Receptor/ß-Arrestin Complex Formation and the Differential Trafficking and Resensitization of ß2-Adrenergic and Angiotensin II Type 1A Receptors
Pieter H. Anborgh,
Jennifer L. Seachrist,
Lianne B. Dale and
Stephen S. G. Ferguson
The John P. Robarts Research Institute and Departments of
Physiology (J.L.S., S.S.G.F.) and Pharmacology and Toxicology
(L.B.D., S.S.G.F.) University of Western Ontario London,
Ontario, Canada N6A 5K8
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ABSTRACT
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ß-Arrestins target G protein-coupled receptors
(GPCRs) for endocytosis via clathrin-coated vesicles. ß-Arrestins
also become detectable on endocytic vesicles in response to angiotensin
II type 1A receptor (AT1AR), but not
ß2-adrenergic receptor
(ß2AR), activation. The carboxyl-terminal
tails of these receptors contribute directly to this phenotype, since a
ß2AR bearing the
AT1AR tail acquired the capacity to stimulate
ß-arrestin redistribution to endosomes, whereas this property was
lost for an AT1AR bearing the
ß2AR tail. Using
ß2AR/AT1AR chimeras,
we tested whether the ß2AR and
AT1AR carboxyl-terminal tails, in part via
their association with ß-arrestins, might regulate differences in the
intracellular trafficking and resensitization patterns of these
receptors. In the present study, we find that ß-arrestin formed a
stable complex with the AT1AR tail in endocytic
vesicles and that the internalization of this complex was dynamin
dependent. Internalization of the ß2AR
chimera bearing the AT1AR tail was observed in
the absence of agonist and was inhibited by a dominant-negative
ß-arrestin1 mutant. Agonist-independent AT1AR
internalization was also observed after ß-arrestin2 overexpression.
After internalization, the ß2AR, but not the
AT1AR, was dephosphorylated and recycled back
to the cell surface. However, the AT1AR
tail prevented ß2AR dephosphorylation and
recycling. In contrast, although the
ß2AR-tail promoted
AT1AR recycling, the chimeric receptor remained
both phosphorylated and desensitized, suggesting that receptor
dephosphorylation is not a property common to all receptors. In
summary, we show that the carboxyl-terminal tails of GPCRs not only
contribute to regulating the patterns of receptor desensitization,
but also modulate receptor intracellular trafficking and
resensitization patterns.
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INTRODUCTION
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G protein-coupled receptor (GPCR) responsiveness to agonist wanes
rapidly with time, a process termed desensitization. GPCR
desensitization occurs as the consequence of rapid receptor
phosphorylation by both second messenger-dependent protein kinases and
G protein-coupled receptor kinases (GRKs) (reviewed in Refs. 1, 2, 3).
GRK-mediated phosphorylation promotes the binding of arrestin proteins
that sterically uncouple receptors from their cognate heterotrimeric G
protein (4, 5). It has been known for some time that desensitized GPCRs
also internalize in clathrin-coated vesicles (6, 7). More recently,
both ß-arrestin1 and ß-arrestin2 were shown to mediate the
internalization of receptors via clathrin-coated pits (8, 9, 10).
ß-Arrestin-dependent receptor internalization via clathrin-coated
vesicles is the consequence of ß-arrestin protein interactions with
both the ß-adaptin subunit of the AP2 adaptor complex and clathrin
(11, 12). In the case of the ß2-adrenergic
(ß2AR)1
and neurokinin 1 receptors, internalization to the endosomal
compartment results in ligand dissociation followed by receptor
dephosphorylation (13, 14, 15, 16, 17). Subsequently, these receptors are recycled
back to the plasma membrane surface as fully functional receptors
(13, 14, 15). This has led to the suggestion that GPCR internalization
represents the predominant mechanism underlying receptor
resensitization. However, this pattern is not universal, since not all
GPCRs recycle back to the cell surface after their internalization. For
example, internalized protease-activated receptors (PAR1) are
specifically sorted to lysosomes and do not recycle (18).
Similar to the ß2AR, the angiotensin II
type 1A receptor (AT1AR) is rapidly
phosphorylated and internalized after its activation with agonist
(19, 20, 21). However, the agonist-stimulated internalization of the
AT1AR appears to be more complex than the
internalization of the ß2AR. While the
predominant mechanism(s) governing AT1AR remain
unclear, AT1AR internalization in HEK 293 and
COS7 cells is not sensitive to inhibition by dominant-negative proteins
of ß-arrestin and dynamin, which interfere with clathrin-coated
vesicle-mediated internalization (9). Furthermore, the
AT1AR colocalizes with ß-arrestin in endocytic
vesicles, a property that is not shared by the
ß2AR (22). Nonetheless, there are conflicting
reports in the literature regarding whether or not
AT1AR internalization can be blocked using
dominant-negative ß-arrestin and dynamin mutants, suggesting that
ß-arrestins and clathrin may play a more substantial role in
regulating AT1AR internalization than originally
envisaged (9, 20, 23).
Recently, using
ß2AR/AT1AR chimeric
receptors and ß-arrestin2 fused to green fluorescent protein (GFP),
we demonstrated that the carboxyl-terminal tails of these receptors
determined whether or not ß-arrestin redistributed to large core
intracellular vesicular structures in response to receptor activation
(22). However, it is unknown whether ß-arrestin is physically
associated with the AT1AR in endocytic vesicles.
Therefore, we tested whether the carboxyl-terminal tails of the
ß2AR and AT1AR, in part
via differences in their ability to form stable complexes with
ß-arrestin, contribute to differences in endocytosis,
dephosphorylation, and plasma membrane-recycling patterns observed for
the ß2AR and AT1AR.
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RESULTS
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Coimmunoprecipitation of
ß2AR-ATCT/ß-Arrestin2 Complexes
Previously, we demonstrated that agonist activation of the
AT1AR, but not the ß2AR,
resulted in the redistribution of ß-arrestin2-GFP to large core
intracellular vesicular structures (22). Furthermore, we demonstrated
that, by replacing the carboxyl-terminal tail of the
ß2AR with the AT1AR tail,
the ß2AR gained the capacity to stimulate the
redistribution of ß-arrestin2-GFP fluorescence to endocytic vesicles
(22). While these observations are suggestive of the formation of
AT1AR/ß-arrestin2-GFP complexes in endosomes,
the evidence supporting this association is circumstantial and does not
demonstrate a physical association between ß-arrestin and the
receptor tail in subcellular membrane locations. Therefore, to
determine whether ß-arrestin2-GFP is physically associated with the
AT1AR carboxyl-terminal tail in endocytic
vesicles, we attempted to coimmunoprecipitate wild-type
ß2AR and ß2AR-ATCT
chimera with ß-arrestin2-GFP from both plasma membrane and light
vesicular membrane fractions. Isoproterenol stimulation of HEK 293
cells expressing ß2AR resulted in a 2.6 ±
1.1-fold increase in ß2AR co-immunoprecipitated
with ß-arrestin2-GFP from the plasma membrane fraction vs.
