Association of ß-Arrestin 1 with the Type 1A Angiotensin II Receptor Involves Phosphorylation of the Receptor Carboxyl Terminus and Correlates with Receptor Internalization
Hongwei Qian,
Luisa Pipolo and
Walter G. Thomas
Molecular Endocrinology Laboratory, Baker Medical Research
Institute, Melbourne 8008, Australia
Address all correspondence and requests for reprints to: Dr. Walter G. Thomas, Molecular Endocrinology Laboratory, Baker Medical Research Institute, PO Box 6492, St. Kilda Road Central, Melbourne Victoria 8008, Australia. E-mail: walter.thomas{at}baker.edu.au
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ABSTRACT
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Arrestins bind to phosphorylated G protein-coupled receptors
and participate in receptor desensitization and endocytosis. Although
arrestins traffic with activated type 1 (AT1A) angiotensin
II (AngII) receptors, the contribution of arrestins to AT1A
receptor internalization is controversial, and the physical association
of arrestins with the AT1A receptor has not been
established. In this study, by coimmunoprecipitating AT1A
receptors and ß-arrestin 1, we provide direct evidence for an
association between arrestins and the AT1A receptor that
was agonist- and time-dependent and contingent upon the level of
ß-arrestin 1 expression. Serial truncation of the receptor carboxyl
terminus resulted in a graded loss of ß-arrestin 1 association, which
correlated with decreases in receptor phosphorylation. Truncation of
the AT1A receptor to lysine325 prevented
AngII-induced phosphorylation and ß-arrestin 1 association as well as
markedly inhibiting receptor internalization, indicating a close
correlation between these receptor parameters. AngII-induced
association was also dramatically reduced in a phosphorylation- and
internalization-impaired receptor mutant in which four serine and
threonine residues in the central portion of the AT1A
receptor carboxyl terminus (Thr332, Ser335,
Thr336, Ser338) were substituted with alanine.
In contrast, substitutions in another serine/threonine-rich region
(Ser346, Ser347, Ser348) and at
three PKC phosphorylation sites (Ser331,
Ser338, Ser348) had no effect on AngII-induced
ß-arrestin 1 association or receptor internalization. While
AT1A receptor internalization could be inhibited
by a dominant-negative ß-arrestin 1 mutant
(ßarr1319418), treatment with hyperosmotic
sucrose to inhibit internalization did not abrogate the differences in
arrestin association observed between the wild-type and mutant
receptors, indicating that arrestin binding precedes, and is not
dependent upon, receptor internalization. Interestingly, a
substituted analog of AngII,
[Sar1Ile4Ile8]-AngII, which
promotes robust phosphorylation of the receptor but does not activate
receptor signaling, stimulated strong ß-arrestin 1 association with
the full-length AT1A receptor. These results identify the
central portion of the AT1A receptor carboxyl terminus as
the important determinant for ß-arrestin 1 binding and
internalization and indicate that AT1A receptor
phosphorylation is crucial for ß-arrestin docking.
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INTRODUCTION
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ß-ARRESTINS WERE IDENTIFIED as proteins
that associated with the phosphorylated form of the
ß2-adrenergic receptor (1)
and quenched signals by sterically hindering the coupling of receptor
to heterotrimeric G protein. A family of arrestin proteins have been
identified [arrestin1 = visual arrestin; arrestin 2 =
ß-arrestin 1 (ßarr1); arrestin 3 = ß-arrestin 2 (ßarr2)],
and they play an important role in regulating the activity of the large
superfamily of G protein-coupled receptors (GPCRs) (2).
High resolution x-ray structures of arrestin (3, 4),
combined with extensive mutagenesis and in vitro assays,
indicate that phosphorylation within the carboxyl-terminal region of
GPCRs acts to disrupt the inactive state of arrestin (5, 6). Ensuing structural rearrangements in arrestin then permit
interactions with additional determinants within the receptor that, in
effect, shield the receptor from further association with G
protein.
In addition to modulating G protein coupling, arrestins can
also bind to components of the internalization machinery, such as
clathrin (7) and adaptins (8), and may act as
adaptors to promote receptor endocytosis. Accordingly, overexpression
of arrestin dominant-negative mutants or arrestin fragments inhibits
the endocytosis of some, but not all, GPCRs (9, 10, 11, 12). In
contrast, overexpression of wild-type ßarr1 or ßarr2 can enhance
the internalization of coexpressed GPCRs (9, 13).
Moreover, the use of green fluorescent protein (GFP)-tagged arrestins
and confocal microscopy has demonstrated that arrestins are recruited
to the cell membrane after receptor activation and are then either
trafficked with receptors to intracellular vesicles or rapidly
separated from internalizing receptors and retained near the cell
membrane (14, 15, 16). Serine- and threonine-rich regions
within the carboxyl terminus of GPCRs appear to control this selective
trafficking of arrestins (14) and whether a particular
GPCR internalizes in an arrestin-, dynamin-, and clathrin-sensitive
manner may be governed by additional regulators, such as the
phosphoprotein, EBP50 (17), GIT1 [GPCR
kinase(GRK)-interactor 1] (18),
N-ethylmaleimide-sensitive protein (19), and
ADP ribosylation factors (20), or by receptor
heterodimerization (21). For some GPCRs, arrestins may
also couple the activated receptors to tyrosine kinase and MAPK
pathways during the process of receptor internalization (22, 23). Thus, arrestins play a central role in GPCR activation and
deactivation.
Angiotensin II (AngII) is an important cardiovascular peptide hormone,
the actions of which are mediated primarily by the type 1 angiotensin
receptor (AT1). The AT1
receptor, which has two subtypes (AT1A and
AT1B) in rodents, is a GPCR that couples
primarily through G
q/11 to activate PLC,
which stimulates PKC and releases calcium from intracellular stores.
After activation, responses to AngII are rapidly terminated, presumably
by receptor phosphorylation and internalization (24). The
carboxyl terminus of the AT1A receptor is the
site of receptor phosphorylation (25, 26) by GRKs and PKC
(27, 28, 29) and is crucial for AngII-mediated receptor
internalization (30, 31). The relationship between
receptor phosphorylation, putative arrestin binding, internalization,
and receptor function remains unclear, and recent evidence from
constitutively active AT1A receptors and
activation-selective analogs of AngII suggests that the processes of
signaling, phosphorylation, and internalization can be differentiated,
supporting the concept of multiple receptor transition states
(32).
The contribution of arrestins to AT1A receptor
desensitization and internalization is also unclear. While
overexpressed arrestins can enhance AT1A receptor
sequestration (10), dominant-negative mutants of arrestin
have little effect on (10), or cause dramatic inhibition
of (33), AT1A receptor endocytosis.
