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J. Biol. Chem., Vol. 277, Issue 45, 43247-43252, November 8, 2002
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2-Adrenergic Receptors Determines Subtype
Specificity of Arrestin Interaction*
,¶
From the Department of Microbiology and Immunology,
Kimmel Cancer Center, Thomas Jefferson University, Philadelphia,
Pennsylvania 19107 and the § Department of Pharmacology,
Vanderbilt University Medical Center, Nashville, Tennessee 37232
Received for publication, July 25, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Nonvisual arrestins (arrestin-2 and -3) serve as
adaptors to link agonist-activated G protein-coupled receptors
to the endocytic machinery. Although many G protein-coupled receptors
bind arrestins, the molecular determinants involved in binding remain
largely unknown. Because arrestins selectively promote the
internalization of the G protein-coupled receptors
(GPCRs)1 transduce
extracellular stimuli into intracellular signaling via coupling to
heterotrimeric guanine nucleotide-binding proteins (G proteins) (1). To
ensure that stimuli are translated into signals of appropriate
magnitude and specificity, these signaling cascades are tightly
regulated. GPCRs are subject to three principal modes of regulation:
desensitization, in which a receptor becomes refractory to continued
stimuli; endocytosis, whereby receptors are removed from the cell
surface; and down-regulation, in which total cellular receptor levels
are decreased (2, 3). Although multiple mechanisms contribute to these
regulatory processes, GPCR phosphorylation by G protein-coupled
receptor kinases (GRKs) and subsequent binding of arrestins plays an
important role in the regulation of many GPCRs (2).
Four mammalian arrestins have been identified with arrestin-1 and -4 being specific to the visual system and arrestin-2 and -3 (also termed
Three Previous studies have demonstrated an important role for the third
intracellular loops of the The subtype-specific differences in arrestin sensitivity for the three
Plasmid Construction--
FLAG-tagged Expression and Purification of GST- Purified Arrestin Binding to GST- Cell Culture and Transfection--
HEK 293 cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 100 µg/ml streptomycin, and 100 units/ml penicillin at
37 °C in a humidified atmosphere containing 5%
CO2. HEK 293 cells grown to 50-75% confluence in 60-mm dishes were transfected with 3 µg of FLAG-tagged wild type or
mutant (KR, RR, 3R, RR/3R) Analysis of Phospho-ERK1/2--
HEK 293 cells in 60-mm dishes
were transfected as described above and then split into three wells of
a poly-l-lysine-coated 12-well dish. The following day, cells were
serum-starved overnight in Dulbecco's modified Eagle's medium
containing 0.5% fetal bovine serum. The next day, cells were
stimulated with 10 µM UK14304 at 37 °C for 0-30 min
and then rinsed with PBS, lysed by addition of SDS sample buffer, and
scraped off the plates. Samples were boiled and then electrophoresed on
10% SDS-polyacrylamide gels. The gels were transferred to
nitrocellulose and blocked for 30 min in Tris-buffered saline
containing 0.1% Tween 20 and 5% non-fat dry milk. Phospho-ERK1/2 was
detected as described previously (25).
Arrestins Differentially Bind to Identification of Arrestin-3 Binding Domains within the
To define specific residues within NT1 and CT3 that contribute to
arrestin binding, a series of alanine point mutants were generated and
tested for their ability to bind arrestin-3. Point mutations introduced
into the NT1 and CT3 are indicated by an asterisk in Fig.
3, A and B,
respectively, whereas double mutations are underlined. These
studies revealed that mutation of basic residues within the NT1 and CT3
constructs resulted in a dramatic reduction in arrestin-3 binding.