cells that were not exposed to agonist (Fig. 1A
). Furthermore, the amount of
ß2AR-ATCT and ß2AR
coimmunoprecipitated with ß-arrestin2-GFP from the plasma membrane
fraction after isoproterenol treatment was not apparently different,
2.2 ± 0.6- vs. 2.6 ± 1.1-fold, respectively
(Fig. 1A
). However, in the absence of isoproterenol, the amount of
ß2AR-ATCT coimmunoprecipitated with
ß-arrestin2-GFP from the plasma membrane fraction was 1.7 ±
0.5-fold greater than that coimmunoprecipitated with the wild-type
receptor (Fig. 1A
). ß2AR coimmunoprecipitated
with ß-arrestin2-GFP from the light vesicular membrane fraction was
not increased by isoproterenol stimulation (Fig. 1B
). However, agonist
treatment of ß2AR-ATCT-expressing cells
resulted in a 2.7 ± 1-fold increase in
ß2AR-ATCT coimmunoprecipitated with
ß-arrestin2-GFP from the vesicular fraction (Fig. 1B
). These
experiments demonstrate that agonist promotes the association of
ß-arrestin2-GFP with both the ß2AR and
ß2AR-ATCT at the plasma membrane, but that the
localization of ß-arrestin2 to endocytic vesicles requires that it
physically associate with the AT1AR
carboxyl-terminal tail of the ß2AR-ATCT
chimera.

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Figure 1. Agonist-Dependent Coimmunoprecipitation of the
ß2AR and ß2AR-ATCT with ß-Arrestin2-GFP
A, The relative amount of ß2AR and
ß2AR-ATCT immunoprecipitated with ß-arrestin2-GFP from
the plasma membrane fraction of HEK 293 cells incubated in the absence
and presence of 10 µM isoproterenol for 45 min at 37 C.
B, The relative amount of of ß2AR and
ß2AR-ATCT immunoprecipitated with ß-arrestin2-GFP from
the light vesicular membrane fraction of HEK 293 cells incubated in the
absence and presence of 10 µM isoproterenol for 45 min at
37 C. The preparation of subcellular membrane fractions and
125I-pindolol binding to receptors coimmunoprecipitated
with ß-arrestin2-GFP using a rabbit polyclonal anti-GFP antibody were
performed as described in Materials and Methods. The
data represent the fold increase in receptor immunoprecipitated when
compared with ß2AR immunoprecipitated with
ß-arrestin2-GFP in the absence of agonist stimulation in each
individual experiment. The data represent the mean ±
SD of three independent experiments. *,
P < 0.05 vs. ß2AR
without agonist.
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Recently, Oakley et al. (24) demonstrated that the
redistribution of ß-arrestin to vesicles with the vasopressin
receptor required GRK-mediated phosphorylation of a cluster of serine
residues within the carboxyl-terminal tail of the receptor. Therefore,
we prepared two AT1AR truncation mutants to
determine whether the deletion of two clusters of putative sites for
GRK-mediated phosphorylation in the carboxyl-terminal tail of the
AT1AR (amino acid residues 326338 and 342348)
contribute to the localization of ß-arrestin to endocytic vesicles.
In response to agonist stimulation, ß-arrestin2-GFP becomes
completely localized to large core vesicles in cells expressing
wild-type AT1AR (Fig. 2A
). In cells expressing an
AT1AR mutant (
339) lacking the final 20 amino
acid residues of the carboxyl-terminal tail, ß-arrestin2-GFP
demonstrates a punctate distribution at the plasma membrane surface and
is localized to small endocytic vesicles after agonist treatment (Fig. 2B
). ß-Arrestin2-GFP distribution after agonist activation of an
AT1AR mutant (
319) lacking the final 40 amino
acid residues of the carboxylterminal tail is limited to a
punctate distribution at the plasma membrane (Fig. 2C
). Each of the
truncated receptors internalized in response to agonist stimulation
(data not shown). Thus, while ß-arrestin2 translocation and
redistribution to coated pits in response to
AT1AR activation does not require the association
of ß-arrestin with the AT1AR carboxyl-terminal
tail, the tail is required for ß-arrestin2-GFP redistribution to
endocytic vesicles.

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Figure 2. Effect of Truncating the AT1AR
Carboxyl-Terminal Tail on Agonist-Stimulated ß-Arrestin2-GFP
Redistribution
ß-Arrestin2-GFP distribution in AT1AR (panel A),
AT1AR- 339 (panel B), and AT1AR- 319 (panel
C) expressing HEK 293 cells after 45 min treatment with 100
nM angiotensin II. HEK 293 cells were transfected
transiently with plasmid cDNAs encoding HA epitope-tagged receptors (10
µg) and pEGFP-N1 ß-arrestin2-GFP (5 µg). Data shown are
representative of three experiments. Bar = 10 µm.
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Clathrin Dependence of AT1AR/ß-Arrestin2
Complex Formation
Since AT1AR endocytosis is mediated by both
dynamin-dependent and -independent mechanisms and is phosphorylation
dependent (9, 20, 23), we tested whether the formation of
AT1AR/ß-arrestin2-GFP complexes in endosomes is
a dynamin-dependent process. In the absence of agonist,
ß-arrestin2-GFP is diffusely localized throughout the cytoplasm of
cells expressing the wild-type AT1AR (Fig. 3A
), and, upon agonist activation,
ß-arrestin2-GFP becomes localized to large core intracellular
vesicles (Fig. 3B
). Unexpectedly, in cells expressing both the
AT1AR and dynamin I-K44A, ß-arrestin2-GFP
fluorescence was localized to the plasma membrane in a punctuated
distribution even in the absence of agonist stimulation (Fig. 3C
).
