From the Howard Hughes Medical Institute Laboratories, Departments of Cell Biology and Medicine, Duke University Medical Center, Durham, North Carolina 27710
Received for publication, February 14, 2001
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ABSTRACT |
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G protein-coupled receptors
(GPCRs)1 comprise a large
gene family of more than 1000 members that regulate a diverse array of physiological functions such as phototransduction, olfaction, neurotransmission, vascular tone, cardiac output, digestion, and pain.
The signaling of GPCRs is intimately controlled by a family of
intracellular proteins termed arrestins that includes visual arrestin,
The association of The ability of the V2R to form a stable complex with In the following study, we investigate the molecular determinants
underlying the formation of stable GPCR- Materials--
Neurotensin was purchased from Peninsula
Laboratories. Oxytocin, angiotensin II, substance P, and isoproterenol
were obtained from Sigma. The anti-hemagglutinin (HA) 12CA5 mouse
monoclonal antibody and the rhodamine-conjugated anti-HA 12CA5 mouse
monoclonal antibody were purchased from Roche Molecular Biochemicals.
[32P]Orthophosphate was purchased from PerkinElmer Life Sciences.
Plasmid DNA--
Construction of plasmids containing the
hemagglutinin epitope-tagged rat NTR, rat angiotensin II type 1A
receptor (AT1AR), and human
The HA-tagged human substance P (NK-1) receptor (SPR) was constructed
as described previously by adding the nine-amino acid HA epitope
(YPYDVPDYA) to the amino-terminal end of the human SPR cDNA (24).
The SPR-383X truncation mutant was generated by replacing nucleotides
ACC encoding Thr-383 of the HA-tagged SPR with nucleotides TGA encoding
a stop codon. The SPR-355X truncation mutant was generated by replacing
nucleotides GAG encoding Glu-355 of the HA-tagged SPR with nucleotides
TGA encoding a stop codon. The SPR-325X truncation mutant was generated
by replacing nucleotides TTC encoding Phe-325 of the HA-tagged SPR with
nucleotides TAG encoding a stop codon. The SPR-AAIAA mutant was
generated by replacing Thr-356, Thr-357, Ser-359, and Thr-360 of the
HA-tagged SPR with alanine residues. The SPR-APAA mutant was generated
by replacing Thr-376, Ser-378, and Ser-379 of the HA-tagged SPR with
alanine residues. Sequences of all DNA constructs were confirmed by DNA sequencing.
Cell Culture and Transfection--
Human embryonic kidney
(HEK-293) cells were provided by the American Type Culture Collection
(ATCC). HEK-293 cells stably expressing the Confocal Microscopy--
Transfected HEK-293 cells were plated
on 35-mm glass-bottom culture dishes. Two hours before the experiment,
the medium was replaced with serum-free medium supplemented with 10 mM HEPES. Confocal microscopy was performed on a Zeiss
laser-scanning confocal microscope (LSM-510) using a heated (37 °C)
microscope stage as described previously (14-16). Images were
collected sequentially using single line excitation (488 nm).
Colocalization of
Analysis of YFP- Receptor Sequestration--
Receptor sequestration was assessed
by flow cytometry as described previously (23). Transfected HEK-293
cells were incubated with or without the appropriate agonist for 30 min. Sequestration was defined as the fraction of total cell surface
receptors that, after exposure to agonist, were removed from the plasma
membrane and, thus, not accessible to antibodies from outside the cell.
Whole Cell Phosphorylation--
Receptor phosphorylation was
performed as described previously (14, 25). In brief, transfected
HEK-293 cells were labeled for 1 h at 37 °C with
[32P]orthophosphate (100 µCi/ml) in phosphate-free
medium. Cells were treated with or without the appropriate agonist for
10 min at 37 °C and then washed three times on ice with ice-cold
phosphate-buffered saline. All cells were scraped in radioimmune
precipitation buffer (150 mM NaCl, 50 mM Tris,
5 mM EDTA, 10 mM NaF, 10 mM
disodium pyrophosphate, 1% Nonidet P-40, 0.5% deoxycholate, 0.1%
SDS) containing a mixture of protease inhibitors and solubilized for
1 h at 4 °C. After centrifugation, supernatants were collected
and assayed for protein concentration (Bio-Rad DC protein assay kit).
HA-tagged receptors were immunoprecipitated at 4 °C using the
anti-HA 12CA5 mouse monoclonal antibody. Equivalent amounts of
receptor, as determined by receptor expression and the amount of
solubilized protein in each sample, were subjected to
SDS-polyacrylamide gel electrophoresis and processed for
autoradiography. Receptor expression was determined on a sample from
each transfection group by flow cytometry analysis as described
previously (26). Receptor phosphorylation was quantitated using a
Molecular Dynamics PhosphorImager and ImageQuant software.
Trafficking of
To determine whether Molecular Determinants Underlying the Trafficking of
To determine whether these clusters are important for the formation of
stable receptor-
For the OTR, mutation of the TSAS cluster to alanine residues
(OTR-AAAA) did not prevent
We next investigated whether the identified serine/threonine clusters
necessary for the formation of stable receptor- Molecular Determinants Underlying the Trafficking of
Our finding that
We next evaluated the phosphorylation status of the wild-type and
mutant SPR in transfected HEK-293 cells. In response to agonist, the
wild-type SPR and SPR-383X truncation mutant were phosphorylated to
similar levels, whereas no phosphorylation was observed for the
SPR-355X truncation mutant (Fig. 7). This
result suggests that the principal site of
agonist-dependent receptor phosphorylation is the TTIST
and/or TPSS cluster (Fig. 3B). Mutation of these clusters to
alanine residues (SPR-AAIAA and SPR-APAA), however, did not appreciably
reduce the level of SPR phosphorylation (Fig. 7). The SPR is
phosphorylated in an agonist-dependent manner by GRKs
rather than second messenger kinases (27), and the abundance of serine
and threonine residues (13 of the last 24 amino acids) downstream of
these two clusters may allow GRK phosphorylation to take place even in
the absence of the preferred site (28, 29).