Indirect evidence for an AngII-induced association of the
AT1A receptor and arrestins comes from the
impressive immunofluorescent colocalization of
AT1A receptors and trafficking GFP-linked ßarr1
and ßarr2 (14, 34, 35). ßarr2-GFP was shown to rapidly
translocate from the cytoplasm to the cell membrane in response to
AT1A receptor activation and then colocalize with
the receptor in endocytic vesicles after extended stimulation via a
mechanism that depended upon the AT1A receptor
carboxyl terminus. Whether arrestins contribute to
AT1A receptor endocytosis or desensitization
(35, 36) or if they merely traffic with the internalizing
receptors remains to be established. Also unresolved is whether
arrestins can physically associate with the AT1A
receptor and the involvement of the AT1A carboxyl
terminus, and its phosphorylation, to this process. Hence, in this
study, we investigated the direct association of ßarr1 with
AngII-activated AT1A receptors and examined the
contribution of the receptor carboxyl terminus, activation status, and
receptor phosphorylation.
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RESULTS
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Assay for AT1A Receptor-ßarr1 Association
To determine whether ßarr1 associates with wild-type and
mutated AT1A receptors (see Fig. 1
), our strategy was to coexpress
hemagglutin (HA)-tagged AT1A receptors and
FLAG-tagged ßarr1 in CHO-K1 cells. After AngII stimulation,
closely associated cellular proteins were covalently cross-linked
with a membrane-permeable, cleavable agent
dithiobis(succinimidyl)propionate (DSP) and
AT1A receptors were immunoprecipitated from cell
lysates with anti-HA monoclonal antibody. Immunoprecipitates were
then Western blotted and probed with an anti-FLAG antibody to detect
coimmunoprecipitation of ßarr1 with the stimulated
AT1A receptor. In preliminary experiments, we
made the unexpected observation (data not shown) that overexpressed
ßarr1 coimmunoprecipitated equally well with the wild-type
NHA-AT1A receptor and a truncated mutant,
NHA-TK325, in which all carboxyl-terminal serine and threonine residues
are removed and which is not phosphorylated in response to AngII
(25). This unanticipated result was also peculiar because
the association was not regulated by AngII stimulation for either
receptor.

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Figure 1. The AT1A Carboxyl Terminus and Various
Truncated and Site-Directed Mutants
Wild-type and mutant receptors were engineered to include an HA epitope
tag at the N terminus (prefix, NHA-) to permit immunoprecipitation. The
top sequence is the carboxyl terminus of the wild-type
AT1A receptor (Leu305) through
Glu359, indicating the boundary of the seventh
transmembrane-spanning helix (TM7) and the start of the carboxyl
terminus (C-T); asterisks label 13 carboxyl-terminal
serine and threonine residues. The three truncated mutants (NHA-TD343,
NHA-TK333, and NHA-TK325) are terminated after Asp343,
Lys333, and Lys325, respectively. Triple or
quadruple alanine mutations (bold/underlined) were
introduced into the carboxyl-terminal region of the AT1A
receptor at
Thr332/Ser335/Thr336/Ser338/Ala,
Ser346/Ser347/Ser348/Ala, and
Ser331/Ser338/Ser348/Ala, which are
abbreviated as NHA-TSTS/A, NHA-SSS/A, and NHA-PKC(SSS)/A,
respectively.
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The constitutive association of ßarr1 with both the wild-type and
truncated AT1A receptors suggested an affinity of
ßarr1 for the basal state of the AT1A receptor,
which was exacerbated by ßarr1 overexpression and was independent of
receptor activation, phosphorylation, or the presence of the carboxyl
terminus. In an attempt to establish a level of ßarr1 expression at
which constitutive (i.e. non-agonist-induced) association
was minimized, we cotransfected variable amounts of FLAG-tagged ßarr1
(0, 0.003, 0.01, 0.03, 0.1, and 0.3 µg DNA/well) into CHO-K1 cells
with a constant amount (0.3 µg DNA/well) of HA-tagged wild-type
AT1A receptor (NHA-AT1A).
Without stimulation by AngII, the transfected cells were directly
treated with DSP for cross-linking, followed by detergent lysis and
receptor immunoprecipitation. Increasing ßarr1 expression was
confirmed by Western blot analysis of cell extracts that were removed
before receptor immunoprecipitation (Fig. 2
, top panel). Detection of
ßarr1 after AT1A receptor immunoprecipitation
(Fig. 2
, middle panel) revealed that, at the highest ßarr1
DNA concentrations (0.1 and 0.3 µg DNA/well), significant amounts of
ßarr1 were coimmunoprecipitated with the unstimulated receptor. Equal
AT1A receptor expression was confirmed by
reprobing with an HA antibody (Fig. 2
, bottom panel).

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Figure 2. Variable Expression of ßarr1 Reveals a
Nonspecific Binding of ßarr1 to the AT1A Receptor
HA-tagged wild-type AT1A receptor (0.3 µg;
NHA-AT1A), a variable amount of ßarr1 (0, 0.003, 0.01,
0.03, 0.1, and 0.3 µg), and empty vector (PCDNA3.1) to a total of 0.6
µg plasmid DNA per well was simultaneously cotransfected into CHO-K1
cells. Without AngII stimulation, the transfected cells were directly
treated with DSP for cross-linking, followed by detergent lysis and
AT1A receptor immunoprecipitation. Ten microliters of cell
extracts, removed before immunoprecipitation, were Western blotted (WB)
and probed with antibodies to the FLAG-epitope (anti-FLAG) to confirm
variable expression of ßarr1 (top panel).
AT1A receptors were immunoprecipitated (IP) with anti-HA
monoclonal antibodies (anti-HA), and immunoprecipitates were resolved
on SDS-PAGE, Western blotted (WB), and probed with biotinylated
anti-FLAG antibodies (anti-FLAG-bio) to determine whether ßarr1 was
coimmunoprecipitated with the receptor (middle panel).
Blots were then stripped and reprobed with antibody to the HA-tag
(anti-HA, 3F10) to confirm receptor expression and immunoprecipitation
(bottom panel). A representative blot from three
separate experiments is shown.