Moreover, in the presence of dynamin I-K44A, no redistribution of
ß-arrestin2-GFP to the endosomal compartment was observed after a
45-min exposure to 100 nM angiotensin II (Fig. 3D
). In
contrast, in cells that expressed the
AT1AR-ß2CT chimera, dynamin I-K44A
overexpression did not alter the cytosolic distribution of
ß-arrestin2-GFP (Fig. 3
, panel E vs. panel G) and did not
prevent AT1AR-ß2CT-mediated ß-arrestin2-GFP
translocation to the cell surface in response to agonist stimulation
(Fig. 3
, F and H). Consistent with the idea that the formation of
ß-arrestin/AT1AR complexes is clathrin
dependent, clathrin immunofluorescence colocalized with
ß-arrestin2-GFP in large core vesicles in
AT1AR- but not
ß2AR-expressing cells (Fig. 4
). Taken together, these observations
indicate that the endocytosis of
AT1AR/ß-arrestin2 complexes is mediated by
clathrin-coated vesicles and not by the putative dynamin-independent
endocytic pathway previously described for the
AT1AR (9). Furthermore, the localization of
ß-arrestin2-GFP to the plasma membrane in the presence of dynamin
I-K44A, in AT1AR- but not
AT1AR-ß2CT-expressing cells, suggests that
ß-arrestins can associate with the AT1AR
carboxyl-terminal tail in the absence of agonist activation.

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Figure 3. Effect of Dynamin I-K44A Overexpression on
ß-Arrestin2-GFP Translocation and Intracellular Redistribution in
Response to AT1AR and AT1AR-ß2CT Activation
ß-Arrestin2-GFP distribution in AT1AR- and
AT1AR-ß2CT-expressing HEK 293 cells in the absence of
dynamin I-K44A before (A and E) or after (B and F) 30 min exposure of
the same cells to 100 nM angiotensin II. Effect of dynamin
I-K44A overexpression on ß-arrestin2-GFP distribution in
AT1AR- and AT1AR-ß2CT-expressing HEK 293
cells before (C and G) or after (D and H) 30 min exposure of the same
cells to 100 nM angiotensin II. HEK 293 cells were
transfected transiently with plasmid cDNAs encoding HA epitope-tagged
receptors (510 µg) and pEGFP-N1 ß-arrestin2-GFP (5 µg) with or
without plasmid cDNAs encoding dynamin I-K44A (5 µg). Data shown are
representative of four experiments. Bar represents 10
µm.
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Figure 4. Agonist-Stimulated Colocalization of
ß-Arrestin2-GFP and Clathrin in HEK 293 Cells
Confocal visualization of the distribution and colocalization (overlay)
of ß-arrestin2-GFP (green) and X-22 monoclonal
antibody-labeled clathrin (red) in HEK 293 cells
expressing ß2AR (panel A) and AT1AR (panel
B). Shown are representative images of cells fixed in 3.6%
paraformaldehyde after a 45-min stimulation with either 10
µM isoproterenol or 100 nM angiotensin II.
Cells were transfected with 10 µg of plasmids encoding either
ß2AR or AT1AR along with 5 µg of
ß-arrestin2-GFP. Data shown are representative of four experiments.
Bar represents 10 µm.
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To further establish that the association of ß-arrestin with the
AT1AR carboxyl-terminal tail mediates the
agonist-independent membrane localization of ßarrestin2-GFP,
we examined whether dynamin I-K44A overexpression altered
ß-arrestin2-GFP distribution in cells expressing either the wild-type
ß2AR or ß2AR-ATCT
chimera. In cells expressing the wild-type ß2AR
with or without dynamin I-K44A, ß-arrestin2-GFP fluorescence is
diffusely distributed throughout the cytoplasm in the absence of
agonist and translocates to the plasma membrane in response to agonist
activation of the receptor (Fig. 5
, AD). However, in cells expressing the
ß2AR-ATCT chimera, ß-arrestin2-GFP is
localized at the plasma membrane surface (>75% of cells) even in the
absence of dynamin I-K44A expression (Fig. 5
, E and G). This
observation is consistent with data presented in Fig. 1
where 1.7
± 0.5-fold more ß2AR-ATCT than
ß2AR is coimmunoprecipitated with
ß-arrestin2-GFP in the absence of agonist stimulation. However, the
redistribution of ß-arrestin2-GFP to large core intracellular
vesicular structures requires agonist activation of the receptor (Fig. 5
, panel E vs. panel F). Similar to what is observed for the
AT1AR, dynamin I-K44A blocked the clathrin-coated
vesicle-mediated endocytosis of
ß2AR-ATCT/ß-arrestin2-GFP complexes (Fig. 5H
).

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Figure 5. Effect of Dynamin I-K44A Overexpression on
ß-Arrestin2-GFP Translocation and Intracellular Trafficking in
Response to either ß2AR or ß2AR-ATCT
Activation
ß-Arrestin2-GFP distribution in both ß2AR- and
ß2AR-ATCT-expressing HEK 293 cells in the absence of
dynamin I-K44A before (A and E) or after (B and F) 30 min exposure of
the same cells to 10 µM isoproterenol. Effect of
dynamin1-K44A overexpression on ß-arrestin2-GFP distribution in both
ß2AR- and ß2AR-ATCT-expressing HEK 293
cells before (C and G) or after (D and H) 30 min exposure of the same
cells to 10 µM isoproterenol. HEK 293 cells were
transfected transiently with plasmid cDNAs encoding pEGFP-N1
ß-arrestin2-GFP (5 µg) and either HA epitope-tagged pcDNA1-Amp
ß2AR (10 µg) or FLAG epitope-tagged pcDNA1-Amp
ß2AR-ATCT (10 µg) with or without plasmid cDNAs
encoding dynamin I-K44A (5 µg). Inset shows the
redistribution of ß-arrestin2-GFP to large-core intracellular
vesicles. Data shown are representative of three independent
experiments. Bar represents 10 µm.
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Agonist-Independent Receptor Internalization
The localization of ß-arrestin2-GFP fluorescence at the plasma
membrane surface in the absence of agonist suggested that the
association of ß-arrestin with the AT1AR
carboxyl terminus might promote agonist-independent
ß2AR-ATCT and AT1AR
internalization. Moreover, agonist-independent internalization of the
AT1AR has previously been reported (25).
Therefore, we tested for the loss of cell surface
ß2AR-ATCT and AT1AR in
the absence of agonist stimulation. Epitope-tagged receptors were
labeled with primary antibody at 4 C for 45 min, and the loss of cell
surface secondary antibody labeling was determined after the warming of
cells to 37 C in the absence of agonist. Significant loss of cell
surface ß2AR-ATCT was observed
(VMAX = 40 ± 6) in the absence of agonist
stimulation and was not increased by the overexpression of
ßarrestin2 (Fig. 6A
). In contrast,
agonist-independent internalization of the wild-type
AT1AR was not observed in the absence of
overexpressed ß-arrestin2 (VMAX = 2 ±
1%) but was observed in the presence (VMAX =
31 ± 9%) of overexpressed ß-arrestin2 (Fig. 6A
). Moreover, no
agonist-independent AT1AR-ß2CT internalization
was observed in the presence or absence of overexpressed ß-arrestin2
(data not shown). To examine whether endogenous ß-arrestins
contributed directly to the agonist-independent loss of cell surface
ß2AR-ATCT, we tested the ability of a
dominant-negative ß-arrestin mutant, ß-arrestin1 (185418),to
inhibit agonist-independent ß2AR-ATCT
endocytosis. When tested, ß-arrestin1 (185418) significantly
retarded the agonist-independent loss of cell surface
ß2AR-ATCT (VMAX = 34
± 4% vs. 56 ± 5%, P < 0.05) (Fig. 6B
). The agonist-independent loss of cell surface
ß2AR-ATCT was unaffected by the treatment of
cells with propranolol (10 µM) (Fig. 6B
). These
experiments demonstrate that ß-arrestin-dependent receptor
internalization can occur in the absence of receptor activation by
agonist.