Specifically, mutation of Arg-201, Arg-204, and Arg-205 in NT1
effectively disrupted arrestin-3 binding, whereas mutation of Arg-207
partially disrupted binding (Fig. 3A). In contrast, mutation
of Lys-200, Ser-202, Asn-203, Pro-205, and Arg-206 in NT1 had no
significant effect on arrestin-3 binding. The placement of essential
residues involved in arrestin binding was interesting given recent
studies demonstrating that mutation of Lys-382 in the third loop of the
parathyroid hormone receptor reduced arrestin-promoted internalization
(40), whereas residues in the LH/CG receptor important for
arrestin-promoted internalization are localized within the analogous
region of the third intracellular loop (38). Mutational analysis of CT3
also revealed the involvement of basic residues in arrestin binding
with mutation of several arginines (358, 359, 360, 365, and 368) as
well as Lys-367, resulting in a significant reduction in arrestin-3
binding (Fig. 3B). In contrast, mutation of Leu-344 and -345 and Gln-355 had a partial effect on binding, whereas Gln-362, Thr-364,
and Glu-366 mutations had no effect on arrestin-3 binding. The
important role of the proximal and distal ends of the third
intracellular loop in arrestin binding is reminiscent of the domains
implicated in G protein binding and activation (27). In fact,
previous studies have identified a BBXXB motif (where B is a
basic residue and X is any residue) in the
To verify that specific arrestin binding mutations made within the NT1
and CT3 constructs were important for arrestin binding in the context
of the whole third loop, a series of GST- Internalization of Wild Type and Mutant Signaling of Wild Type and Mutant Conclusions--
Our results on the NT1 region of the
At least two sites within arrestin are involved in GPCR binding, a
phosphorylation recognition site that binds to
phosphoserines/threonines on the receptor and an activation recognition
site that binds the agonist-bound conformation of the receptor (31).
Arrestin binding to phosphoserines/threonines is thought to destabilize the arrestin polar core and promote secondary high affinity binding to
the receptor and binding to the phospholipid bilayer (42). In our
studies, the third loops were not phosphorylated; however, arrestin
binding sites that would normally be inaccessible in the unactivated
holo-receptor are likely available for arrestin binding in the isolated
third intracellular loops. One possible reason why the
It is important to note that although the RR and RR/3R mutant
In summary, we have identified two discrete arrestin-3 binding domains
within the 
2b- and
2c-adrenergic receptors (ARs) while having no effect on
the 2aAR, here we used 2ARs to identify
molecular determinants involved in arrestin binding. Initially, we
assessed the ability of purified arrestins to bind glutathione
S-transferase fusions containing the third intracellular
loops of the 2aAR, 2bAR, or
2cAR. These studies revealed that arrestin-3 directly binds to the 2bAR and 2cAR but not the
2aAR, whereas arrestin-2 only binds to the
2bAR. Truncation mutagenesis of the 2bAR
identified two arrestin-3 binding domains in the third intracellular
loop, one at the N-terminal end (residues 194-214) and the
other at the C-terminal end (residues 344-368). Site-directed
mutagenesis further revealed a critical role for several basic residues
in arrestin-3 binding to the 2bAR third intracellular
loop. Mutation of these residues in the holo-2bAR and
subsequent expression in HEK 293 cells revealed that the mutations had
no effect on the ability of the receptor to activate ERK1/2. However,
agonist-promoted internalization of the mutant 2bAR was
significantly attenuated as compared with wild type receptor. These
results demonstrate that arrestin-3 binds to two discrete regions
within the 2bAR third intracellular loop and that
disruption of arrestin binding selectively abrogates agonist-promoted
receptor internalization.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-arrestin-1 and -2) being ubiquitously expressed (4-7). Arrestin
binding to activated-phosphorylated GPCRs results in the physical
uncoupling of receptor from G protein, a process that functions to
terminate agonist-mediated signaling. The two nonvisual arrestins also
directly interact with clathrin (8), the adaptor protein AP2 (9), and
phosphoinositides (10) to promote GPCR internalization. Indeed,
nonvisual arrestins have been implicated in the desensitization and
internalization of a wide variety of GPCRs including members of the
class 1 (rhodopsin-like) and class 2 (secretin-like) families (2, 11).
Nevertheless, despite numerous studies that demonstrate an essential
role for arrestins in the regulation of GPCR signaling and trafficking, the precise molecular determinants within GPCRs required for arrestin binding have not been thoroughly characterized.