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Agonist-Dependent Association of ßarr1 with the AT1A
Receptor
Having established a level of ßarr1 expression (<0.1 µg
DNA/well) that minimized the amount of agonist-independent association,
we next investigated whether AngII could induced
ßarr1-AT1A receptor association and determined
if the carboxyl terminus was involved in the association. For this,
ßarr1 (0.06 µg DNA/well) was transiently cotransfected with vector,
HA-tagged wild-type AT1A receptor
(NHA-AT1A, 0.30 µg DNA/well) or NHA-TK325 (0.30
µg DNA/well). Forty eight hours later, the transfected cells were
stimulated (or not stimulated) with AngII (100 nM, 10 min)
followed by DSP cross-linking, immunoprecipitation, and Western
blotting. Figure 3A
(top
panel) shows that when cells were cotransfected with both
AT1A receptor and ßarr1, the amount of ßarr1
coimmunoprecipitated with the receptor was significantly increased
after agonist stimulation. Truncation of the AT1A
receptor (NHA-TK325) abolished AngII-induced arrestin association. Both
the wild-type and truncated receptor, together with ßarr1, displayed
a similar level of expression among the different groups tested (Fig. 3A
, middle and bottom panels).

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Figure 3. Association of ßarr1 with the AT1A
Receptor After Agonist Stimulation Depends on the Carboxyl Terminus and
Is Time Dependent
A, CHO-K1 cells were cotransfected with HA-tagged wild-type
AT1A receptor (NHA-AT1A) or truncated
AT1A receptor (NHA-TK325) and ßarr1 (0.06 µg DNA/well).
Cells were stimulated with AngII (100 nM) for 10 min,
followed by cross-linking with DSP, detergent lysis, and
immunoprecipitation (IP) of AT1A receptors.
Immunoprecipitates were resolved on SDS-PAGE, Western blotted (WB), and
probed with biotinylated anti-FLAG antibodies (anti-FLAG-bio) to
visualize ßarr1 coimmunoprecipitation with the receptor (top
panel). Blots were stripped and reprobed with anti-HA antibody
(3F10) to confirmed receptor expression (middle panel),
and samples of cell extracts before immunoprecipitation were probed
with anti-FLAG antibodies to confirm equal expression of ßarr1
between groups (bottom panel). B, Cells coexpressing the
wild-type NHA-AT1A receptor and ßarr1 were stimulated
with 100 nM AngII for the indicated time and processed for
receptor immunoprecipitation and probed with anti-FLAG antibodies.
Results are representative blots from four separate experiments.
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Next, we determined whether the agonist-regulated association of
ßarr1 with the receptor was time-dependent. Cells cotransfected
with ßarr1 and wild-type AT1A receptor were
treated with AngII for 0, 0.5, 1, 5, 10, and 30 min, respectively, and
ßarr1 coimmunoprecipitated with the receptor at each time point.
Figure 3B
shows that the level of the association was slightly
increased above basal at 0.5 min, the earliest time point tested,
followed by a greater increase at 1 min; maximal association was seen
at 5 min, and this was maintained to 30 min. The kinetics of ßarr1
association correlate well with the phosphorylation and internalization
(Ref. 25 and Fig. 4
, C and
D) kinetics previously determined for the AT1A
receptor. Based on this experiment and previous information, we used a
maximal 10-min stimulation with AngII for further
ßarr1-AT1A receptor association experiments.
Together, these results demonstrate that at moderate levels of
expression, ßarr1 can associate with the AT1A
receptor in an agonist-, time-, and carboxyl-terminal-dependent
manner.

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Figure 4. The Central Region of the AT1A Receptor
Carboxyl Terminus Is Important for AngII-Stimulated ßarr1 Association
A, CHO-K1 cells were cotransfected with vector, HA-tagged wild-type
AT1A receptor (NHA-AT1A), or various truncated
versions of the receptor (NHA-TD343, NHA-TK333 and NHA-TK325), and
FLAG-tagged ßarr1. The transfected cells were stimulated (or not
stimulated) with AngII (100 nM, 10 min) followed by DSP
cross-linking, detergent lysis, and immunoprecipitation (IP) of
AT1A receptors and probed for ßarr1 coimmunoprecipitation
(top panel). Blots were stripped and reprobed to confirm
receptor expression (middle panel), and cell extracts
were tested for ßarr1 expression (bottom panel). B,
AngII-induced ßarr1 association for the various receptor constructs
was quantified from four experiments and presented as means ±
SE. (**, P < 0.001 compared with
wild-type receptor, NHA-AT1A). C, Receptor phosphorylation
determined by incorporation of 32P into receptors after
AngII stimulation (100 nM, 10 min) and immunoprecipitation,
SDS-PAGE, and phosphoimaging. Data are representative of three separate
experiments. D, Internalization kinetics for wild-type and truncated
receptors determined by acid-sensitive binding of
125I-AngII. Results are the means ± SE
from three experiments.
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The ßarr1-AT1A Receptor Interaction Involves
Phosphorylation of the Receptor Carboxyl Terminus
To further analyze the contribution of the
AT1A receptor carboxyl terminus to ßarr1
association, we compared ßarr1 binding to full-length receptors and a
series of mutant receptors truncated at the carboxyl terminus (listed
in Fig. 1
). HA-tagged wild-type AT1A receptor
(NHA-AT1A), NHA-TD343 with a deletion of 16 amino
acids, NHA-TK333 with a deletion of 26 amino acids, or NHA-TK325 with a
deletion of 34 amino acids were transiently cotransfected with ßarr1
into CHO-K1 cells. The transfected cells were stimulated (or not
stimulated) with AngII (100 nM, 10 min), and the receptors
were immunoprecipitated and probed for ßarr1 association. As shown in
Fig. 4A
(top panel), serial truncation at the carboxyl
terminus caused a commensurate decrease in ßarr1 association with the
receptor. The binding of ßarr1 to the receptors was quantified and is
shown in Fig. 4B
. NHA-TD343, with a truncation to
Asp343, showed a reduction in the association
that was not statistically different from that of wild-type receptor.
However, the association of ßarr1 with the NHA-TD333 and NHA-TK325
mutants was significantly reduced by greater than 70% and 85%,
respectively. The results suggest that the carboxyl terminus of the
AT1A receptor is an important determinant for
ßarr1 binding. Interestingly, the level of association correlated
with the degree of receptor phosphorylation (Refs. 25 and
26 and Fig. 4C
) and internalization (Refs. 25
and 26 and Fig. 4D
) in that the more truncated mutants
(NHA-TK333 and NHA-TK325) showed dramatically reduced phosphorylation,
arrestin binding, and internalization compared with the wild-type and
NHA-TD343 receptors.
Serine/threonine-rich clusters at an appropriate distance (3040 amino
acids) from the cell membrane in the carboxyl terminus of GPCRs play an
important role in ß-arrestin trafficking to the receptor, presumably
through direct interaction between phosphorylated receptor and
ß-arrestin (34). Oakley et al.