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Figure 6. Agonist-Independent Endocytosis of the
AT1AR and ß2AR-ATCT in the Presence and
Absence of Overexpressed Wild-Type and Dominant-Negative ß-Arrestins
A, Time course for the agonist-independent internalization of the
AT1AR (circles) and ß2AR-ATCT
(squares) in the absence (open symbols)
and presence of overexpressed ßarrestin2 (filled
symbols). B, Time course for the agonist-independent
endocytosis of the ß2AR-ATCT in the absence or presence
of either ß-arrestin1 (185418), or 10 µM propranolol.
Cell surface receptors were labeled with primary antibody on ice as
described in the Materials and Methods and then warmed
to 37 C for the times indicated. Agonist-independent internalization is
defined as a loss of antibody-labeled cell surface receptor sites in
the absence of agonist stimulation. HEK 293 cells were transfected
transiently with plasmid cDNA containing 12CA5-AT1AR (12
µg) and FLAG-ß2AR-ATCT (12 µg) and either empty
vector (10 µg), wild-type ß-arrestin2 (10 µg), or ß-arrestin1
mutants (10 µg). The data represent the mean ± SE
of three independent experiments normalized to the loss of
ß2AR cell surface receptor in the absence of agonist
stimulation.
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Wild-Type and Chimeric Receptor Recycling
While it is well documented that internalized
ß2AR are recycled back to the cell surface
(13, 14, 15), it is less clear whether this occurs for the
AT1AR. Therefore, using both wild-type and
chimeric ß2AR and AT1AR,
we examined whether differences exist in the relative ability of these
receptors to recycle and whether this might correlate with their
observed capacity to form complexes with ß-arrestin complexes in
endosomes. When tested, we found that, unlike the
ß2AR, AT1AR were not
efficiently recycled back to the cell surface after the washout of
agonist even in the absence of ß-arrestin overexpression (Fig. 7
, A and B). However, when the
AT1AR carboxyl-terminal tail was replaced with
the ß2AR tail, plasma recycling of the
AT1AR was observed (Fig. 7B
), whereas the
replacement of the ß2AR carboxyl-terminal tail
with the AT1AR tail prevented plasma membrane
recycling of the ß2AR (Fig. 7A
). These results
suggest that the plasma membrane recycling of
ß2AR and AT1AR is
negatively correlated with the formation of receptor/ß-arrestin
complexes in endosomes.

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Figure 7. Role of the ß2AR and
AT1AR Carboxyl-Terminal Tails in Receptor Recycling
Time courses for the recycling of the wild-type ß2AR and
the ß2AR-ATCT chimera (panel A) and the wild-type
AT1AR and the AT1AR-ß2CT chimera (panel B) to
the cell surface after agonist stimulation for 30 min with 10
µM isoproterenol and 100 nM angiotensin II,
respectively. Cells were washed twice on ice and then incubated at 37 C
in the absence of agonist for the times indicated in the figure.
Receptor recycling was measured as a time-dependent return of cell
surface immunofluorescence after agonist removal. HEK 293 cells were
transfected transiently with plasmid cDNA containing the
12CA5-ß2AR (250 ng), 12CA5-AT1AR (12 µg),
FLAG-ß2AR-ATCT (12 µg), and 12CA5
AT1AR-ß2CT (12 µg) along with either empty vector (1
µg) or ß-arrestin2 (1 µg) in the case of the
ß2AR-ATCT. The extent of internalization after 30 min
exposure to agonist for each receptor in these experiments was as
follows: ß2AR = 27 ± 2%,
ß2AR-ATCT = 28 ± 2%, AT1AR =
72 ± 4%, and AT1AR-ß2CT = 66 ± 1% of
cell surface receptors. The data represent the mean ±
SE of three experiments.
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Wild-Type and Chimeric Receptor Dephosphorylation
In the case of the ß2AR, internalization
is required for receptor dephosphorylation and resensitization (14, 17). However, it is unknown whether internalized
AT1AR are dephosphorylated. Therefore, we
examined the whole cell phosphorylation and dephosphorylation of the
ß2AR and AT1AR in the
absence of ß-arrestin overexpression. Both receptors were effectively
phosphorylated in response to agonist stimulation (Fig. 8
, A and B). When agonist was removed and
cells were allowed to recover in agonist-free media for 20 min, marked
dephosphorylation was observed for the wild-type
ß2AR, but not the wild-type
AT1AR (Fig. 8
, A and B). To test whether
ß-arrestin dissociation from the ß2AR was
required for dephosphorylation, similar to that observed previously for
rhodopsin (26), we tested the dephosphorylation of the
ß2AR-ATCT chimera. We find that the
ß2AR-ATCT chimera, which exhibits the capacity
to form a complex with ß-arrestin in endosomes, did not become
dephosphorylated when allowed to recover in agonist-free medium (Fig. 8A
). However, the AT1AR-ß2CT chimera, which
does not exhibit the capacity to internalize with ß-arrestin
bound, also did not become dephosphorylated (Fig. 8B
). The lack
of AT1AR-ß2CT dephosphorylation indicates that
the lack of AT1AR dephosphorylation may be
independent of ß-arrestin complex formation. This suggests that
receptor dephosphorylation is not common to all GPCRs.

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Figure 8. Whole-Cell Phosphorylation and Dephosphorylation of
Wild-Type and Chimeric ß2AR and AT1AR
HEK 293 transfected to overexpress ß2AR and
ß2AR-ATCT (panel A) and AT1AR and
AT1AR-ß2CT (panel B) were labeled with 100 µCi/ml
[32P]orthophosphate for 1 h in phosphate-free
medium. Cells were pretreated for 10 min in serum-free medium at 37 C
in the absence (N, naive) or presence (D, desensitized; R,
resensitized) of either 10 µM isoproterenol or 100
nM angiotensin II, washed three times, and either allowed
to resensitize (R) for 20 min at 37 C or kept on ice (N, D).