2-adrenergic receptors, 2aAR,
2bAR, and 2cAR, have been identified
(12-14). 2ARs are activated by the catecholamines epinephrine and norepinephrine and regulate sympathetic outflow and
cardiovascular function in vivo (15-17). All three
2AR subtypes primarily couple to the Gi family of G
proteins and modulate a variety of signaling pathways including
activation of phospholipase A2 (18), phospholipase D (19),
and extracellular regulated kinases ERK1/2 (20-22) and inhibition of
adenylyl cyclase (23). 2AR signaling is also subject to
dynamic regulation. The 2aAR and 2bAR are
subject to agonist-dependent phosphorylation by GRKs (24).
Moreover, previous studies have revealed a role for arrestins in
agonist-promoted internalization of 2ARs with arrestin-3 promoting internalization of the 2bAR and
2cAR and arrestin-2 selectively promoting
internalization of the 2bAR. Interestingly, 2aAR internalization was not promoted by either
arrestin, suggesting arrestin/receptor binding specificity (25).
2ARs in mediating
protein-protein interaction. These loops are quite large (>150 amino
acids) and include sites for GRK phosphorylation (26), Gi activation
(27), and binding of 14-3-3 (28), sphinophilin (29), and arrestin (30).
Although arrestin binding to GPCRs is dependent on both the
phosphorylation and activation state of the receptor (31), the receptor
domains that mediate the agonist-dependent nature of
arrestin binding have not been thoroughly characterized. Because third
intracellular loops mediate agonist-dependent binding and activation of heterotrimeric G proteins (32, 33), it seems likely that
specific regions of the third intracellular loop might also confer the
agonist dependence and selectivity of arrestin binding. Indeed, the
third intracellular loop has been implicated in arrestin interaction
for a number of GPCRs including rhodopsin (34),
2a-adrenergic (30), M2 and M3
muscarinic (30), 
-opioid (35), 5-hydroxytryptamine2A
(36), CXCR4 (37), and the luteinizing hormone/choriogonadotropin
(LH/CG) receptor (38).
2ARs provides a useful model to identify specific regions involved in arrestin binding. To address this issue, we studied
arrestin binding to a series of glutathione S-transferase (GST) fusion proteins containing various regions of the third intracellular loops of the three 2AR subtypes. Our
results revealed arrestin binding specificity that recapitulates the
arrestin selectively observed previously in 2AR
trafficking (25). Truncation and site-directed mutagenesis revealed
that arrestin-3 binds to two discrete regions within the
2bAR third intracellular loop. Moreover, disruption of
arrestin binding selectively abrogated agonist-promoted internalization
of the 2bAR. These studies help to address questions of
specificity in GPCR/arrestin interaction that ultimately will lead to a
better understanding of the role of arrestins in regulating receptor-mediated signaling.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2AAR,
2BAR, and 2CAR were cloned into
pcDNA3 as described previously (25). The third intracellular loops of the 2aAR (residues 218-374), 2bAR
(residues 194-368), and 2cAR (residues 232-379) were
amplified by PCR using the full-length receptors as template.
The PCR products were cut with EcoRI/XhoI (2bAR and 2cAR) or
EcoRI/SalI (2aAR), gel-purified,
inserted into the plasmid pGEX4T-2 in-frame with GST, and sequenced.