(34) nominated two regions in the
AT1A receptor
(Thr332Ser335Thr336Ser338
and
Ser346Ser347Ser348)
as potential candidates for ß-arrestin binding. To test the
importance of these separate sites on arrestin binding, we examined
ßarr1 coimmunoprecipitation for wild-type receptor and mutated
receptors (NHA-TSTS/A, NHA-SSS/A). In addition, we and others
(27, 28, 29, 37, 38) have previously demonstrated that PKC is
a major kinase that phosphorylates and regulates the
AT1A receptor. A combined mutation of
Ser331, Ser333, and
Ser338 to alanine [NHA-PKC(SSS/A)] causes about
60% decrease in AngII-dependent phosphorylation (29).
Hence, we also tested the binding of arrestin to NHA-PKC(SSS/A). As
shown in Fig. 5
, A and B, compared with
the wild-type AT1A receptor, the level of
AngII-stimulated binding of ßarr1 above basal was similar in the
NHA-SSS/A mutant, was slightly, but not significantly, decreased in the
NHA-PKC(SSS/A) mutant, and was dramatically reduced by about 84% in
the NHA-TSTS/A mutant. The expression level of either receptor
(wild-type and mutants) or ßarr1 were similar among the different
groups (Fig. 5A
, middle and bottom panel). The impaired
capacity of the NHA-TSTS/A mutant suggests that
Thr332, Ser335,
Thr336, and Ser338 at the
carboxyl terminus of the AT1A receptor is a
critical site for ßarr1-receptor interaction. Previously, we have
shown that the NHA-TSTS/A mutant displays a markedly decreased
AngII-stimulated receptor phosphorylation (Ref. 25 and
Fig. 5C
) and significantly reduced receptor internalization (Ref.
25 and Fig. 5D
). For the NHA-PKC(SSS/A) mutant,
phosphorylation was reduced by about 60% but this receptor displays
full internalization (Ref. 29 and Fig. 5D
). In addition,
the NHA-SSS/A mutant showed an equivalent phosphorylation and
internalization to the wild-type AT1A receptor
after AngII stimulation (Fig. 5
, C and D). Taken together, and despite
the caveat that the efficiency of DSP cross-linking may differ for the
various AT1A receptor mutants due to slight
differences in the relative conformation of intracellular regions,
these results strongly indicate that the region from
Thr332 to Ser338 is most
important for ß-arrestin association and receptor internalization,
presumably via phosphorylation in this region.

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Figure 5. Effect of Mutating Serine- and Threonine-Rich
Regions of the AT1A Receptor Carboxyl Terminus on
AngII-Induced ßarr1 Binding, Receptor Phosphorylation, and
Internalization
A, The association of ßarr1 with wild-type (NHA-AT1A) and
carboxyl-terminally mutated receptors was examined in response to AngII
stimulation (100 nM, 10 min) (top panel),
and receptor expression was confirmed by probing for the HA epitope
(middle panel). ßarr1 expression was examined in cell
extracts before immunoprecipitation (bottom panel). B,
AngII-induced ßarr1 association for the various receptor constructs
was quantified from seven experiments and presented as means ±
SE (*, P < 0.05 compared with
wild-type receptor, NHA-AT1A). C, Receptor phosphorylation
determined by incorporation of 32P into receptors after
AngII-stimulation (100 nM, 10 min) and immunoprecipitation,
SDS-PAGE, and phosphoimaging. Data are representative of three separate
experiments. D, Internalization kinetics for wild-type and truncated
receptors determined by acid-sensitive binding of
125I-AngII. Results are the means ± SE
from three experiments.
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Correlation and Independence of AT1A Endocytosis and
ßarr Association
The above data are consistent with the idea that the central
carboxyl terminus is phosphorylated after AngII stimulation and serves
to bind arrestin and mediate AT1A receptor
internalization. However, because the various
AT1A receptor mutants tested for arrestin binding
possess differing capacities to internalize, one alternative
interpretation could be that the internalization process itself
stabilizes the receptor-ßarr1 complex. Hence, receptor mutants with
affected internalization may have reduced ßarr association. To
examine this, we sought to inhibit wild-type and mutant
AT1A receptor internalization and examine ßarr1
association. If internalization is necessary to stabilize ßarr1
binding, then we would predict that abolishing internalization with
hyperosmotic sucrose (39, 40) would inhibit
AT1A-ßarr1 association and abrogate the
differences observed between the wild-type and mutant receptors. As
shown in Fig. 6A
, pretreatment of cells
expressing wild-type and mutated AT1A receptors
with sucrose (0.45 M, 30 min, 37 C) led to a marked and
equivalent inhibition of receptor internalization. In contrast, sucrose
treatment, while having a slight inhibitory effect on overall arrestin
binding (not shown), did not alter the pattern of differences in
arrestin association between the wild-type and mutant receptors. Thus,
these data suggest that internalization is not obligate for arrestin
binding, although we cannot rule out some stabilization of arrestin
binding during internalization.

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Figure 6. Differences in AngII-Induced Arrestin Association
Between Wild-Type and Mutant AT1A Receptors Persists in the
Presence of Hyperosmolar Sucrose That Blocks AT1A Receptor
Internalization
A, CHO-K1 cells transfected with wild-type or mutant AT1A
receptors were treated with 0.45 M sucrose at 37 C for 30
min before determination of receptor internalization by acid-sensitive
binding of 125I-AngII (1 nM, 10 min). Results
are the means ± SE (n = 3). B, CHO-K1 cells
cotransfected with wild-type or mutant AT1A receptor and
FLAG-tagged ßarr1 were treated with 0.45 M sucrose (37 C,
30 min) before AngII stimulation (100 nM, 10 min). After
cross-linking with DSP and detergent lysis, AT1A receptors
were immunoprecipitated (IP) with anti-HA antibody and resolved on
SDS-PAGE, Western blotted (WB), and then probed with biotinylated
anti-FLAG antibodies (anti-FLAG-bio) to visualize ßarr1
coimmunoprecipitation with the receptor. Data are representative of
three experiments.
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Dominant-negative mutants of arrestin have been used to inhibit
endogenous arrestin action and identify an arrestin-mediating endocytic
pathway. Zhang et al. (10) reported that
ßarr1V53D inhibited
ß2-adrenergic but not AT1
receptor internalization in HEK-293 cells. In contrast, very recently,
Gáborik et al. (33) demonstrated that the
overexpressed ßarr1V53D in CHO-K1 cells
inhibited AT1A receptor internalization.