Representative autoradiographs and phosphorimager analysis of the
mean ± SEM of three independent experiments
demonstrating the whole-cell phosphorylation of the ß2AR
and ß2AR-ATCT (panel A) and AT1AR and
AT1AR-ß2CT (panel B) are shown. Each lane was loaded with
equivalent amounts of receptor protein as described in Materials
and Methods. The phosphorimager data are normalized to receptor
phosphorylation in the absence of agonist. *, P <
0.05 vs. desensitized wild-type receptor
phosphorylation.
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AT1AR Desensitization and
Resensitization
AT1AR-mediated GFP-protein kinase C
(PKC)ßII translocation was previously demonstrated to serve as an
effective measure of homologous AT1AR
desensitization (27). This assay was used to determine receptor
desensitization/resensitization in the present experiments for two
reasons: 1) the sensitivity of the assay and 2) the determination of
receptor desensitization/resensitization is not confounded by the
accumulation of intracellular second messengers in response to the
desensitizing stimulus. In cells expressing either the wild-type or
chimeric AT1AR, angiotensin II stimulated the
plasma membrane translocation of GFP-PKCßII (Fig. 9A
). In both cases, the GFP-PKCßII
translocation response desensitized rapidly (Fig. 9A
). However, while
GFP-PKCßII translocation was reestablished in cells that were allowed
to recover for 20 min in agonist-free media (Fig. 9B
), the
magnitude of GFP-PKCßII plasma membrane translocation in response to
agonist remained attenuated for both the AT1AR
and the AT1AR-ß2CT chimera when compared with
untreated control cells (Fig. 9C
).

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Figure 9. Activation, Desensitization, and Resensitization of
AT1AR- and AT1AR-ß2CT-Mediated PKC-GFP
Translocation
A, Time course for the activation and desensitization of
AT1AR and AT1AR-ß2CT-mediated GFP-PKCßII
membrane translocation in responses to 100 nM angiotensin
II. Data shown are representative of seven independent experiments. B,
Resensitization of AT1AR- and
AT1AR-ß2CT-mediated PKC-GFP translocation responses to
100 nM angiotensin II after the washout of agonist and a
30-min recovery of cells in agonist-free media. The data are
representative of five to six different experiments. C, Quantification
of the relative magnitude of AT1AR- and
AT1AR-ß2CT-stimulated GFP-PKCßII translocation
responses in control cells and cells allowed to resensitize 30 min in
the absence of agonist after the preexposure of cells to 100
nM angiotensin II. The relative GFP-PKCßII fluorescence
intensity and duration of membrane translocation were measure using the
LSM-510 image analysis software and the normalized data presented as
comparison with data obtained from control AT1AR-expressing
cells. The data for each curve represent the mean ±
SEM of the data obtained from 13 different
AT1AR-expressing cells from seven distinct experiments and
1720 AT1AR-ß2CT-expressing cells from five to six
independent experiments. *, P < 0.05 vs.
control. Bar represents 10 µm.
|
|
 |
DISCUSSION
|
---|
Our findings provide direct biochemical and cell biological
evidence that the redistribution of ß-arrestin2 to large-core
intracellular vesicular structures in response to
AT1AR activation involves the formation of a
stable complex between the carboxyl-terminal tail of the receptor and
ß-arrestin2. In contrast, the ß2AR
carboxyl-terminal tail does not support the formation of intracellular
receptor/ß-arrestin complexes. We find that the stable association of
ß-arrestin with the AT1AR tail not only
mediates the agonist-stimulated internalization of the
AT1AR via clathrin-coated pits but also has the
potential to allow the agonist-independent internalization of the
AT1AR via clathrin-coated vesicles. Furthermore,
while internalized ß2AR is recycled back to the
cell surface, internalized AT1AR are retained
within the cell and do not recycle. However, the relative ability of
these receptors to recycle appears to be regulated by their
carboxyl-terminal tails and is correlated with the ability of the
receptor tails to form stable complexes with ß-arrestin. In the case
of the ß2AR, ß-arrestin dissociation was
required for receptor dephosphorylation in the endosomal compartment
but, surprisingly, dephosphorylation of the AT1AR
did not occur even under conditions where no ß-arrestin association
with the receptor in endosomes was expected to occur. This observation
suggests that receptor dephosphorylation may not represent a property
common to all GPCRs. Taken together, these data support the hypothesis
that receptor carboxyl-terminal tails not only regulate the
desensitization of GPCRs, but also play an essential role in regulating
differences in receptor trafficking and resensitization patterns. In
part, this may be the consequence of their relative capacity to form
stable complexes with ßarrestin proteins.
To our knowledge, the present experiments provide the first evidence
implicating ß-arrestins in promoting agonist-independent GPCR
endocytosis. Although agonist-stimulated GPCR internalization is now a
well established phenomenon (1, 2, 3), the concept that GPCRs internalize
in an agonist-independent manner is less well accepted.
Agonist-independent receptor internalization has now been reported for
a growing number of GPCRs, including the AT1AR
(25, 28, 29). While we observed agonist-independent
AT1AR internalization only after ß-arrestin
overexpression, Hein et al. (25) documented
agonist-independent AT1AR internalization in HEK
293 cells in the absence of ß-arrestin overexpression. This
discrepancy suggests that there exists heterogeneity in ß-arrestin
expression levels between different HEK 293 cell cultures. Nonetheless,
it is likely that the agonist-independent internalization of some
GPCRs will be found to be ß-arrestin independent.
While our data show that ß-arrestins can associate with receptors in
the absence of agonist occupancy, agonist stimulation clearly
stabilizes the complex formed between the receptor and ß-arrestin,
since ß-arrestin redistribution to endosomes was observed only in the
presence of agonist (Fig. 4
, compare panels E and F). The stabilization
of the receptor/ß-arrestin complex by agonist is likely the
consequence of both receptor isomerization to a "high-affinity"
ligand binding state and GRK-mediated phosphorylation of the receptor
C-terminal tail. This is consistent with a recent report demonstrating
that vasopressin V2 receptor-stimulated redistribution of
ß-arrestin to endosomes involved GRK2 phosphorylation of a triplet
motif of serine residues in the carboxyl-terminal tail of the receptor
that presumably stabilized interactions of ß-arrestin with the
receptor (24). In the case of PAR1, the mutation of putative receptor
phosphorylation sites in the carboxyl-terminal tail of PAR1 prevented
agonist-stimulated PAR1 internalization, whereas
agonist-independent PAR1 internalization was unaffected by these
mutations (28). Our data suggest that, under the appropriate
conditions, the agonist-independent association of ß-arrestin with
both the ß2AR-ATCT mutant and
AT1AR is facilitated by a receptor conformation
promoted by the AT1AR carboxyl-terminal tail. The
agonist-independent association of ßarrestin with these
receptors is not supported by the ß2AR
carboxyl-terminal tail, which may negatively regulate the association
of ß-arrestin in the absence of agonist stimulation. The
higher-affinity agonist-independent interactions with the
ß2AR-ATCT mutant may reflect receptor-specific
differences in the contribution of other intracellular domains to
ß-arrestin interactions. Nonetheless, for the
AT1AR, ß-arrestin regulates both
agonist-stimulated and agonist-independent internalization. It is
possible that the role of GRK phosphorylation in the formation of
stable receptor/ßarrestin complexes may represent the molecular
event distinguishing agonist-stimulated PAR1 internalization to
lysosomes from the agonist-independent PAR1 trafficking (28).