The following 2bAR third loop truncation mutants were
made and cloned into pGEX4T-2 using the same strategy:
N-terminal (NT) (residues 194-292), NT1 (194-214), NT2 (206-292),
C-terminal (CT) (293-368), CT1 (293-312), CT2 (313-358), and CT3
(344-368). 2bAR point mutations were constructed by
oligonucleotide-directed PCR mutagenesis in either the NT1 construct
(K200A, R201A, K200A/R201A, S202A, N203A, R204A, R205A, R204A/R205A,
P207A, and R208A), the CT3 construct (L344A/L345A, R347A, Q355A,
W356A/W357A, R358A, R359A, R360A, Q362A, T364A, R365A, E366A, K367A,
and R268A), or the third loop construct (K200A/R201A (KR), R204A/R205A
(RR), R358A/R359A/R360A (3R), and RR/3R). PCR products were cut with
EcoRI/XhoI, ligated into
EcoRI/XhoI-digested pGEX4T-2, and sequenced. The
K200A/R201A, R204A/R205A, and R358A/R359A/R360A mutations were also
generated in full-length 2bAR by two-step PCR
using the expand high fidelity PCR system according to the
manufacturer's recommendations. Briefly, using
pcDNA3-2bAR as template, an N-terminal fragment of
the mutant 2bAR was created using a cytomegalovirus
(5'-tgt acg gtg gga ggt-3') sense primer and a mutagenic
antisense primer, and a C-terminal fragment of the mutant
2bAR was generated using a mutagenic sense primer and an
Sp6 (5'-gat aag ata tca cag tgg att tac-3') antisense primer. Mutagenic
primers used were: KR sense (5'-ctg atc gcc gca gcc agc aac cgc-3') and
antisense (5'-gcg gtt gct ggc tgc gta gat cag-3'); RR sense (5'-cgc agc
aac gcc gca ggt ccc agg-3') and antisense (5'-cct ggg acc tgc ggc gtt gct gcg-3'); 3R sense (5'-ggg cag tgg tgg gct gca gcg gcg cag ctg acc
cgg-3') and antisense (5'-ggt cag ctg cgc cgc tgc agc cca cca ctg
ccc-3'). PCR products were purified, and N-terminal and C-terminal
products (100 ng of each) were then used as template using the
cytomegalovirus and Sp6 primers. PCR products were cut with
EcoRI/XhoI, purified, and ligated into
pcDNA3. An RR/3R mutation in full-length 2bAR was
generated as described above using RR as template for the first round
of PCR and then proceeding as specified above for the 3R mutation.
2AR Fusion
Proteins--
BL21 (De3) lysS cells transformed with a
GST-2AR fusion construct were grown overnight at
37 °C, diluted 1:100 into LB containing ampicillin, grown for 3 h at 37 °C, and then induced with 0.1 mM
isopropyl-thiogalactyl-pyranosidase for 2 h at 30 °C. Cells were pelleted (3000 × g for 30 min) and washed with
phosphate-buffered saline (PBS) containing 1 mM
dithiothreitol and protease inhibitors (1 mM
phenylmethylsulfonyl fluoride, 0.2 mg/ml benzamidine, 20 µg/ml
leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin). Cells were
pelleted, resuspended in 1-3 ml of PBS plus protease inhibitors (per
100 ml of culture), lysed by incubation for 10 min on ice with 1 mg/ml
lysozyme, and then aliquoted (0.4 ml), frozen, and stored at 
80 °C
until needed. Aliquots were thawed on ice, Triton X-100 (2% final) and
sarcosyl (0.5% final) were added, and the cells were frozen, thawed,
and centrifuged for 1 h (30,000 rpm in TLA-45 rotor). The
supernatant (~200 µl) was then incubated with 200 µl of 50%
glutathione-agarose bead slurry for 1 h at 4 °C, and the beads
were washed twice with PBS containing protease inhibitors and 1%
Triton X-100 and resuspended in 200 µl of PBS with protease
inhibitors. To quantify protein amounts, 20 µl of resuspended beads
were incubated with SDS sample buffer and centrifuged, and the
supernatant was electrophoresed on a 10% SDS-polyacrylamide gel. The
gel was stained with Coomassie Blue, and protein levels were quantified
using bovine serum albumin as standard.
2AR Fusion
Proteins--
GST-2AR fusion proteins (typically ~500
ng/incubation) bound to glutathione-agarose were washed with arrestin
binding buffer (20 mM Tris-HCl, pH 7.4, 100 mM
NaCl, 0.04% Triton X-100, 1 mM dithiothreitol, and
protease inhibitors) and then incubated with 300 ng of purified bovine
arrestin-1, -2, or -3 in arrestin binding buffer for 1 h at
4 °C. Incubation mixtures were centrifuged (1000 × g) for 1 min and washed three times in binding buffer, and
the proteins were released with SDS sample buffer. Samples were
centrifuged, and the supernatants were electrophoresed on a 10%
SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose and
detected by immunoblotting using a general arrestin antibody (F4C1,
1:5000 dilution), an arrestin-3-specific antibody (182, 1:5000), or an arrestin-2-specific antibody (178, 1:5000). GST bound to
glutathione-agarose was used in all experiments as a control.