Trafficking of a
Differences in the ability of
The agonist-activated In the present study, we identify a conserved motif in the
carboxyl-terminal tail of GPCRs that mediates the
agonist-dependent formation of stable receptor- The serine/threonine clusters mediating the formation of stable
receptor--Arrestins bind agonist-activated G
protein-coupled receptors (GPCRs) and mediate their desensitization and
internalization. Although
-arrestins dissociate from some receptors
at the plasma membrane, such as the
2 adrenergic receptor, they
remain associated with other GPCRs and internalize with them into
endocytic vesicles. Formation of stable receptor-
-arrestin complexes
that persist inside the cell impedes receptor resensitization, and the
aberrant formation of these complexes may play a role in GPCR-based
diseases (Barak, L. S., Oakley, R. H., Laporte, S. A.,
and Caron, M. G. (2001) Proc. Natl. Acad. Sci.
U. S. A. 98, 93-98). Here, we investigate the molecular
determinants responsible for sustained receptor/
-arrestin interactions. We show in real time and in live human embryonic kidney
(HEK-293) cells that a
-arrestin-2-green fluorescent protein conjugate internalizes into endocytic vesicles with agonist-activated neurotensin-1 receptor, oxytocin receptor, angiotensin II type 1A
receptor, and substance P receptor. Using receptor mutagenesis, we
demonstrate that the ability of
-arrestin to remain associated with
these receptors is mediated by specific clusters of serine and
threonine residues located in the receptor carboxyl-terminal tail.
These clusters are remarkably conserved in their position within the
carboxyl-terminal domain and serve as primary sites of
agonist-dependent receptor phosphorylation. In addition, we identify a
-arrestin mutant with enhanced affinity for the
agonist-activated
2-adrenergic receptor that traffics into endocytic
vesicles with receptors that lack serine/threonine clusters and
normally dissociate from wild-type
-arrestin at the plasma membrane.
By identifying receptor and
-arrestin residues critical for the
formation of stable receptor-
-arrestin complexes, these studies
provide novel targets for regulating GPCR responsiveness and treating
diseases resulting from abnormal GPCR/
-arrestin interactions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin-1, and
-arrestin-2 (1, 2). Arrestins bind
agonist-activated GPCRs at the plasma membrane that have been
phosphorylated by G protein-coupled receptor kinases (GRKs) on serine
and threonine residues located in the third intracellular loop or
carboxyl-terminal tail (3-5). The association of a single arrestin
with a GRK-phosphorylated receptor uncouples the receptor from its cognate G protein, resulting in the termination of GPCR signaling, a process termed desensitization (3-7). The nonvisual arrestins,
-arrestin-1 and
-arrestin-2, then target desensitized receptors to clathrin-coated pits for endocytosis by functioning as
adaptor proteins that link the receptor to components of the endocytic
machinery such as AP-2 and clathrin (8-11). The internalized receptors
are dephosphorylated in endosomes and recycled back to the cell surface
fully resensitized (12-14).
-arrestins with agonist-activated receptors at
the plasma membrane is a feature common to almost all GPCRs (15, 16).
However, the fate of these receptor-
-arrestin complexes differs
markedly among receptors (14, 16).
-Arrestins dissociate from some
receptors, such as the
2-adrenergic receptor (
2AR), at or near
the plasma membrane and are excluded from receptor-containing endocytic
vesicles. In contrast,
-arrestins remain associated with other
receptors, such as the vasopressin V2 receptor (V2R), and traffic with
them into endocytic vesicles. The stability of the
receptor-
-arrestin complex appears to regulate the rate of receptor
resensitization (14). Receptors that dissociate from
-arrestin at or
near the plasma membrane are rapidly dephosphorylated and recycled,
whereas receptors that remain associated with
-arrestin are slowly
dephosphorylated and recycled.
-arrestin is
mediated by a specific cluster of GRK -phosphorylated serine residues
in the receptor carboxyl-terminal tail (14). Whether a similar motif is
necessary for other GPCRs to remain associated with
-arrestin is
unknown. Identifying such residues is a critical goal of current
research not only because the strength of the receptor/
-arrestin
interaction controls GPCR responsiveness but also because the
inappropriate formation of these complexes may underlie the pathology
of several GPCR-based diseases. For example, a nonsignaling V2R mutant
associated with nephrogenic diabetes insipidus is complexed with
-arrestin in endocytic vesicles in the absence of agonist (17). The
normal plasma membrane localization and the ability of this
constitutively desensitized receptor to signal can be restored by
eliminating the cluster of serine residues that promotes the high
affinity V2R/
-arrestin interaction (17). Moreover, several recent
studies report that the continued association between rhodopsin and
visual arrestin may underlie some forms of retinal degeneration
(18-20).