Moreover, expression of ßarr11349 markedly
inhibited AT1 receptor internalization. To
further examine whether AT1 receptor
internalization is arrestin/clathrin mediated, we constructed
ßarr1319418, a mutant that lacks a capacity
to bind to receptor but possesses motifs for interaction with AP2
(41) and clathrin (11), thereby inhibiting
the capacity of endogenous arrestin to functionally couple to
endocytosis. As shown in Fig. 7
, when
ßarr319418 was cotransfected with HA-tagged
wild-type AT1A receptor
(NHA-AT1A) in CHO-K1 cells, the overexpressed
ßarr319418 markedly reduced
AT1A receptor internalization. Thus, our
immunoprecipitation (Figs. 3
, 4
, and 5
) and
ßarr1319418 inhibitory (Fig. 7
) data,
combined with those of Gáborik et al.
(33), strongly suggest that the AT1
receptor binds to arrestin to internalize via a clathrin-dependent
pathway.

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Figure 7. ßarr1319418 Inhibits
Internalization of the AT1A Receptor
A, Internalization kinetics (acid-sensitive binding of
125I-AngII) were determined for CHO-K1 cells cotransfected
with wild-type AT1A receptor (NHA-AT1A) and
either vector (pcDNA6/Myc-His A) or the
carboxyl-terminal arrestin fragment (ßarr1319418);
results are the means ± SE (n = 4). B,
Expression of full-length ßarr1 and ßarr1319418
confirmed by Western blotting. Cell extracts (5 µl) were Western
blotted (WB) and probed with anti-FLAG antibodies for wild-type ßarr1
and with anti-MYC antibodies for ßarr1319418.
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[Sar1Ile4Ile8]-AngII Promotes
AT1A-ßarr1 Association
The AT1A receptor is thought to transit
through multiple conformational states in the process of receptor
activation and regulation (i.e. signaling, phosphorylation,
and internalization) (32). We have shown previously that
an analog of AngII,
[Sar1Ile4Ile8-AngII,
that does not activate signaling through the inositol phosphate
pathway, can nevertheless induce a robust (
85% of AngII)
phosphorylation of the wild-type AT1A receptor
(32). To examine whether receptor phosphorylation is
sufficient for ß-arrestin association, we tested the capacity of
[Sar1Ile4Ile8]-AngII
to induce an association of ßarr1 with the wild-type
AT1A receptor. Shown in Fig. 8
is the association of ßarr1 with the
wild-type AT1A receptor stimulated by either
AngII or
[Sar1Ile4Ile8]-AngII
at concentrations 100 times the dissociation constant for the receptor
(100 nM for AngII and 30
µM for
[Sar1Ile4Ile8]-AngII).
Both AngII and
[Sar1Ile4Ile8]-AngII
induced ßarr1 binding to the receptor. Compared with AngII,
[Sar1Ile4Ile8]-AngII
stimulated slightly less ßarr1 association (Fig. 8A
), although this
decrease was not statistically significant, which parallels the
slightly reduced receptor phosphorylation in response to this analog
(32). The physical association of ßarr1 with the
AT1A receptor induced by
[Sar1Ile4Ile8]-AngII
suggests that receptor phosphorylation is sufficient for ßarr1
binding, although the active conformation of the receptor presumably
enhances the affinity of the interaction.

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|
Figure 8. Receptor Phosphorylation Is Sufficient to Promote
ßarr1 Binding to the AT1A Receptor
A, ßarr1 association with the wild-type receptor
(NHA-AT1A) was determined in response to AngII (100
nM) or
[Sar1Ile4Ile8]-AngII (30
µM) stimulation for 10 min. B, Quantification of ßarr1
association from 14 separate experiments is presented as means ±
SE. The slightly reduced association after
[Sar1Ile4Ile8]-AngII stimulation
was not significant, compared with AngII.
|
|
 |
DISCUSSION
|
---|
The major finding of the present study is that ßarr1 associates
with the AT1A receptor after AngII stimulation.
To stabilize the receptor-arrestin complex during immunoprecipitation,
we used a covalent, reversible cross-linking agent, DSP, as previously
described (22, 42), which considerably improved the
reproducibility of the procedure. Interestingly, vast overexpression of
ßarr1 initially masked the agonist- and time-dependent association of
receptor and ß-arrestin. This nonspecific association (in the absence
of AngII stimulation) could be alleviated by careful titration of
cellular ßarr1 levels to reveal a robust, carboxyl-terminal- and
phosphorylation-dependent binding of arrestin to the activated
AT1A receptor. Such nonspecific binding of
arrestin to the basal (inactive) state of the receptor has been
reported (5). Its lack of regulation, in our study, by
receptor stimulation or the carboxyl terminus, indicates an inherent
affinity of ß-arrestin for the cytoplasmic loops of the
AT1A receptor. As cautioned by Mundell and
Benovic (36), and exemplified by our observations, care
should be taken in the interpretation of results from studies in which
arrestin function is elucidated from untitrated arrestin
overexpression.
Having established a reproducible coimmunoprecipitation approach for
examining AngII-stimulated ßarr1 binding to the
AT1A receptor, we next explored the contribution
of the receptor carboxyl terminus and its phosphorylation to this
association. Truncation of the AT1A receptor to
remove only 16 amino acids from the receptor carboxyl-terminus did not
significantly reduce AngII-induced arrestin binding compared with the
full-length receptor, and this correlated with little effect of this
truncation on receptor phosphorylation or internalization. In contrast,
larger truncations of the receptor tail (to
Lys333 and to Lys325)
produced proportionally greater decreases in ßarr1 association with
corresponding reductions in receptor phosphorylation and
internalization. Together, these results indicate a high degree of
correlation between the level of receptor phosphorylation and the
degree of ßarr1 binding and identify the central region of the
carboxyl terminus (Lys333 to
Asp343) as most important for AngII-induced
phosphorylation and ß-arrestin binding, as well as internalization.
While we and others have previously identified this central region as
important for phosphorylation and internalization (25, 26), the present study is the first to correlate these
regulatory aspects with the degree of ß-arrestin binding. This
correlation, as well as the rapid time-dependent ßarr1 association,
the persistence of ßarr1-AT1A interactions in
the presence of hyperosmotic sucrose that destabilizes clathrin cages
and prevents internalization, and the inhibition of
AT1A receptor endocytosis by the
ßarr1319418 mutant, strongly implicates
AngII-induced ß-arrestin binding in the initiation and/or support of
AT1A receptor endocytosis.