2bAR using 10 µl of
FuGENE-6 reagent according to the manufacturer's protocol. Briefly,
cells were incubated with the FuGENE-DNA mixture for 5 h and then
split into poly-l-lysine-coated 12-well dishes (for ERK1/2 assays) or
24-well dishes (for enzyme-linked immunosorbent assay). Enzyme-linked immunosorbent assays were performed 24 h after transfection as described previously (25), whereas ERK1/2 assays were done 48 h
after transfection.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2AR Third
Intracellular Loops--
Previous studies have demonstrated
subtype-specific differences in arrestin-promoted internalization of
2ARs with arrestin-3 enhancing internalization of
2bAR and 2cAR and arrestin-2 only acting
on the 2bAR (25). To explore the mechanistic basis of arrestin binding selectivity for 2ARs, we initially
focused on the third intracellular loop of the 2ARs. The
first and second intracellular loops of the 2ARs share
sequence homology; however, the third intracellular loops have very
divergent sequences, and this region likely directly contributes to
arrestin binding specificity (27, 30). The third intracellular loops of
the 2aAR (residues 218-374), 2bAR
(residues 194-368), and 2CAR (residues 232-379) were
expressed as GST fusion proteins, purified on glutathione-agarose, and
then used in direct binding assays with purified arrestin-1, -2, or -3 (Fig. 1A). Arrestin-1 did not
bind to any of the 2AR fusion proteins (data not shown),
arrestin-2 bound only to the GST-2bAR third loop, and
arrestin-3 bound to both the GST-2bAR and the
GST-2cAR third loops but not to the 2aAR
(Fig. 1B). A dose-response analysis was next performed to
determine whether there were binding differences between arrestin-2 and
-3 and the 2bAR. Arrestin-3 was found to bind much more
effectively to the 2bAR as compared with arrestin-2 with
~20-fold more binding at the highest concentrations of arrestin (Fig.
1C). Overall, these results largely recapitulate the
selectivity of arrestins in promoting internalization of the
2ARs (25) and suggest that this selectivity is mediated
by differences in arrestin binding to the third intracellular loops of
these receptors.
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Fig. 1.
In vitro binding of arrestins to
GST- 2AR third loop fusion
proteins. In A, 2AR third loops were
expressed as GST fusions in bacteria and purified on
glutathione-agarose beads. Proteins were electrophoresed on a 10%
SDS-polyacrylamide gel and visualized by Coomassie Blue staining. Shown
are 1 µg of purified GST or GST fusions containing the third
intracellular loop of the 2aAR (2A),
2bAR (2B), or 2cAR
(2C) and purified arrestin-1, -2, and -3. In B,
500 ng of purified GST-2AR fusion proteins were
incubated with 300 ng of purified arrestin-2 or arrestin-3 as described
under "Experimental Procedures." Bound proteins were eluted by
addition of SDS sample buffer and then electrophoresed on a 10%
SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with
an arrestin monoclonal antibody (F4C1). The experiment was repeated a
total of three times with similar results. In C, various
concentrations of arrestin-2 and arrestin-3 (0.02-0.34
µM) were incubated with 250 ng of GST-2bAR
third loop fusion protein as described under "Experimental
Procedures." Proteins were separated on a 10% SDS-polyacrylamide
gel, transferred to nitrocellulose, and blotted with arrestin-2- or
arrestin-3-specific polyclonal antibodies and goat anti-rabbit
secondary antibody. Blots were quantified by densitometric scanning and
compared with a purified arrestin protein standard. Each point
represents the mean ± S.E. of four to five independent
experiments.
2BAR Third Loop--
In an effort to identify
specific arrestin binding domains, we further investigated the
interaction of arrestin-3 with the 2bAR third loop.