In addition to the AT1AR, receptor-mediated
redistribution of ß-arrestin to intracellular endocytic structures
has recently been reported to occur in response to neurotensin,
vasopressin V2, neurokinin 1, TRH, and protease receptor activation
(22, 24, 30, 31, 32). However, while each of these receptors is presumed to
be associated with ß-arrestin in endocytic vesicles, there is no
evidence supporting a direct interaction between any of these receptors
and ß-arrestin in endosomes. In the present study, we show that
ß-arrestin2 is physically associated with the
AT1AR carboxyl-terminal tail and that the
AT1AR tail, through its association with
ß-arrestin, potentially prevents receptor recycling.
Nonetheless, it remains unclear whether ß-arrestin acts as a
retention signal preventing receptor recycling. However, except for the
neurokinin 1 receptor (15, 32), poor recycling to the plasma membrane
is a common property shared by receptors that stimulate ß-arrestin
redistribution to intracellular vesicles (24, 30, 31, 33). The fact
that the neurokinin 1 receptor recycles back to the cell surface
suggests that either this receptor is internalized to a distinct
endosomal compartment or that the neurokinin 1 receptor does not
physically associate with ß-arrestin in endocytic vesicles. Moreover,
it is also likely that the specific association of distinct GPCRs with
additional components of the endosomal compartment may also contribute
to differences in both the intracellular trafficking patterns and
extent of plasma membrane recycling for each of these GPCRs. The
finding that, unlike observed for the vasopressin V2 receptor (24), the
ß2AR tail does not completely rescue
AT1AR recycling may suggest that other receptor
domains contribute to the intracellular retention of the
AT1AR. The answers to these questions remain to
be determined.
In the present study, we provide evidence that in response to agonist,
ß-arrestin is physically associated rather than colocalized with the
AT1AR in endocytic vesicles. First, we show that
the ß2AR bearing the
AT1AR tail can be coimmunoprecipitated with
ß-arrestin from the light vesicular membrane pool. Second, the loss
of all potential sites for GRK-mediated AT1AR
phosphorylation does not prevent ß-arrestin association with
AT1AR but completely prevents the localization of
ß-arrestin to endocytic vesicles. Third, the coexpression of the
dynamin I-K44A inhibitor of clathrin-mediated endocytosis prevented the
redistribution of ß-arrestin to endocytic vesicles. Finally, by
switching the carboxyl-terminal tails between the
AT1AR and the
ß2AR, we reversed the ability of these
receptors to internalize with ß-arrestin without preventing their
ability to stimulate ß-arrestin translocation. These findings are
consistent with other data showing that phosphorylation is required for
the agonist-dependent redistribution of ß-arrestin with both the
vasopressin V2 receptor and AT1AR to endocytic
vesicles (Ref. 24 and R. H. Oakley and M. G. Caron,
personal communication). Taken together, these observations suggest
that the signal for stable GPCR/ß-arrestin association involves
phosphorylation and that ß-arrestins remain associated with the
receptors as they traffic through the endosomal compartment. Future
experiments, using bioluminescent resonance energy transfer (BRET) will
be required to determine whether the association of ß-arrestin with
receptors in the endosomal compartment is either static or dynamically
regulated (34).
Recently, Oakley et al. (35) analyzed arrestin translocation
in response to the activation of multiple different GPCRs. In doing so,
they identified two distinct classes of GPCRs: class A GPCRs
(e.g. ß2AR), receptors that interact
with ß-arrestin2 with greater affinity than ß-arrestin1 and do not
interact with visual arrestin, and class B GPCRs (e.g.
AT1AR), receptors that bind equally well to both
ß-arrestin isoforms and also associated with visual arrestins.
Furthermore, using ß2AR/vasopressin V2 receptor
chimeras, Oakley et al. (35) demonstrated that the
carboxyl-terminal tails of the receptors determined the affinity of
receptors for ß-arrestins and visual arrestins. Interestingly, each
of the class B receptors promotes the redistribution of ß-arrestin to
endocytic vesicles (24). In the case of at least two class B GPCRs, the
vasopressin V2 receptor and AT1AR, GRK-mediated
phosphorylation is required for ß-arrestin redistribution to
endocytic vesicles. Our observation that deleting the tail of the
AT1AR to remove putative sites for GRK-mediated
phosphorylation (residues 326338) does not prevent ß-arrestin
translocation in response to AT1AR activation
suggests that phosphorylation stabilizes the complex between the
receptor and ß-arrestin. Thus, multiple domains contribute to the
association of ß-arrestins with GPCRs, but the tail appears to
regulate the affinity of these interactions. Consequently, the observed
differences in ß-arrestin affinity that is observed for class A
vs. class B receptors may be determined by differences in
the patterns and/or extent of GRK-mediated phosphorylation.
An important facet of ß2AR internalization is
to promote receptor dephosphorylation and resensitization. We find that
the internalization of ß-arrestin with the
ß2AR-ATCT prevented the dephosphorylation of
the ß2AR chimera, indicating that receptor dephosphorylation in the
endosomal compartment appears to require the dissociation of the
receptor/ß-arrestin complex (Fig. 8
). However, the
AT1AR-ß2CT chimera, which does not internalize
with ß-arrestin bound, was not dephosphorylated. It is proposed that
acidification of receptors in endosomes induces a conformational change
in the receptor that supports the association of a GPCR-specific
phosphatase (16, 36). This suggests that, in addition to the
dissociation of the receptor/ß-arrestin complex, receptors must
exhibit the capacity to serve as phosphatase substrates. It would
appear that either the AT1AR lacks domains
required for the binding of phosphatases or acidification in endocytic
vesicles does not induce receptor conformational changes in
AT1AR structure required for phosphatase
association. Alternatively, the lack of
AT1AR-ß2CT dephosphorylation might be the
consequence of a fortuitous interaction between intracellular domains.
However, all other receptor properties can be exchanged normally
between the receptors.
