1 mutant that
phospho-
-arrestin1 now acts essentially as a dominant-negative
inhibitor of receptor internalization (as does S412D
-arrestin1
(6)).
To determine whether the effect of MEK mutants was specifically due to
altered
-arrestin1 function, we also tested their effects on
receptor sequestration mediated by
-arrestin2. Interestingly, this
enhancement was not significantly affected by either MEK1 mutant in
cells overexpressing
-arrestin2 (Fig. 4B). This result suggests that ERKs can modulate the function of
-arrestin1 but not
-arrestin2. Although
-arrestin2 is also a phosphoprotein in cells
(data not shown), it has no site corresponding to Ser-412 of
-arrestin1. This suggests that ERKs are not the kinases that phosphorylate
-arrestin2 in cells.
A Model for Negative Feedback Regulation of
-Arrestin1 Function
by ERK-mediated Phosphorylation--
Fig.
5 provides a model for the feedback
regulation of
-arrestin1 function by ERK-mediated phosphorylation of
Ser-412. Cytosolic
-arrestin1, which is predominately phosphorylated
at Ser-412 (6), is recruited to the plasma membrane upon agonist
stimulation. Membrane-bound
-arrestin1 is dephosphorylated by as yet
unknown phosphatases. Although dephosphorylation of
-arrestin1 is
not required for its receptor binding, it is required for several of
its other functions including Src recruitment (15) and clathrin binding
(6). These events in turn are necessary for GPCR-mediated activation of
the Ras-dependent ERK pathway (13). Once activated, the
ERKs are able to phosphorylate
-arrestin1 at Ser-412, thereby reducing these functions and, in a feedback regulatory fashion, reducing further ERK signaling.

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Fig. 5.
A model for the negative feedback regulation
of -arrestin1 function by ERK-mediated
phosphorylation. Cytosolic -arrestin1 is predominantly
phosphorylated at Ser-412. It is dephosphorylated when recruited to the
plasma membrane in response to agonist stimulation. Dephosphorylated
-arrestin1 binds to Src and also targets the agonist-occupied,
GRK-phosphorylated GPCR to the clathrin-coated pits for
internalization. ERK is activated subsequent to the receptor
internalization event. Afterward, the activated ERK phosphorylates
-arrestin1 at Ser-412, reduces its ability to bind Src (15) and
clathrin (6), and thereby attenuates ERK signaling. Ultimately the
receptors are dephosphorylated and recycled back to the plasma membrane
for resensitization. A, agonist; GRK2, G
protein-coupled receptor kinase 2; (+), stimulatory effect;
( ), inhibitory effect.
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|
 |
ACKNOWLEDGEMENTS |
The model shown in this paper was kindly
provided by Dr. Stuart Maudsley. The expression vectors of MEK1(K97A)
and MEK1(S218D/S222D) mutants were generous gifts from Dr. Edwin G. Krebs and Dr. Raymond. L. Erikson, respectively. We thank Drs. Yehia
Daaka, Julie A. Pitcher, and Randy Hall for helpful discussions. We
also thank Donna Addison and Mary Holben for excellent secretarial assistance.
 |
FOOTNOTES |
*
This work was supported by the Howard Hughes Medical
Institute and by Grant HL16037 from the National Institutes of Health.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.
Investigator of the Howard Hughes Medical Institute. To whom
correspondence should be addressed: Howard Hughes Medical Inst., Dept.
of Medicine and Biochemistry, Duke University Medical Center, Box 3821, Durham, NC 27710. E-mail: lefko001{at}mc.duke.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
GPCR, G
protein-coupled receptor;
ERK, extracellular signal-regulated kinase(s);
PAGE, polyacrylamide gel electrophoresis.
 |
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