Initially, the third loop was bisected into NT (residues
194-292) and CT (residues 293-368) pieces and tested for arrestin
binding (Fig. 2A). These
studies revealed that both the NT and CT regions of the
2bAR third loop bind arrestin-3, albeit not as well as
the intact third loop (Fig. 2B). Truncation mutagenesis of
the NT construct revealed that arrestin-3 binding was primarily
localized to the first ~20 residues as NT1 (residues 194-214) bound
arrestin-3 as well as NT, whereas NT2 (residues 206-292) did not bind
(Fig. 2B). Similar analysis of CT revealed that arrestin-3
binding was primarily localized to the last ~25 amino acids since CT3
(residues 344-368) bound arrestin-3, whereas CT1 (residues 293-312),
which contains a long stretch of acidic residues implicated previously
in receptor desensitization (39), and CT2 (residues 313-358) did not
(Fig. 2B). These results suggest that the proximal and
distal ends of the third intracellular loop of the 2bAR
contain the major arrestin-3 binding domains.
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Fig. 2.
In vitro binding of arrestin-3
with specific domains of the 2bAR
third loop. A, schematic representation of the
GST-2bAR third loop constructs used in binding assays
with purified arrestin-3. Relative arrestin-3 binding to
2bAR third loop segments is represented as +, with ++++
indicating the strongest binding. In B,
GST-2bAR third loop fusions (500 ng) were incubated with
300 ng of purified arrestin-3, and bound proteins were eluted,
electrophoresed on a 10% SDS-polyacrylamide gel, transferred to
nitrocellulose, and probed with an arrestin-3-specific antibody. Shown
is a representative blot from three independent experiments.
2AR involved in Gi activation with Lys-367 and Arg-368
contributing to this motif (27, 32). This suggests that significant
overlap between the arrestin-3 and Gi binding sites on the
2bAR will likely contribute to the mechanism by which
arrestin mediates desensitization (i.e. G protein
uncoupling).
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Fig. 3.
In vitro binding of arrestin-3
with site-directed mutants of the N-terminal and C-terminal regions of
the 2bAR third loop.
N-terminal (residues 194-214) (A) and C-terminal (residues
344-368) (B) domains of the 2bAR third loop
were mutated to determine specific residues involved in arrestin-3
interaction. Residues marked with an asterisk were mutated
to alanine, whereas double mutations to alanine are
underlined. The various GST fusion proteins (~500 ng) were
incubated with 300 ng of arrestin-3, and bound arrestin was eluted,
electrophoresed on a 10% SDS-polyacrylamide gel, transferred to
nitrocellulose, and probed with an arrestin-3-specific antibody. Blots
shown are representative of three to four independent
experiments.
2bAR third loop
mutants were generated. Four different GST-2bAR fusions incorporating either the K200A/R201A (KR), R204A/R205A (RR),
R358A/R359A/R360A (3R), or RR/3R mutations were used in binding assays
with purified arrestin-3 and compared with the wild type third loop.
The ability of the KR and 3R mutants to bind arrestin-3 was modestly
reduced as compared with the wild type 2bAR (Fig.
4A). However, RR and RR/3R
mutant binding to arrestin-3 was strongly attenuated with an 80-90%
reduction in binding. To detect potential binding differences between the RR and RR/3R mutations, a dose-response analysis was performed. The RR mutation alone had a very similar binding pattern to
RR/3R with the R204A/R205A mutation almost completely disrupting arrestin-3 binding to the 2bAR third loop (Fig.
4B). Taken together, these results suggest that the
2bAR third intracellular loop contains two arrestin-3
binding domains with the N-terminal region playing the predominant
role.
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Fig. 4.
In vitro binding of arrestin-3
with site-directed mutants of the
2bAR third loop.
GST-2bAR third loop fusion proteins were mutated to
K200A/R201A (KR), R204A/R205A (RR),
R358A/R359A/R360A (3R), and RR/3R as described under
"Experimental Procedures." In A, equal amounts of
GST-third loop fusion proteins (~ 500 ng) were incubated with 300 ng
of purified arrestin-3, and bound arrestin was detected as described
under "Experimental Procedures." Blots were quantified by
densitometric scanning, and then the data were normalized using
arrestin-3 binding to the wild type (WT) 2bAR
third loop as 100%. Data shown are mean ± S.E. of four to nine
independent experiments. Statistical analysis was performed using an
unpaired t test (*, p < 0.001 versus WT). B, wild type, RR, and RR/3R third
loops were used in an arrestin-3 dose-response analysis. 250 ng
(~0.04 µM) of each GST third loop fusion were incubated
with arrestin-3 (0.02-0.22 µM), and bound arrestin was
detected as described under "Experimental Procedures." Gels were
quantified by densitometric scanning and compared with purified
arrestin-3 protein standards. Data represent the mean ± S.E. of
from four to five independent experiments.