-arrestin complexes that
persist inside the cell. We demonstrate in real time and in live cells
that the ability of
-arrestin to remain associated with a variety of
GPCRs is mediated by specific clusters of phosphorylated serine and
threonine residues strategically positioned within the receptor
carboxyl-terminal tail. The requirement for these phosphorylated
serine/threonine clusters can be bypassed, however, by mutations in
-arrestin that enhance its affinity for agonist-activated GPCRs.
Identification of the molecular determinants underlying the formation
of stable receptor-
-arrestin complexes provides novel targets for
manipulating the affinity of these two proteins and regulating GPCR responsiveness.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2AR have been described previously
(21-23).
-Arrestin-2 with GFP conjugated to its carboxyl terminus
(
arr2-GFP) and
-arrestin 1 with yellow fluorescent protein (YFP)
conjugated to its amino terminus (YFP-
arr1) have been described
previously (15, 16). All other constructs were generated by polymerase
chain reaction following standard protocols. The YFP-
arr1-383T
truncation mutant was constructed by replacing nucleotides GAT encoding
Asp-383 with nucleotides TAG encoding a stop codon in YFP-
arr1. The
NTR-AMAA mutant was generated by replacing Ser-407, Ser-409, and
Ser-410 of the HA-tagged NTR with alanine residues. The NTR-AAA mutant was generated by replacing Ser-415, Thr-416, and Ser-417 of the HA-tagged NTR with alanine residues. The HA-tagged human oxytocin receptor (OTR) was constructed by inserting the nine-amino acid sequence (YPYDVPDYA) recognized by the anti-HA 12CA5 mouse monoclonal antibody immediately after the amino-terminal initiator methionine of
the human OTR cDNA. The OTR-AAAA mutant was generated by replacing Thr-360, Ser-361, and Ser-363 of the HA-tagged OTR with alanine residues. The OTR-AAA-1 mutant was generated by replacing Ser-368, Ser-369, and Ser-370 of the HA-tagged OTR with alanine residues. The
OTR-AAA-2 mutant was generated by replacing Ser-377, Ser-378, and
Ser-379 of the HA-tagged OTR with alanine residues. The AT1AR-AALAA mutant was generated by replacing Ser-328, Ser-329, Ser-331, and Thr-332 of the HA-tagged AT1AR with alanine residues. The AT1AR-AALA mutant was generated by replacing Ser-335, Thr-336, and Ser-338 of the
HA-tagged AT1AR with alanine residues. The AT1AR-AAA mutant was
generated by replacing Ser-346, Ser-347, and Ser-348 of the HA-tagged
AT1AR with alanine residues. The V2R-AAA-1 mutant was generated by
replacing Ser-362, Ser-363, and Ser-364 of the HA-tagged V2R with
alanine residues as previously described (14).
2AR have been described
previously (16). Cells were grown in Eagle's minimal essential medium
with Earle's salt supplemented with 10% (v/v) heat-inactivated fetal
bovine serum and gentamicin (100 µg/ml). Transient transfections were
performed using a modified calcium phosphate coprecipitation method as
described previously (25). Twenty-four hours after transfection, cells were split into appropriate plates, and experiments were performed the
following day.
arr2-GFP with rhodamine-labeled receptors
was performed on transfected cells pre-incubated in serum-free medium
containing a rhodamine-conjugated anti-HA 12CA5 mouse monoclonal
antibody (1:100) for 45 min at 37 °C. Cells were then washed three
times with serum-free medium, treated with the appropriate agonist at
37 °C for 30 min, and imaged by confocal microscopy.
Arr2-GFP and
rhodamine-labeled receptor fluorescence were performed using dual
excitation (488, 568 nm) and emission (515-540 nm, GFP; 590-610 nm,
rhodamine) filter sets.
arr1 and YFP-
arr1-383T translocation to the
2AR was performed as described previously (16). In brief, HEK-293
cells stably expressing the
2AR were transiently transfected with
either YFP-
arr1 or YFP-
arr1-383T. Cells expressing low and
equivalent levels of YFP fluorescence were selected. The relative level
of YFP fluorescence (in intensity per pixel) was measured using the
range of interest analysis provided with the microscope software.
Settings on the microscope (laser power, pinhole size, detector gain,
amplifier offset, amplifier gain, etc.) were held constant within and
between experiments to ensure that cells expressing similar amounts of
the two
-arrestin proteins were compared. A saturating concentration
of isoproterenol was applied directly over the selected cells
immediately after the second image within a time series. The relative
level of YFP-
-arrestin fluorescence (in intensity per pixel) was
measured in a fixed area of the cytoplasm over the duration of the
experiment using the range of interest analysis. Data were analyzed
using a "plateau with exponential decay" nonlinear regression
function in GraphPad Prism.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Arrestin with the Agonist-activated NTR-1, OTR,
AT1AR, and SPR into Endocytic Vesicles--
We employed a functional
-arrestin-2-green fluorescent protein conjugate (
arr2-GFP) (15)
to visualize the association of
-arrestin with the agonist-occupied
NTR-1, OTR, AT1AR, and SPR in live HEK-293 cells. In the absence of
agonist,
-arrestin was uniformly distributed in the cytoplasm of
cells, as indicated by the homogeneous
arr2-GFP fluorescence (Fig.