The data presented in this paper suggest the following model for the
intracellular trafficking and resensitization of the
ß2AR and AT1AR (Fig. 10
). In the absence of inhibitors, such
as dynamin I-K44A, agonist stimulation promotes the desensitization and
internalization of both the ß2AR and
AT1AR by a common mechanism, GRK phosphorylation
and ß-arrestin binding. The AT1AR internalizes
with ß-arrestin bound, whereas ß-arrestin dissociates from the
ß2AR shortly after the formation of endocytic
vesicles. In the case of the ß2AR,
acidification of the receptor in endocytic vesicles enhances receptor
dephosphorylation by a membrane-bound receptor-specific phosphatase
(16, 36), after which the receptor is rapidly recycled back to the cell
surface. In the case of the AT1AR, internalized
receptor is not dephosphorylated in endocytic vesicles due to both the
continued association of the receptor with ß-arrestin and the
inability of the receptor to serve as a phosphatase substrate. The
resensitization of AT1AR-mediated responses is
mediated by either the delayed recycling of internalized receptors back
to the cell surface, the surface reexpression of receptors that
were internalized in the absence of agonist stimulation, or the
mobilization of newly synthesized receptors to the cell surface (25, 28).

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|
Figure 10. Proposed Model for Difference in the Intracellular
Trafficking and Surface Reexpression of ß2AR and
AT1AR
Agonist activation of ß2AR and AT1AR in
HEK 293 cells leads to their desensitization and internalization by a
common mechanism involving GRK phosphorylation and ßarrestin binding.
Both receptors are internalized by clathrin-coated pits, and
internalized ß2AR is dephosphorylated and rapidly
recycled back to the cell surface, whereas the AT1AR
internalizes with ßarrestin and is neither dephosphorylated nor
recycled. AT1AR resensitization is either mediated by the
slow recycling of receptor back to the cell surface, de
novo receptor synthesis, or the recruitment of a reserve pool
of intracellular receptors. ßArr, ßarrestin; G, G protein; GRK, G
protein-coupled receptor kinase; and GRP, G protein-coupled receptor
phosphatase; A, agonist.
|
|
In summary, while many of the molecular mechanisms first
described using the ß2AR as a model apply to
other GPCRs (1, 2, 3), the recent characterization of a diverse variety of
GPCRs has revealed clear differences in the intracellular trafficking
agendas and resensitization patterns of many GPCRs (9, 18, 22, 24, 30, 31, 32, 33, 35, 37). In the present study, we have demonstrated that the
carboxyl-terminal receptor domains not only contribute to the
endocytosis of the ß2AR and
AT1AR, but that the stable association of
ß-arrestin with the AT1AR carboxyl-terminal
tail contributes to observed differences in the intracellular
trafficking, surface reexpression, and resensitization patterns of
these receptors. Therefore, while GPCR activation, desensitization, and
internalization may involve common mechanisms, observed differences in
receptor responsiveness may involve differences in the intracellular
trafficking patterns of GPCRs that result from differences in their
capacity to form stable complexes with their regulatory proteins.
 |
MATERIALS AND METHODS
|
---|
Materials
Human embryonic kidney cells (HEK 293) were provided by the
American Type Culture Collection (ATCC,
Manassas, VA). Tissue culture media and FBS were obtained from
Life Technologies, Inc. (Gaithersburg, MD). Isoproterenol
was purchased from Research Biochemicals International
(Natick, MA). Mouse anti-HA 12CA5 and anti-Flag monoclonal
antibodies were obtained from Roche Molecular Biochemicals
(Indianapolis, IN) and Research Diagnostic, Inc. (Flanders, NJ),
respectively. 125I-Pindolol was purchased from
NEN Life Science Products (Boston, MA). fluorescein
isothiocyanate (FITC)-conjugated goat antimouse secondary antibody and
angiotensin II and all other biochemical reagents were purchased from
Sigma.
DNA Construction
All recombinant cDNA procedures were carried out following
standard protocols. The ßarrestin1 mutant, 185418, was constructed
by PCR. 5'-Oligonucleotide primers introduced an amino-terminal
EcoRI restriction site, minimal Kozak sequence, and
initiation ATG at the appropriate site of ß-arrestin1, and
3'-oligonucleotide primers introduced a carboxyl-terminal
XhoI restriction site, stop codon, and Flag-epitope tag
sequence (DYKDDDDK) at the C terminus of ß-arrestin1. The
AT1AR carboxyl-terminal tail truncation mutants
were also constructed by PCR. 3'-Oligonucleotide primers were used to
introduce a stop codon after tyrosine residues 319 and 339 followed by
a XbaI restriction site. The 5'-oligonucleotide was
identical to the AT1AR sequence upstream of an
unique EcoRI site. The resulting PCR products were digested
with EcoRI and XbaI and subcloned into the
pcDNA3.1 AT1AR plasmid construct digested with
the same enzymes. The sequence integrity of the ß-arrestin and
AT1AR mutants was confirmed by DNA sequencing.
All other cDNA constructs used have been reported previously (8, 22, 27).
Cell Culture and Transfection
HEK 293 cells were grown in Eagles minimal essential medium
with Earles salt (MEM) supplemented with 10% (vol/vol)
heat-inactivated FBS and gentamicin (100 µg/ml). The cells were
seeded at a density of 2.5 x 106/100-mm
dish and transiently transfected with the cDNAs described in the figure
legends by a modified calcium phosphate method. After transfection
(
18 h) the cells were reseeded into 35-mm glass-bottomed culture
dishes (MarTek) for confocal studies or six-well Falcon dishes.
Receptor Expression
Receptor expression was 1,0003,000 fmol/mg whole-cell protein
for confocal studies and 1,0001,500 fmol/mg whole-cell protein for
internalization and phosphorylation studies.
AT1AR expression was matched to
ß2AR expression by flow cytometry, and
ß2AR expression was measured by saturating
125I-pindolol binding (8, 9, 38).
Subcellular Fractionation
HEK293 cells transfected with either
ß2AR or ß2AR-ATCT were
plated in 100-mm dishes, treated as described in the figure legends,
1.8 x 107 cells were scraped in 6 ml of 10
mM Tris, pH 7.4, with 1 mM EDTA, dounce
homogenized on ice (20 strokes), and spun at 500 x g
for 10 min to remove unbroken cells and nuclei. The supernatant was
loaded on a sucrose cushion [5 ml 35% (wt/vol) sucrose in 10
mM Tris, pH 7.4, with 1 mM
EDTA] and centrifuged for 90 min at 150,000 x g at 4
C. The plasma membrane pellets were solubilized for 2 h in 1 ml of
digitonin buffer [20 mM Tris-HCl, pH 8.0, 1%
(wt/vol) digitonin, 20% (vol/vol) glycerol, 300
mM NaCl, 1 mM EDTA
containing 0.1 mM phenylmethylsulfonyl fluoride,
10 µg/ml leupeptin, 5 µ g/ml aprotinin, and 1 µg/ml pepstatin).