2bARs in HEK
293 Cells--
We next incorporated the various third loop mutations
(KR, RR, 3R, and RR/3R) into the holo-2bAR. Because
arrestins are involved in agonist-promoted internalization of the
2bAR (25), we anticipated that disrupting arrestin
binding to the 2bAR third intracellular loop would
attenuate receptor internalization. HEK 293 cells expressing FLAG-tagged wild type or mutant 2bARs were incubated
with agonist for 30 min and then analyzed for cell surface receptors by
enzyme-linked immunosorbent assay (Fig.
5). Internalization of the wild type 2bAR was ~30% after agonist treatment, consistent
with previous studies of 2bAR internalization in HEK 293 cells (41). Internalization of the KR and 3R mutant receptors was very
similar to that of the wild type 2bAR, consistent with
the in vitro data showing that these mutations did not
severely disrupt arrestin binding. In contrast, internalization of the
RR mutant was reduced ~50%, whereas the RR/3R mutant was decreased
~65% as compared with the wild type receptor. These data suggest
that disrupting arrestin binding to the third intracellular loop of the
2bAR has an inhibitory effect on agonist-promoted
receptor internalization. These results also help to confirm the
important role of arrestins in mediating internalization of the
2bAR.
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Fig. 5.
Agonist-promoted
internalization of wild type and mutant
2bARs expressed in HEK 293 cells.
HEK 293 cells transfected with FLAG-tagged wild type (WT) or
mutant (KR, RR, 3R, and
RR-3R) 2bARs were treated with 100 µM epinephrine for 30 min, and the loss of cell surface
receptors was quantified by enzyme-linked immunosorbent assay as
described under "Experimental Procedures." Data shown represent the
mean ± S.E. of three to nine independent experiments. Statistical
analysis was performed using an unpaired t test (*,
p < 0.001 versus WT).
2bARs in HEK 293 Cells--
To ensure that the various mutations did not directly
affect signaling of the 2bAR, we next analyzed the
ability of the wild type and mutant 2bARs to activate
ERK1/2. Our previous studies demonstrated that all three
2AR subtypes activate ERK1/2 in an agonist-dependent manner via a pathway that is Gi- and
Ras-dependent but arrestin- and internalization-independent
(25). HEK 293 cells expressing wild type or mutant (RR, 3R, RR/3R)
2bARs were incubated with agonist for 0, 5, or 30 min
and then analyzed for ERK activation by immunoblotting for
phospho-ERK1/2. All receptors activated ERK1/2 from 5- to 7-fold after
a 5-min treatment with agonist, suggesting that the mutations that
inhibit arrestin binding and receptor internalization have no
significant effect on 2bAR activation of signaling (Fig.
6, A and B).
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Fig. 6.
Agonist-promoted activation of ERK1/2 by wild
type and mutant 2bARs in HEK 293 cells. In A, HEK 293 cells transfected with FLAG-tagged
wild type (WT) or mutant (RR and
RR-3R) 2bARs were serum-starved, treated with
10 µM UK14304 for 0, 5, or 30 min, and then analyzed for
phospho-ERK1/2 as described under "Experimental Procedures." In
B, blots were quantified by densitometric scanning and
normalized to unstimulated cells. Data are the mean ± S.E. of
five independent experiments.
2bAR suggest the importance of a BXXBB
binding motif that is essential for arrestin-3 binding. Mutation of
Arg-201, Arg-204, or Arg-205 completely disrupted arrestin-3 binding,
whereas mutation of surrounding residues had minimal effect on arrestin
binding (Fig. 3). Interestingly, the analogous region of the
2cAR, but not the 2aAR, can also directly bind arrestin-3 (data not shown). Although the three
2ARs share significant homology within the N-terminal 20 residues of the third intracellular loops, a key basic residue present
in both the 2bAR (Arg-201) and the 2cAR
(Arg-234) is replaced with Gln-221 in the 2AAR (Fig.