1, 0 min). The addition of
agonist promoted the rapid redistribution of
-arrestin from the
cytoplasm to each of the receptors at the plasma membrane (Fig. 1,
2 min). The punctate pattern of
arr2-GFP fluorescence at
the plasma membrane reflects its localization with receptors in
clathrin-coated pits (9, 15, 21). With a longer agonist exposure,
arr2-GFP was observed to redistribute from the plasma membrane to
endocytic vesicles in each of the receptor-expressing cells (Fig. 1,
30 min). These vesicles, which were first detected within
3-5 min of agonist addition, grew in size and number and were still
observed after 1 h of agonist treatment.
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Fig. 1.
-Arrestin traffics with the
agonist-activated NTR-1, OTR, AT1AR, and SPR into endocytic
vesicles. HEK-293 cells were transiently transfected with
arr2-GFP and either the NTR-1, OTR, AT1AR, or SPR. The distribution
of
arr2-GFP was visualized before and after treatment with the
appropriate agonist. Shown are representative confocal microscopic
images of
arr2-GFP fluorescence in the same HEK-293 cells treated
with agonist for 0, 2, and 30 min at 37 °C.
-arrestin colocalized with the receptor
in these endocytic vesicles, we examined the distribution of receptor
and the distribution of
arr2-GFP in the same living HEK-293 cells
before and after agonist treatment. In the absence of agonist, receptor
immunofluorescence was present at the plasma membrane and
arr2-GFP
fluorescence was uniformly distributed in the cytoplasm, and no
colocalization was observed (data not shown). However, after a 30-min
treatment with agonist, receptor immunofluorescence (red)
and
arr2-GFP fluorescence (green) showed extensive
colocalization (yellow) in endocytic vesicles (Fig. 2). These results demonstrate that
-arrestin binds the agonist-activated NTR-1, OTR, AT1AR, and SPR at
the plasma membrane and traffics with them into endocytic vesicles.
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Fig. 2.
-Arrestin colocalizes with the
NTR-1, OTR, AT1AR, and SPR in endocytic vesicles in live HEK-293
cells. HEK-293 cells were transiently transfected with
arr2-GFP and either the NTR-1, OTR, AT1AR, or SPR. Cell surface
receptors were pre-labeled with the rhodamine-conjugated anti-HA mouse
monoclonal antibody and treated for 30 min with the appropriate agonist
at 37 °C. Shown are representative confocal microscopic images of
receptor immunofluorescence (red) and
arr2-GFP
fluorescence (green). Colocalization (yellow) of
arr2-GFP with the receptor is indicated in the overlay.
-Arrestin
with the NTR-1, OTR, and AT1AR into Endocytic Vesicles--
The
ability of the V2R to form a stable complex with
-arrestin that is
preserved in endocytic vesicles is mediated by a specific cluster of
three serine residues that resides in the receptor carboxyl-terminal
tail and is phosphorylated in response to agonist (14). The
carboxyl-terminal tails of the NTR-1, OTR, and AT1AR are very similar
in size to the V2R tail and contain multiple clusters of potential
phosphate acceptor sites (Fig.
3A). Clusters were defined as
serine/threonine residues occupying three out of three, three out of
four, or four out of five consecutive positions. Based on this
criterion, the NTR-1 contains two clusters (SMSS and STS), the OTR
contains three clusters (TSAS, SSS, and SSS), and the AT1AR contains
three clusters (SSLST, STLS, and SSS).
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Fig. 3.
The carboxyl-terminal tails of the NTR-1,
OTR, AT1AR, and SPR contain multiple clusters of serine and threonine
residues. A, amino acid composition of the V2R, NTR-1,
OTR, and AT1AR carboxyl-terminal tails beginning with the conserved
NPXXY motif, which marks the end of the seventh
transmembrane domain. Serine and threonine residues occupying three out
of three, three out of four, or four out of five consecutive positions
were designated as clusters (underlined). Receptor mutants
resulting from the mutation of individual clusters to alanine residues
are indicated below each wild-type receptor. The V2R cluster mutants
have been described previously and are shown here for comparison (14).
B, amino acid composition of the SPR carboxyl-terminal tail
beginning with the conserved NPXXY motif. Clusters, as
defined above, were removed by truncation or alanine substitution, and
the resulting receptor mutants are indicated below the wild-type SPR.
For both A and B, the specific clusters
identified in this study that mediate the formation of stable
receptor- -arrestin complexes are designated with a line
above the cluster, and putative sites of palmitoylation are indicated
with an asterisk.
-arrestin complexes that persist inside the cell, we
selectively mutated each cluster to alanine residues (Fig.
3A). The resulting receptor mutants were expressed in
HEK-293 cells and found to undergo agonist-dependent
sequestration at levels comparable to that observed for their wild-type
counterparts (data not shown). We next assessed the distribution of the
arr2-GFP fusion protein in cells expressing each receptor mutant
before and after a 30-min treatment with agonist. For the NTR-1,
mutation of the proximal SMSS cluster to alanine residues (NTR1-AMAA)
did not affect the ability of
arr2-GFP to traffic with this receptor into endocytic vesicles (Fig.
4A). In marked contrast,
however, mutation of the more distal STS cluster to alanine residues
(NTR1-AAA) essentially abolished the ability of
arr2-GFP to
internalize with the receptor (Fig. 4A).
arr2-GFP still
translocated to the NTR1-AAA mutant at the plasma membrane upon agonist
addition; however, even with a prolonged agonist incubation,
-arrestin did not redistribute with the receptor into endocytic
vesicles but rather remained at the plasma membrane in clathrin coated pits (Fig. 4A).