The 35% sucrose interface (light vesicular fraction) was recovered,
diluted in 10 mM Tris, pH 7.4, with 1
mM EDTA and pelleted at 436,000 x
g for 90 min in an Optima TL ultracentrifuge (Beckman
Coulter, Inc., Fullerton, CA). The light vesicular pellets were
subsequently solubilized for 2 h in 1 ml of digitonin buffer.
Receptor Coimmunoprecipitation
Plasma membrane and light vesicular membrane fraction lysates
were cleared by centrifugation at 436,000 x g for 20
min at 4 C and incubated (1:500 dilution) with rabbit anti-GFP
polyclonal antibody (Molecular Probes, Inc., Eugene, OR)
overnight along with 100 µl of a 20% slurry of protein A Sepharose
beads (Pharmacia Biotech, Piscataway, NJ) in digitonin
buffer plus 3% BSA. The beads were recovered by centrifugation and
washed three times in PBS, pH 7.4, and incubated in 200 µl of PBS
containing a saturating concentration of
125I-pindolol (
2 nM) at
37 C for 2 h. The beads were recovered by centrifugation and
washed three times with PBS pH 7.4. The washed beads were counted in a
-counter. Nonspecific 125I-pindolol binding to
the beads was less than 10% of specific
125I-pindolol binding to
ß2AR in the absence of agonist (data not
shown).
Agonist-Independent Internalization Assays
The agonist-independent internalization of cell surface
receptors was measured by prelabeling cell surface epitope-tagged
receptors with primary mouse antiepitope tag antibody (1:500 dilution)
on ice for 45 min and then warming cells to 37 C in the absence of
agonist for the times indicated in the figure legends. Cells were then
transferred back to ice and labeled with the secondary FITC-conjugated
antimouse antibody (1:250 dilution) for 45 min. Receptor
internalization was defined as the fraction of total cell receptors
lost from the cell surface and thus not available to secondary
antibodies outside the cell.
Receptor Recycling Assays
Cells expressing epitope-tagged receptors were treated with or
without agonist for 30 min at 37 C, washed three times with serum-free
media, and either kept on ice or allowed to recover to 37 C for the
times indicated in the figure legends. The cells were antibody stained,
and the cell surface receptor expression was determined by flow
cytometry. Receptor recycling was defined as a recovery of cell surface
receptors accessible to antibodies outside the cell after the removal
of agonist when compared with the cell surface expression of receptors
in matched controls that were not exposed to agonist.
Confocal Microscopy
Confocal microscopy was performed on a LSM-510 laser scanning
microscope (Carl Zeiss, Thornwood, NY) using a Zeiss 63x
1.3 NA oil immersion lens. HEK 293 cells expressing
ß2AR, AT1AR, and
ß2AR/AT1AR chimeras with
and without dynaminI-K44A and low levels of either ß-arrestin2-GFP or
PKCßII-GFP were plated on 35-mm glass-bottomed culture dishes and
kept warm at 37 C in serum-free MEM on a heated microscope stage as
described previously (22). Clathrin staining of HEK 293 cells grown on
cover slips and fixed with 3.7% paraformaldehyde in PBS with 0.2%
Triton X-100 for 20 min was performed using the monoclonal antibody X22
(ATCC) in conjunction with a Texas red-conjugated goat
antimouse secondary antibody (Molecular Probes, Inc.).
Colocalization studies of ß-arrestin2-GFP and rhodamine-labeled
clathrin fluorescence were performed using dual excitation (488, 543
nm) and emission (505530 nm, GFP; 560 nm, rhodamine) filter sets.
Specificity of labeling and absence of signal cross-over were
established by examination of single-labeled samples.
Whole-Cell Phosphorylation
Receptor phosphorylation was performed as described previously
(38). In brief, the intracellular ATP pool was
[32P] labeled by incubating transfected cells
seeded in six-well dishes with
[32P]orthophosphate (100 µCi/ml) in
phosphate- and serum-free medium for 45 min at 37 C. Cells were then
treated with or without 1 µM isoproterenol for 10 min at 37 C and
washed three times with ice-cold PBS. Resensitized cells were allowed
to recover for 20 min at 37 C in agonist-free PBS. The cells were
solubilized in radioimmune precipitation buffer (150 mM
NaCl, 50 mM Tris, 5 mM EDTA, 10 mM
NaF, 10 mM Na2pyrophosphate, 1%
NP-40, 0.5% deoxycholate, 0.1% SDS, 0.1 mM
phenylmethysulfonyl fluoride, 10 µg/ml leupeptin, 5 µg/ml
aprotinin, 1 µg/ml pepstatin A, pH 7.4), and epitope-tagged receptors
were immunoprecipitated as described previously (38). In each
experiment, equivalent amounts of receptor protein, as determined by
receptor expression and the amount of solubilized protein, were
subjected to SDS-PAGE followed by autoradiography. The extent of
receptor phosphorylation was quantitated using a phosphorimaging system
and ImageQuant software (Molecular Dynamics, Inc.,
Sunnyvale, CA).
Data Analysis
The mean and SEM are expressed for values obtained
for the number of separate experiments indicated. Statistical
significance was determined by an unpaired two-tailed t
test. Time course data were analyzed using PRISM software
(GraphPad Software, Inc., San Diego, CA).
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank Drs. S. A. Laporte, M. G.
Caron, and D. J. Kelvin for helpful discussions and critical
reading of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Stephen S. G. Ferguson, Robarts Research Institute, 100 Perth Drive, P.O. Box 5015, London, Ontario, Canada N6A 5K8. E-mail: ferguson{at}rri.on.ca
P.H.A. is the recipient of a Merck Frosst Canada Postdoctoral
Fellowship. L.D. is the recipient of a John P. Robarts Research
Institute studentship. S.S.G.F. is the recipient of a McDonald
Scholarship Award from the Heart Stroke Foundation of Canada. This work
was supported by Heart and Stroke Foundation of Ontario Grants NA-3349
and T 4047 and Medical Research Council of Canada Grant MA-15506.
1 Abbreviations used in this paper:
AT1AR, angiotensin type 1A receptor;
AT1AR-ß2CT, AT1AR-based chimera with the
ß2AR carboxyl-terminal tail; ß2AR,
ß2-adrenergic receptor; ß2AR-ATCT,
ß2AR-based chimera with the AT1AR
carboxyl-terminal tail. 
Received for publication May 5, 2000.
Revision received August 9, 2000.
Accepted for publication August 23, 2000.
 |
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