Oakley et al. (34) recently demonstrated that a
ß-arrestin-GFP chimera trafficked to the plasma membrane and then
into endocytic vesicles with activated V2 vasopressin receptors, while
the same chimera was retained near the cell membrane and did not follow
the activated ß2-adrenergic receptor into
endosomes. The endocytic translocation of ß-arrestin was contingent
upon clusters of phosphorylated serine/threonine residues within the V2
receptor carboxyl terminus. Swapping of carboxyl termini between the V2
and ß2 receptors revealed that both the
presence and the contextual placement of these clusters (3040 amino
acids relative to the cytoplasmic face of the membrane) was critical
for ß-arrestin endocytic trafficking as well as a resistance to
receptor dephosphorylation, recycling, and resensitization. Moreover,
such clusters were noted in appropriate positions in the carboxyl
termini of the neurotensin 1 and the AT1A
receptors (34), which, like the V2 vasopressin receptor,
also traffic ß-arrestins into endocytic vesicles (14).
Thus, a major aim of our study was to examine the relative
contribution of these two potential candidate clusters
(Thr332Ser335Thr336Ser338
and
Ser346Ser347Ser348)
to arrestin binding. We therefore tested alanine mutants of these
regions (NHA-TSTS/A and NHA-SSS/A, respectively) for their capacity to
inhibit AngII-induced ßarr1-receptor association as well as receptor
phosphorylation and internalization. We observed dramatic decreases in
ßarr1 binding to the NHA-TSTS/A mutant that correlated with a similar
large inhibition of AngII-stimulated phosphorylation and
internalization. In contrast, mutations of the more distal serine
cluster
(Ser346Ser347Ser348)
did not significantly affect ßarr1 binding or receptor
phosphorylation and internalization. Consistent with the idea that a
GRK- (not PKC-) mediated phosphorylation in the central portion of the
receptor carboxyl terminus recruits arrestins to activated
AT1A receptors, a receptor mutated at three PKC
phosphorylation sites [NHA-PKC(SSS/A)] retained full capacity for
ßarr1 binding. As previously noted (29), substitution of
the PKC phosphorylation sites significantly inhibits AngII-induced
receptor phosphorylation (see also Fig. 6
), suggesting that the
reported role of PKC phosphorylation on receptor regulation and
desensitization (27, 28, 29, 37, 38) may occur independently
of ß-arrestin. Indeed, the NHA-PKC(SSS/A) mutant displays
wild-type-like receptor internalization (Ref. 29 ; see also
Fig. 6
). Together, these results identify the central region of the
AT1A carboxyl terminus
(Thr332Ser335Thr336Ser338)
as the crucial determinant for GRK-mediated phosphorylation,
ß-arrestin binding, and internalization.
As introduced earlier, ambiguity has existed as to the role of
arrestins and other mediators of clathrin-mediated endocytosis
(e.g. the guanosine triphosphatase, dynamin) in
AT1A receptor internalization. Dominant-negative
mutants of ß-arrestin (ßarr1V53D) and dynamin
(K44A), which inhibit the internalization of other GPCRs, were reported
to have marginal effects on AT1A receptor
internalization (10), suggesting that the
AT1A receptor internalizes independently of
ß-arrestin and dynamin. Recently, Claing et al.
(18) suggested that GPCR internalization can be
finger-printed based on sensitivity to overexpression of the GIT1
protein, ß-arrestin, and dynamin dominant-negative mutants and by
inhibitors of clathrin-cage formation. Internalization of the
AT1A receptor was insensitive to GIT1
overexpression, which correlated with an apparent divergence from the
arrestin-, dynamin-, and clathrin-dependent pathway. Finally, an
arrestin-antisense strategy was ineffective in blocking desensitization
of an endogenous AT1A
receptor-G
i signal in HEK293 cells
(36). In direct contrast, a series of recent studies
(14, 33, 35, 43, 44), including the present study, argue
persuasively in favor of an arrestin-, dynamin-, and clathrin-dependent
mechanism of AT1A receptor internalization.
Indeed, there is irrefutable evidence from confocal microscopy
(14, 35, 43) for an AngII- and
AT1A-directed trafficking of ßarr2-GFP in
HEK293 cells. ßarr2-GFP colocalized with an epitope-tagged
AT1A receptor and both translocated to a common
endocytic vesicle (14). That this trafficking was
dependent on the AT1A receptor carboxyl terminus
was convincingly demonstrated using a chimeric
ß2-adrenergic receptor containing the
AT1A receptor tail, which gained the capacity to
translocate ßarr2-GFP to endocytic vesicles, in contrast to the
parent ß2-adrenergic receptor. Moreover, AngII
stimulation of carboxyl terminally truncated AT1A
receptors led to ßarr2-GFP translocation to plasma membrane-localized
coated pits but did not promote redistribution to endosomes
(35). In the same study (35), agonist
(isoproterenol)-induced coimmunoprecipitation from endocytic vesicles
of ßarr2-GFP with a chimeric ß2-adrenergic
receptor containing the AT1A receptor carboxyl
terminus was reported. This chimeric receptor also displayed
agonist-independent internalization that could be significantly
inhibited by a dominant-negative arrestin mutant
(ßarr1185418). Moreover, dominant-negative
mutants of arrestin (ßarr1V53D and
ßarr11349) as well as guanosine
triphosphatase- and phosphatidylinositol-insensitive versions of
dynamin have been recently reported to dramatically inhibit
AngII-stimulated internalization of the wild-type
AT1A receptor (33, 44). Finally,
cellular context may also be important given that the dynamin I K44A
mutant can indeed potently inhibit AT1A
internalization when the AT1A receptor is
coexpressed with the bradykinin B2 receptor, ostensibly as a
consequence of AT1A/B2 receptor
heterodimerization (21). In support of these studies, our
data strongly suggest that ß-arrestin can directly interact with the
AT1A receptor (via phosphorylation of the central
carboxyl terminus) and thereby regulate receptor endocytosis,
presumably via an arrestin-directed route. Our demonstration that
AT1A receptor internalization is inhibited by
coexpression of ßarr1319418 is also
consistent with an arrestin-, dynamin-, and clathrin-mediated pathway
for AT1A receptor endocytosis.
Finally, ß-arrestin trafficking is thought to involve recruitment to
the cell membrane and dephosphorylation (12), followed by
binding to the phosphorylated carboxyl terminus of an activated GPCR
and secondary interactions with other regions of the receptor. It is,
however, still not clear what drives ß-arrestin recruitment to the
membrane and whether this involves receptor-mediated signaling,
phosphatase activation, or if agonist-mediated receptor phosphorylation
itself is sufficient to serve this process. To test if receptor
phosphorylation is sufficient to drive ßarr1 binding to the
AT1A receptor, we stimulated cells expressing the
NHA-AT1A receptor and FLAG-ßarr1 with an analog
of AngII,
[Sar1Ile4Ile8]-AngII.