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Fig. 4.
Trafficking of
-arrestin with the NTR-1, OTR, and AT1AR into
endocytic vesicles is mediated by specific clusters of serine and
threonine residues in the receptor carboxyl-terminal tail. HEK-293
cells were transiently transfected with
arr2-GFP and either the
wild-type or mutant NTR-1 (A), OTR (B), or AT1AR
(C). Shown are representative confocal microscopic images of
arr2-GFP distribution in cells expressing the wild-type or mutant
receptors ~30 min after treatment with the appropriate agonist at
37 °C.
-arrestin from trafficking with this
receptor into endocytic vesicles (Fig. 4B). However, when either one of the two serine triplets was mutated to alanine residues (OTR-AAA-1 and OTR-AAA-2), the ability of
arr2-GFP to internalize with the receptor was eliminated (Fig. 4B). Two adjacent
clusters of serine and threonine residues were also found to meditate
the ability of the AT1AR to form a stable complex with
-arrestin. As
shown in Fig. 4C, mutation of the SSLST or STLS cluster to alanine residues (AT1AR-AALAA and AT1AR-AALA) essentially abolished the
ability of
arr2-GFP to traffic with the AT1AR into endocytic vesicles, whereas mutation of the more distal SSS cluster to alanine residues (AT1AR-AAA) had no effect. These results demonstrate that
specific clusters of serine and threonine residues located in the
receptor carboxyl-terminal tail promote the formation of high affinity
receptor-
-arrestin complexes that remain together inside the cell
after receptor endocytosis. In the absence of this motif,
receptor-
-arrestin complexes are presumably less stable and
dissociate at or near the plasma membrane, and
-arrestin is excluded
from receptor containing endocytic vesicles.
-arrestin complexes
were actually phosphorylated in response to agonist. For these
experiments, whole cell phosphorylations were performed on HEK-293
cells expressing wild-type or mutant receptors. Compared with their
wild-type counterparts, the agonist-induced phosphorylation of the
NTR1-AAA mutant was reduced by 95% (Fig.
5A), the agonist-induced phosphorylation of the OTR-AAA-1 and OTR-AAA-2 mutants was each reduced
by 32% (Fig. 5B), and the agonist-induced phosphorylation of the AT1AR-AALAA and AT1AR-AALA mutants was each reduced by ~67%
(Fig. 5C). These results show that the clusters of serine and threonine residues necessary for the continued association of
-arrestin with the NTR-1, OTR, and AT1AR are important sites of
agonist-dependent receptor phosphorylation. In addition,
because clusters not essential for the high affinity interaction may
also be phosphorylated (NTR-AMAA and AT1AR-AAA mutants), these results suggest that the phosphorylated clusters responsible for the stable receptor-
-arrestin complex must be properly positioned within the
receptor carboxyl-terminal tail.
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Fig. 5.
Agonist-dependent phosphorylation
of the wild-type and mutant NTR-1, OTR, and AT1AR. HEK-293 cells
were transiently transfected with the wild-type or mutant NTR-1
(A), OTR (B), or AT1AR (C). After
treating the transfected cells for 10 min at 37 °C with vehicle
(CON) or the appropriate agonist (neurotensin
(NT), oxytocin (OT), or angiotensin II
(AII)), receptors were immunoprecipitated using the anti-HA
mouse monoclonal antibody and assayed for phosphorylation as described
under "Experimental Procedures." The right panels
show representative autoradiographs, and the left
panels show the mean ± S.E. of three to four independent
experiments quantified by PhosphorImager analysis.
-Arrestin
with the SPR into Endocytic Vesicles--
The SPR carboxyl-terminal
tail is much longer than the tails of the V2R, NTR-1, OTR, and AT1AR
and contains four clusters of serine and threonine residues (TTIST,
TPSS, SSRS, and SFSS) (Fig. 3B). To determine if one or more
of these clusters mediates the ability of the SPR to form a stable
complex with
-arrestin, we initially truncated the receptor at
position 383 (SPR-383X) to eliminate the two most distal clusters and
at position 355 (SPR-355X) to eliminate all four clusters (Fig.
3B). The agonist-activated SPR-383X mutant recruited
arr2-GFP into endocytic vesicles just as well as the wild-type SPR
(Fig. 6, compare upper left
and upper middle panels). In contrast, the ability of the
SPR-355X mutant to recruit
arr2-GFP into endocytic vesicles was
severely compromised (Fig. 6, compare upper left and
upper right panels). A small amount of
-arrestin
internalized with the SPR-355X mutant; however, much more
-arrestin
was now found at the plasma membrane in clathrin-coated pits and in a
uniform distribution back in the cytoplasm. These results suggested
that the TTIST and/or TPSS clusters play an important role in promoting
a high affinity SPR·
-arrestin complex; therefore, we selectively
mutated each of these clusters to alanine residues (SPR-AAIAA and
SPR-APAA) (Fig. 3B). Trafficking of
arr2-GFP with the
SPR-APAA mutant was indistinguishable from its trafficking with the
wild-type SPR (Fig. 6, compare upper left and lower
middle panels). In contrast, the ability of
-arrestin to
traffic with the SPR-AAIAA mutant into endocytic vesicles was impaired,
as indicated by the reduced amount of
-arrestin in endocytic
vesicles and the increased amount at the plasma membrane (Fig. 6,
compare upper left and lower left images). Thus,
it appears that the TTIST cluster is important for the formation of
high affinity SPR·
-arrestin complexes that persist inside the cell
after receptor endocytosis.