7). The absence of this particular basic
residue within the 2AAR may disrupt arrestin binding. It
is also interesting to note that mutation of Arg-239 (the last B in the
BXXBB motif) within the NT1 region of the
2cAR completely disrupts arrestin-3 binding (data not
shown), further establishing the importance of basic residues within
this region for arrestin binding. Several recent studies have also
suggested a role for receptor third intracellular loops in arrestin
binding (30, 34-38). For example, the third loops of the -opioid
receptor (35) and the LH/CG receptor (38) can directly bind arrestins,
and third loop peptides from the LH/CG receptor inhibit receptor
desensitization by sequestering arrestin-2 (38). The domains involved
in these interactions share similar homology and generally contain
several basic residues as well as serines and/or threonines (Fig. 7).
Although the -opioid receptor studies suggested a role for two
serines in arrestin binding (35), all of these domains contain the
BXXBB motif and help to establish the importance of such a
motif in arrestin binding.
View larger version (10K):
[in a new window]
Fig. 7.
Sequence alignment of residues involved in
receptor-arrestin binding. An alignment of the amino acid
sequences of the 2AR third loops, -opioid
receptor third loop (DOR), and human LH/CG receptor third
loop is shown. Residues indicated in green result in the
loss of arrestin binding when mutated, whereas residues indicated in
red do not appear to contribute to arrestin binding. Basic
residues within the boxes represent the proposed
BXXBB arrestin binding domain.
2bAR binds arrestin-2 and -3 with higher affinity than
the 2cAR may be the acidic stretch of amino acids
located within the 2bAR third loop. This acidic stretch
is involved in mediating receptor desensitization (39) and may act to
destabilize the arrestin polar core and promote binding much like
phosphorylation does.
2bARs demonstrated a defect in agonist-promoted receptor
internalization, the inhibition was not complete. This suggests either
the incomplete disruption of arrestin binding within the third
loop (as seen in our GST binding studies) or a role for additional
arrestin binding sites on the receptor. Additional potential arrestin
binding sites on 2ARs include the second intracellular
loop, which contains the DRY motif that has been implicated in arrestin
association in several GPCRs (43-45). Further studies involving
2AR domains may reveal insight as to how arrestin
regulates both internalization and signaling of receptors as recent
studies propose that arrestin binding to different regions of a
receptor can regulate different functions (40).
2bAR third intracellular loop. Specifically Arg-201, Arg-204 and Arg-205, and to a lesser extent Arg-358, Arg-359,
and Arg-360, are important for arrestin binding in vitro. Mutation of these residues severely impairs
agonist-dependent receptor internalization, suggesting the
critical nature of arrestin in this process. Interestingly, arrestin-3
directly interacts with the basic residues of the G protein activation
motif located in the third loop, suggesting a model by which arrestin
binding displaces G protein, thus attenuating signaling in addition to linking the receptor to clathrin-coated pits.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Adriano Marchese for critical review of the manuscript, RoseAnn Stracquatanio for purification of arrestin polyclonal antibodies, Dr. Larry Donoso for the arrestin monoclonal antibody F4C1, and members of the Benovic laboratory for helpful discussions.
| |
FOOTNOTES |
|---|
* This work was supported in part by National Institutes of Health Grant GM47419 (to J. L. B.) and Grants EY11500 and GM63097 (to V. V. G.) and by National Institutes of Health Training Grant T32-AI07523 (to J. L. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Thomas Jefferson University, 233 South 10th St., Philadelphia, PA 19107. Tel.: 215-503-4607; Fax: 215-923-1098; E-mail: jeff.benovic@mail.tju.edu.
Published, JBC Papers in Press, August 29, 2002, DOI 10.1074/jbc.M207495200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; GST, glutathione S-transferase; HEK, human embryonic kidney; PBS, phosphate-buffered saline; AR, adrenergic receptor; LH/CG, luteinizing hormone/choriogonadotropin receptor; NT, N-terminal; CT, C-terminal; ERK, extracellular signal-regulated kinase.
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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