We have previously shown that this analog can induce a significant
phosphorylation of the AT1A receptor despite an
inability to activate phospholipase-mediated signaling
(32), consistent with the idea that
[Sar1Ile4Ile8]AngII
is unable to promote the active, signaling conformation in the
receptor. We observed a clear association of ßarr1 with the
AT1A receptor after
[Sar1Ile4Ile8]-AngII
stimulation that was similar to that produced by AngII. Moreover,
truncation of the receptor to Lys325, which
retains signaling (i.e. can attain the active state) but not
phosphorylation, showed no association with ßarr1 after AngII (Fig. 7
) or
[Sar1Ile4Ile8]-AngII
stimulation (data not shown). These observations suggest the crucial
requirement for ß-arrestin recruitment and binding is receptor
phosphorylation and not the active, signaling state of the receptor or
a signal emanating from the receptor. Nevertheless, the activated
conformation of the receptor almost certainly contributes to high
affinity binding of arrestin to the phosphorylated receptor, although
our use of cross-linker to trap arrestin on the receptor precludes any
significant analysis of relative affinities. Despite the capacity of
[Sar1Ile4Ile8]-AngII
to induce robust phosphorylation and significant ßarr1 binding to the
AT1A receptor, it is intriguing that
[Sar1Ile4Ile8]-AngII
does not promote strong AT1A receptor
internalization (32), as measured by an assay that
involved ligand stimulation, surface receptor stripping, and rebinding
with 125I-AngII. One interpretation could be
that, in the absence of an active receptor conformation or an
accompanying signal, ß-arrestin binding alone is not sufficient to
instigate receptor endocytosis. Alternatively, more direct methods for
determining receptor internalization (e.g. confocal
microscopy of GFP-labeled receptors and arrestins), which do not rely
on indirect stimulation/rebinding assays, may reveal slower, yet
significant, AT1A receptor internalization in
response to
[Sar1Ile4Ile8]-AngII
stimulation.
In conclusion, coimmunoprecipitation of phosphorylated
AT1A receptors and ßarr1 involves key
serine/threonine residues in the central portion of the receptor
carboxyl-terminus and correlates with the capacity of receptors to
rapidly internalize in response to AngII and for receptors to traffic
with ß-arrestin inside the cell (14). Future studies
will further examine the interplay between the active, signaling, and
phosphorylated states of the AT1A receptor,
ß-arrestin binding and trafficking, and receptor internalization,
desensitization, and resensitization. The receptor mutants described in
the present study, as well as constitutively active versions of the
AT1A receptor that are poorly phosphorylated
(32), and G protein-uncoupled AT1A
receptor mutants [e.g. Asp74
(45) and Tyr302 (46)]
that internalize with wild type-like kinetics will prove useful in
these endeavors. In conjunction with confocal microscopy, the
coimmunoprecipitation approach used here will provide important
insights into the identity and hierarchy of proteins that interact with
the AT1A receptor during the cycle of receptor
activation and deactivation.
 |
MATERIALS AND METHODS
|
---|
Materials
CHO-K1 cells were obtained from the American Type Culture Collection (Manassas, VA). Dr. R. J. Lefkowitz and Dr.
M. G. Caron (Duke University Medical Center, Durham, NC) kindly
supplied FLAG epitope-tagged versions of ß-arrestin. The ExSite
Mutagenesis kit was purchased from Stratagene (La Jolla,
CA) and DNA modifying enzymes were from Promega Corp.
(Madison, WI). 5'-Phosphorylated oligonucleotides,
-MEM, OPTI-MEM,
HBSS, okadaic acid, and lipofectAMINE were purchased from Life Technologies, Inc. (Gaithersburg, MD), while protein A-agarose
and anti-HA high-affinity rat monoclonal antibody (3F10) were from
Roche Molecular Biochemicals (Indianapolis, IN). The 12CA5
monoclonal antibody was purified from hybridoma culture media using an
influenza HA antigen (HA)-peptide affinity column. Anti-FLAG M2
monoclonal antibody and anti-FLAG biotinylated M2 monoclonal antibody
were from Sigma (St. Louis, MO), and anti-MYC antibody was
from Calbiochem (La Jolla, CA). The avidin-horseradiash
peroxidase conjugate was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). Antirat IgG-horseradish peroxidase was
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA). SuperSignal West Pico Chemiluminescent substrate and the
cross-linker, DSP, were purchased from Pierce Chemical Co.
(Rockford, IL). 125I-Ang II (specific
activity > 2,000 Ci/mmol) was provided by Austin Biomedical
Services (Melbourne, Australia) and
32P-orthophosphate was from ICN Biochemicals, Inc. AngII and
[Sar1Ile4Ile8]-AngII
were from Auspep Pty. Ltd. (Melbourne, Australia). All other chemicals
were from Sigma or BDH Laboratory Supplies (Kilsyth,
Victoria, Australia).
Plasmid DNA Constructs
The construction of an HA epitope-tagged wild-type
AT1A receptor, three carboxyl-terminal truncated
versions (NHA-TD343, deletion of 16 amino acids; NHA-TK333, deletion of
26 amino acids; NHA-TK325, deletion of 34 amino acids), and two
point-mutated versions (NHA-TSTS/A, T332/S335/T336/S338/A and
NHA-PKC(SSS/A), S331/S338/S348/A) have been described previously
(25, 29). Another mutant NHA-SSS/A, characterized by three
consecutive substitutions of serine residues
(Ser346, Ser347,
Ser348) with alanine (see Fig. 1
), was generated
using PCR-based site-directed mutagenesis (ExSite). 5'-Phosphorylated
oligonucleotides used for mutagenesis were (5'-3'):
S346/347AS:
p-GCTGCGGCCAAAAAGCCTGCG
(sense)
S346/347AA:
p-TGCCATGTTATCCGAAGGCCG
(antisense)
The primers were designed to generate a triple (S346/S347/S348A) mutant
when the single mutant (S348A) (29) was used as a
template. A silent PvuII (bold italic, formed
after ligation of PCR product generated by primers S346/347AS and
S346/347AA) restriction site was introduced to assist with the
screening for mutated clones. Mutations from the original sequence are
underlined. The mutant receptor was sequenced to confirm the
entire coding region and the relevant nucleotide mutations.
The dominant-negative ßarr1 (ßarr1319418)
was constructed using PCR from pcDNA3-FLAG-ßarr1, a gift from Dr.
R. J. Lefkowitz (Duke University Medical Center, Durham, NC). A
sense primer
(5'-ACAAGCTTACCATGGTTTCCTACAAAGTCAAAGTGAAGCTG-3')
was designed to include a HindIII site (italics)
and a minimal Kozak consensus sequence (underlined) around
an initiator methionine, while the antisense primer
(5'-AGTGGGCCCTCTGTTGTTGAGGTGTGGAGA-3') included an
ApaI site (italics). The PCR fragment was
digested with HindIII and ApaI and ligated to
pcDNA6/Myc-His A vector (Invitrogen, San Diego,
CA) cut with the same enzymes. Positive clones were selected and
sequenced to confirm the integrity of the construct.