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Fig. 6.
Trafficking of
-arrestin with the SPR into endocytic vesicles is
mediated by a specific cluster of serine and threonine residues in the
receptor carboxyl-terminal tail and by additional residues outside the
receptor carboxyl-terminal tail. HEK-293 cells were transiently
transfected with
arr2-GFP and either the wild-type or mutant SPR.
Shown are representative confocal microscopic images of the
arr2-GFP
distribution in cells expressing the wild-type or mutant SPR after a
30-min treatment with 1 µM substance P at 37 °C.
arr2-GFP still traffics, albeit weakly, with the
SPR-355X mutant into endocytic vesicles suggests that other receptor
residues may also be involved in promoting a stable SPR·
-arrestin complex. To investigate the contribution made by the remaining serine
and threonine residues in the tail of the SPR-355X mutant, we truncated
the receptor at position 325 (SPR-325X) (Fig. 3B). This
receptor mutant is missing almost the entire carboxyl-terminal tail and
is devoid of serine and threonine residues downstream of the
NPXXY motif. However, as shown in the lower right image of
Fig. 6, the agonist-activated SPR-325X mutant still recruited a small
amount of
arr2-GFP into endocytic vesicles. These data suggest that
residues outside the SPR carboxyl-terminal tail are responsible for the
residual amount of
-arrestin recruited into endocytic vesicles with
the SPR-355X and SPR-AAIAA mutants (Fig. 6).
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Fig. 7.
Agonist-dependent phosphorylation
of the wild-type and mutant SPR. HEK-293 cells were transiently
transfected with the wild-type or mutant SPR. After treating the
transfected cells for 10 min at 37 °C with vehicle (CON)
or substance P (SP), receptors were immunoprecipitated using
the anti-HA mouse monoclonal antibody and assayed for phosphorylation
as described under "Experimental Procedures." The upper
panel shows a representative autoradiograph, and the lower
panel shows the mean ± S.E. of two independent experiments
quantified by PhosphorImager analysis.
-Arrestin 1 Truncation Mutant with GPCRs
Lacking Serine and Threonine Clusters into Endocytic Vesicles--
If
the importance of serine/threonine clusters in the formation of stable
GPCR·
-arrestin complexes is simply to enhance the affinity of the
receptor/
-arrestin interaction, then it might be possible to enhance
the affinity of this interaction in some other way. Removal of the last
36 amino acids of
-arrestin-1 produces a truncated protein
(
arrestin-1-383T) that desensitizes the
2AR more rapidly than
the wild-type
-arrestin-1 (30). This difference in desensitization
kinetics suggests that the mutant
-arrestin-1 has higher affinity
for the
2AR than does wild-type
-arrestin-1. We measured the
relative affinity of these two proteins for the
2AR by fusing the
YFP to the amino terminus of
-arrestin-1 (YFP-
arr1) and
-arrestin-1-383T (YFP-
arr1-383T). We then compared the ability
of these YFP-tagged
-arrestins to bind the agonist-activated
2AR
in real time and in live cells. As shown in Fig.
8A, both
-arrestins
redistributed from the cytoplasm to the receptor at the plasma membrane
upon agonist addition. However, as indicated by the increase in
fluorescence at the plasma membrane, YFP-
arr1-383T translocated
faster and to a greater extent than YFP-
arr1. In fact, the robust
translocation of
-arrestin-1-383T cleared out the cytoplasm and
revealed the pool of YFP-
arr1-383T left behind in the nucleus.
View larger version (60K):
[in a new window]
Fig. 8.
-Arrestin-1-383T binds
the
2AR with higher affinity than
-arrestin-1. HEK-293 cells stably
overexpressing the
2AR were transiently transfected with YFP-
arr1
or YFP-
arr1-383T. Single cells expressing equivalent amounts of the
two
-arrestin isoforms were selected as described under
"Experimental Procedures." A, distribution of
YFP-
arr1 and YFP-
arr1-383T as visualized with the confocal
microscope before (0 s) and after (25 and 240 s) treatment with 10 µM isoproterenol. B, quantitation of
YFP-
arr1 and YFP-
arr1-383T translocation to the
2AR.
-Arrestin fluorescence in the cytoplasm of cells was measured from
confocal images collected every 24.8 s (scan time = 3.9 s). The agonist was added (arrow) immediately after the
second scan. Data represent the mean ± S.E. of three to four
independent experiments (n = 22-26 cells) and were
analyzed using a plateau with exponential decay nonlinear regression
function in GraphPad Prism.
-arrestin-1 and
-arrestin-1-383T
to translocate to the
2AR were quantitated by measuring the
time-dependent loss of
-arrestin fluorescence from the
cytoplasm after treatment with agonist. Analysis of these data revealed three major differences in the translocation profiles (Fig.
8B). First,
-arrestin-1-383T began translocating sooner
than
-arrestin-1. The delay in time between agonist addition
(arrow) and the initial loss of
-arrestin from the
cytoplasm was less than 1 s for YFP-
arr1-383T and ~12 s for
YFP-
arr1. Second,
-arrestin-1-383T translocated faster than
-arrestin-1. The half-life of
-arrestin depletion from the
cytoplasm was 27.6 ± 0.9 s for YFP-
arr1-383T and 41 ± 1.9 s for YFP-
arr1. Third,
-arrestin-1-383T translocated
to a much greater extent than
-arrestin-1. The fraction of
cytoplasmic
-arrestin that translocated to the plasma membrane was
73.4 ± 0.4% for YFP-
arr1-383T and only 31.6 ± 0.3%
for YFP-
arr1. These results demonstrate that the
-arrestin-1-383T truncation mutant has a much higher affinity for
the
2AR than the wild-type
-arrestin-1.
2AR, which lacks serine/threonine clusters in
its carboxyl-terminal tail, dissociates from
-arrestin at the plasma
membrane and internalizes without
-arrestin into endocytic vesicles
(14). However, this receptor can be made to form a stable complex with
-arrestin that persists inside the cell after receptor endocytosis
by adding a serine cluster to its carboxyl-terminal tail (14). If the
molecular mechanism underlying the formation of stable
receptor-
-arrestin complexes is simply a high affinity
receptor/
-arrestin interaction, then the increased affinity of the
-arrestin-1-383T truncation mutant for the
2AR may allow it to
remain associated with this receptor independent of serine/threonine
clusters. We tested this hypothesis by assessing the distribution of
YFP-
arr1 and YFP-
arr1-383T in cells expressing the
2AR after
a 30-min treatment with agonist. As expected, YFP-
arr1 dissociated
from the receptor at the plasma membrane and was observed in
clathrin-coated pits (Fig. 9A,
left panel). In marked contrast, YFP-
arr1-383T
trafficked with the
2AR into endocytic vesicles (Fig. 9A,
right panel). We also tested whether YFP-
arr1-383T could
form a stable complex with the NTR-AAA and V2R-AAA-1 mutants since
these receptors are missing the specific serine cluster needed to
promote a high affinity receptor/
-arrestin interaction. As shown in
Fig. 9B, YFP-
arr1-383T remained associated with these
receptor mutants and trafficked with them into endocytic vesicles.
View larger version (71K):
[in a new window]
Fig. 9.
-Arrestin-1-383T traffics with
GPCRs lacking serine and threonine clusters into endocytic
vesicles. A, HEK-293 cells stably overexpressing the
2AR were transiently transfected with YFP-
arr1 or
YFP-
arr1-383T. Shown are representative confocal microscopic images
of the distribution of YFP-
arr1 and YFP-
arr1-383T after a 30-min
treatment with 10 µM isoproterenol at 37 °C.
B, HEK-293 cells were transiently transfected with
YFP-
arr1 or YFP-
arr1-383T and either the NTR-AAA mutant
(upper panel) or the V2R-AAA-1 mutant (lower
panel). Shown are representative confocal microscopic images of
the distribution of YFP-
arr1 and YFP-
arr1-383T after a 30-min
treatment with the appropriate agonist at 37 °C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin
complexes that persist inside the cell after GPCR internalization. This
motif consists of serine and threonine residues that are clustered
together within the receptor carboxyl-terminal tail and serve as
primary sites of agonist-dependent receptor
phosphorylation. In the absence of this motif, the
receptor/
-arrestin interaction is less stable, and the
agonist-occupied receptors dissociate from
-arrestin at or near the
plasma membrane. We also identify a mutation in
-arrestin that
allows it to form stable complexes with agonist-activated GPCRs that
lack serine/threonine clusters. These studies, by identifying receptor
and
-arrestin residues critical for the formation of high affinity
receptor-
-arrestin complexes, provide novel targets for regulating
GPCR responsiveness and treating diseases resulting from abnormal
GPCR/
-arrestin interactions.
-arrestin complexes show a significant degree of conservation in their relative position within the receptor
carboxyl-terminal tail. For example, the clusters are 36, 45, 38/47,
25/32, and 50 amino acids downstream of the NPXXY motif for
the V2R, NTR-1, OTR, AT1AR, and SPR, respectively (Fig. 3). For the
subgroup of receptors with carboxyl-terminal tails of similar length
and containing putative sites of palmitoylation (V2R, NTR-1, and OTR),
the conservation of the cluster location is even more remarkable. The
serine/threonine clusters are 36, 45, and 38/47 amino acids downstream
of the NPXXY motif; 19, 26, and 20/29 amino acids downstream
of the putative sites of palmitoylation; and 7, 7 and 10/19 amino acids
upstream from the end of the carboxyl-terminal tail (Fig.
3A). Because the cluster motif is essentially absent from
the carboxyl-terminal tails of GPCRs that form unstable complexes with
-arrestin (Fig. 10A), it
should be possible to predict whether a given receptor will bind
-arrestin with low or high affinity by simply analyzing the amino
acid sequence of the receptor carboxyl-terminal tail. This in turn will
provide valuable information as to how the cellular complement of
arrestin isoforms might regulate the pattern of receptor
desensitization, sequestration, and resensitization (16, 31).
View larger version (20K):
[in a new window]
Fig. 10.
Serine/threonine clusters in the
carboxyl-terminal tails of other GPCRs. A, amino acid
composition of the 2AR,
1B-adrenergic receptor
(
1BAR), dopamine D1A receptor (D1AR), µ opioid receptor (MOR), and endothelin type A receptor
(ETAR) carboxyl-terminal tails beginning with the conserved
NPXXY motif. Each of these agonist-activated receptors
dissociates from
-arrestin at or near the plasma membrane (16).
B, amino acid composition of the proteinase-activated
receptor 2 (PAR2) carboxyl-terminal tail beginning with the
conserved NPXXY motif. This receptor has recently been
reported to form stable complexes with
-arrestin that persist inside
the cell after receptor endocytosis (39, 40). For both A and
B, clusters of serine/threonine residues are indicated
(underlined).