Transient Transfection of CHO-K1 Cells
CHO-K1 cells were maintained in
-MEM containing FBS (10%),
penicillin G sodium (100 µg/ml), streptomycin sulfate (100 µg/ml),
and amphotericin (0.25 µg/ml, Life Technologies, Inc., Gaithersburg, MD) (complete media), seeded
in 12-well culture plates, and grown in complete media until 7080%
confluent. Cells were transiently transfected with a total 0.6 µg
plasmid DNA/well, using lipofectAMINE, as previously described
(25). For cotransfection, 0.3 µg DNA/well of either
HA-tagged wild-type, truncated, or mutated AT1A
receptor were used simultaneously with various amount of FLAG-tagged
ßarr1 up to 0.3 µg DNA/well, and various amounts of pRc/CMV (empty
vector) to a total of 0.6 µg DNA/well. After 5 h exposure to
DNA-lipofectAMINE complexes, cells were washed and grown in complete
media for a further 48 h.
Coimmunoprecipitation Assay for AT1A Receptor and
ß-Arrestin1 Association
The procedure for immunoprecipitation of transiently transfected
HA-tagged AT1A receptors in CHO-K1 cells
(25) was modified to include a cross-linking step
(22, 42) for coimmunoprecipitation of
AT1A receptors and ßarr1, using the reversible
cross-linker, DSP. Transfected cells in 12-well plates were
serum-starved for 16 h and stimulated by the agonist (Ang II, 100
nM) or an analog
([Sar1Ile4Ile8]-AngII,
30 µM) for 10 min at 37 C. In some experiments, the cells
were treated with 0.45 M sucrose (30 min, 37 C), before
agonist stimulation, to determine the effect of inhibiting
internalization on arrestin association. For the time course
experiment, stimulation by AngII was for 0, 0.5, 1, 5, 10, and 30 min
at 37 C. After stimulation, DSP was added to a final concentration of
2.5 mM, and the plates were incubated for an additional 20
min at room temperature. The plates were placed on ice, washed twice
with 1 ml/well of HBSS (4 C), and solubilized by the addition of RIPA
buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 2
mM EDTA, 50 mM sodium fluoride, 1% Triton
X-100, 0.5% sodium deoxycholate, 0.1% SDS) containing both
phosphatase inhibitors (0.5 mM okadaic acid, 10
mM sodium pyrophosphate) and proteinase inhibitors (1
µg/ml of aprotinin, 5 µg/ml of leupeptin, and 1 µg/ml of
pepstatin). Plates were rocked at 4 C for 1 h, and the cell
lysates were harvested, clarified by centrifugation (14,000 x
g for 15 min) and precleared by the addition of protein
A-agarose and BSA. Precleared lysates were incubated with 2 µg of
affinity-purified 12CA5 antibody and 20 µl of protein A-agarose and
agitated overnight at 4 C to coimmunoprecipitate the epitope-tagged
AT1A receptors and associated, cross-linked
proteins. The immunoprecipitates were washed six times, resuspended in
an urea-based SDS sample buffer containing 10%
ß-mercaptoethanol, and incubated at 37 C for 90 min to cleave the
DSP cross-linker and extract the receptor and any associated proteins.
Samples were resolved by 10% SDS-PAGE, Western blotted, and probed
with a biotinylated anti-FLAG antibody and an avidin-horseradish
peroxidase conjugate to identify coimmunoprecipitated arrestin protein.
Blots were then stripped and reprobed with a rat monoclonal anti-HA
(3F10) antibody and an antirat IgG horseradish peroxidase conjugate to
confirm receptor expression. Samples representing 0.03% of the total
cell extract before immunoprecipitation were also Western blotted and
probed with an anti-FLAG antibody and an antimouse IgG horseradish
peroxidase conjugate to confirm arrestin expression. All blots were
developed using enhanced chemiluminescence. Images were analyzed using
Scion image software (Frederick, MD) and differences between
group compared using t tests (P < 0.05 was
considered significant).
AT1A Receptor Phosphorylation
In parallel to the ßarr1 association experiments, the
AngII-induced phosphorylation of wild-type, truncated, and
alanine-mutated AT1A receptors was
determined. The procedure for the phosphorylation assay has been
described previously (25). Briefly, CHO-K1 cells,
transiently transfected with HA-tagged wild- type
AT1A, truncated, or alanine-mutated receptor,
were incubated for 1 h with 0.4 mCi/ml
32Pi and stimulated (or not
stimulated) with AngII (100 nM, 10 min). Cells were lysed,
and AT1A receptors were immunoprecipitated with
the 12CA5 monoclonal antibody and resolved on SDS-PAGE. Gels were
fixed, dried, and placed against type BAS-IIIs phosphoimaging plates
(Fuji Photo Film Co., Ltd., Tokyo, Japan), which
were read in a FUJIX Bio-imaging Analyzer BAS 1000 (Fuji Photo Film Co., Ltd.) and analyzed using MacBAS v1.0
software (Fuji Photo Film Co., Ltd.).
AT1A Receptor Internalization
Internalization kinetics for the wild-type and mutated
AT1A receptors were determined from
acid-insensitive 125I-AngII receptor binding, as
previously described (25). In some experiments, the
transfected CHO-K1 cells were treated with 0.45 M sucrose
(30 min, 37 C) before agonist stimulation to examine the capacity of
hyperosmotic sucrose to inhibit AT1A receptor
internalization.
 |
ACKNOWLEDGMENTS
|
---|
We are indebted to Drs. R. J. Lefkowitz and M.G. Caron for
supplying FLAG-tagged ß-arrestin constructs. We thank the members of
the Molecular Endocrinology Laboratory for comments and assistance.
 |
FOOTNOTES
|
---|
This work was supported by A National Heart Foundation of Australia
Grant-in-Aid to W.G.T. (G99M0301) and a National Health and Medical
Research Council of Australia Block Grant to the Baker Medical Research
Institute.
Abbreviations: AngII, Angiotensin II; AT1A,
angiotensin II type 1A receptor; ßarr, ß-arrestin; DSP,
dithiobis(succinimidyl)propionate; GFP, green fluorescent protein;
GIT1, GRK-interactor 1; GPCR, G protein-coupled receptor; GRK, GPCR
kinase; HA, hemagglutin; NHA-AT1A, N-terminal HA-tagged
AT1A receptor.
Received for publication December 18, 2000.
Accepted for publication June 27, 2001.
 |
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