Binding of -arrestin to agonist-activated GPCRs at the plasma
membrane is thought to involve multiple interactions (2, 32). A large
region within the amino-terminal half of
-arrestin, termed the
activation recognition domain, binds the second and/or third
intracellular loops of the receptor (33). This is followed by the
binding of a smaller, positively charged region in the central portion
of
-arrestin, termed the phosphorylation recognition domain, to the
GRK-phosphorylated receptor carboxyl-terminal tail (34).
Phosphorylation of a cluster of serine and threonine residues would
produce a localized concentration of negative charges, and the ionic
interaction between the cluster of negative charges in the receptor
carboxyl-terminal tail and the cluster of positive charges in the
phosphorylation recognition domain of
-arrestin might be sufficient
to stabilize the receptor-
-arrestin complex. Alternatively,
engagement of the phosphorylated serine/threonine cluster may induce a
conformational change in
-arrestin that allows a subsequent high
affinity interaction between the receptor and
-arrestin to take
place (35). In favor of the latter model is our finding that the
-arrestin-1-383T truncation mutant remains associated with GPCRs
lacking serine/threonine clusters. Removal of the
-arrestin-1
carboxyl terminus may constitutively expose the high affinity receptor
binding site and allow the mutant
-arrestin to bind GPCRs in the
absence of phosphorylation (30).
The stability of the GPCR·-arrestin complex has been shown to
regulate the kinetics of receptor resensitization. For example, the
ability of
-arrestin to remain associated with the V2R impairs the
efficient recycling of this receptor back to the plasma membrane (14).
In agreement with these findings, we show that the NTR-1 and SPR form
stable complexes with
-arrestin, and both these receptors have been
reported to recycle poorly (36, 37). However, the stability of the
receptor-
-arrestin complex may also be involved in initiating and/or
regulating other signaling pathways. Recent studies have demonstrated
that
-arrestin-bound internalized receptors are important for
GPCR-mediated activation of mitogen-activated protein kinases.
-Arrestin appears to function as a molecular scaffold that organizes
and recruits components of the mitogen-activated protein kinase cascade
after binding the agonist-activated GPCR (44). For example, stimulation
of the AT1AR activates the c-Jun amino-terminal kinase 3 (JNK3), which
colocalizes with the receptor and
-arrestin in endocytic vesicles
(38). In addition, activation of the extracellular signal-regulated
kinases 1 and 2 (ERK1/2) by the proteinase-activated receptor 2 requires the association of receptor,
-arrestin, and ERK1/2 in
endocytic vesicles (39). Proteinase-activated receptor 2 has been
reported to form stable complexes with
-arrestin that persist inside
the cell (39, 40), and its carboxyl-terminal tail contains three
serine/threonine clusters occupying positions very similar to those
occupied by the clusters found in the tails of the V2R, NTR, and OTR
(Fig. 10B, compare with Fig. 3A). For both the
AT1AR and proteinase-activated receptor 2, the prolonged association
between receptor,
-arrestin, and mitogen-activated protein kinase
may ensure the proper localization, specificity, and/or duration of the
mitogen-activated protein kinase response.
In summary, we have identified a motif in the carboxyl-terminal tail of
GPCRs that promotes the formation of stable receptor--arrestin complexes. In addition, we have shown that similar stable
receptor-
-arrestin complexes can be achieved with GPCRs lacking this
motif by mutations in
-arrestin that enhance its affinity for
receptors. Understanding the molecular determinants underlying high
affinity GPCR/
-arrestin interactions may be useful in a variety of
pathophysiological conditions. For example, reducing the affinity of
the receptor-
-arrestin complex may restore signaling to
nonfunctional V2R mutants that are constitutively bound to
-arrestin
and responsible for some forms of nephrogenic diabetes insipidus (17).
Alternatively, increasing the affinity of the receptor/
-arrestin
interaction may be an effective treatment for certain forms of
hyperthyroidism and familial precocious puberty that result from
constitutively active GPCRs (41, 42). Finally, modulating the stability
of GPCR·
-arrestin complexes may enhance the efficacy of
GPCR-acting drugs by altering the kinetics of receptor desensitization
and/or resensitization (43).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Marc Thibonnier, Case Western Reserve University, for the oxytocin receptor cDNA; Dr. Jürgen Wess, National Institutes of Health, for the vasopressin V2 receptor cDNA; and Dr. Shigetada Nakanishi, Kyoto University for the neurotensin receptor.
![]() |
FOOTNOTES |
---|
Recipient of a fellowship award from the Canadian Institutes of
Health Research.
§ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, Box 3287, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-5433. Fax: 919-681-8641. E-mail: caron002@mc.duke.edu
Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.M101450200
This work was supported in part by National Institutes of Health Grants NS 19576 (to M. G. C.) and HL 61365 (to L. S. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G
protein-coupled receptor;
GRK, G protein-coupled receptor kinase;
2AR,
2-adrenergic receptor;
V2R, vasopressin V2 receptor;
NTR-1, neurotensin-1 receptor;
OTR, oxytocin receptor;
AT1AR, angiotensin II
type 1A receptor;
SPR, substance P receptor;
GFP, green fluorescent
protein;
YFP, yellow fluorescent protein;
HA, hemagglutinin;
HEK, human
embryonic kidney..
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