From the Departments of Cell Biology and Physiology
and § Pathology and the
University of New Mexico
Cancer Research and Treatment Center, University of New Mexico Health
Sciences Center, Albuquerque, New Mexico 87131 and the
¶ Department of Pharmacology, Vanderbilt University Medical
Center, Nashville, Tennessee 37232
Received for publication, May 13, 2002, and in revised form, October 15, 2002
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ABSTRACT |
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Arrestins regulate the signaling and
endocytosis of many G protein-coupled receptors (GPCRs). It has been
suggested that the functions of arrestins are dependent upon both the
number and pattern of phosphorylation sites present in an activated
GPCR. However, little is currently known about the relationships
between the sites of receptor phosphorylation, the resulting affinities of arrestin binding, and the ensuing mechanisms of receptor regulation for any given GPCR. To investigate these interactions, we used an
active truncated mutant of arrestin (amino acids 1-382) and phosphorylation-deficient mutants of the N-formyl peptide
receptor (FPR). In contrast to results with wild type arrestins, the
truncated arrestin-2 protein bound to the unphosphorylated wild type
FPR, although with lower affinity and a low affinity for the agonist as
revealed by competition studies with heterotrimeric G proteins. Using
FPR mutants, we further demonstrated that the phosphorylation status of
serines and threonines between residues 328-332 is a key determinant
that regulates the affinity of the FPR for arrestins. Furthermore, we
found that the phosphorylation status of serine and threonine residues
between amino acids 334 and 339 regulates the affinity of the receptor
for agonist when arrestin is bound. These results suggest that the
agonist affinity state of the receptor is principally regulated by
phosphorylation at specific sites and is not simply a consequence of
arrestin binding as has previously been proposed. Furthermore, this is
the first demonstration that agonist affinity of a GPCR and the
affinity of arrestin binding to the phosphorylated receptor are
regulated by distinct receptor phosphodomains.
The regulation of G protein-coupled receptors
(GPCRs)1 is a high fidelity
mechanism that involves, at a minimum, receptor phosphorylation, arrestin binding, and internalization. Agonist activation leads to the
rapid phosphorylation of serines and threonines in the GPCR carboxyl
terminus and/or intracellular loops through the activity of a G
protein-coupled receptor kinase (GRK) (1-5). Phosphorylation partially
quenches heptahelical signaling, as heterotrimeric G proteins display
lower affinity for certain phosphorylated receptors (6, 7). Subsequent
binding of arrestins to activated phosphorylated GPCRs further
desensitizes them as a result of the steric preclusion of G protein
binding. In addition, arrestin binding can direct the translocation of
phosphorylated GPCRs to the endocytic machinery (8-10) as well as
target numerous kinases to GPCR-based scaffolds (9, 11-15). Many GPCRs
resensitize over time in a process presumably dependent upon ligand and
arrestin dissociation and the actions of phosphatase(s) (16-18).
Notwithstanding this basic model, there is great diversity in the
processing of GPCRs with respect to sites of phosphorylation and the
ensuing mechanisms of desensitization and internalization (8). Thus, it
is likely that multiple biological models are necessary to account for
the complex mechanisms of GPCR processing.
Surprisingly little is known regarding arrestin's mechanism of
activation through its interactions with GPCRs and the reciprocal effects of arrestin binding on receptor properties. It has been suggested that the ionic interactions of polar residues in the "phosphate-sensing" core of arrestin with phosphoamino acids of an
activated G protein-coupled receptor are required for stable complex
formation (18-22). Presumably, phosphoreceptor binding disrupts a
delicate charge balance and causes reorientation of the inactive
arrestin molecule around a "fulcrum" in the molecule (20, 23). It
has been put forward that the binding of activated arrestins to
phosphorylated receptors preferentially stabilizes a receptor
conformation that displays enhanced ligand affinity (7, 24). Despite
these advances, a single model that accounts for the coordinated
actions of multiple receptor and arrestin domains in terms of complex
formation and agonist affinity regulation has yet to be described.
Furthermore, the precise correlation between the type, number, and
location of receptor phosphorylation sites and their role in
determining arrestin binding affinity as well as the regulation of
receptor ligand binding affinity has yet to be elucidated.
Carboxyl-terminal truncation of arrestin-2 has been characterized as an
"activating" mutation (19, 25-27). It is believed that the tail of
arrestin stabilizes the basal inactive state by intramolecular
interactions with the polar core domain. Truncation reduces the
activation energy of visual arrestin by ~50%, in theory, by the
removal of stabilizing interactions that maintain the molecule in an
inactive conformation (28, 29). Arrestin truncation has been
demonstrated to lead to partial phosphorylation- and activation-independence with a number of GPCRs, including receptors from which the carboxyl terminus, containing critical phosphorylation sites, has been removed (25). We have recently demonstrated that the
affinity of truncated arrestin-2 for a carboxyl-terminal peptide of the
N-formyl peptide receptor (FPR) is significantly higher than
that of wild type arrestin-2 (30). Thus, it is likely there is a
complex interplay of multiple receptor and arrestin domains. However,
it has yet to be fully determined how multiple, distinct mutations can
render arrestins phosphorylation-independent.
We have previously demonstrated that FPR internalization,
mitogen-activated protein kinase (MAPK) signaling, and
chemotaxis can proceed through arrestin-independent mechanisms
(31-33). We have also described the characteristics of G protein- and
arrestin-FPR complexes in both cell-based and cell-free systems using
flow cytometry, spectrofluorometry, and confocal microscopy (7, 34,
35). Surprisingly, our work has suggested that phosphorylation of the
wild type FPR is sufficient to prevent G protein binding in
vitro (7). Recently, we also demonstrated that two partial phosphorylation-deficient mutants of the FPR are unable to interact with wild type arrestins (36). Presumably, the absence of key phosphates in either of two distinct serine and threonine clusters in
the carboxyl terminus of these receptors completely inhibited their
ability to bind arrestins. Despite this inability, these receptors were
fully proficient in terms of their signaling and internalization.
In the current report, we examined the interactions of a
truncated arrestin-2 mutant (amino acids 1-382) with both wild type and phosphorylation-deficient forms of the FPR. We initially
demonstrated both high and low affinity binding of the mutant arrestin
to the phosphorylated and nonphosphorylated wild type receptor,
respectively. Furthermore, we showed that the relative agonist affinity
of such complexes was entirely dependent upon the phosphorylation
status of the receptor. Through subsequent reconstitutions with
phosphorylation-deficient FPRs, we identified regions of receptor
phosphorylation regulating the affinity of arrestins for the FPR as
well as sites critical for high agonist affinity complex formation. To
our knowledge, these studies have allowed, for the first time,
conclusive discrimination of phosphorylation sites regulating arrestin
binding from sites regulating agonist affinity of the receptor.
Reagents--
fNle-Leu-Phe-Nle-Tyr-Lys-Alexa546 was synthesized
as follows. Alexa Fluor 546 carboxylic acid succinimidyl ester
(Molecular Probes) and fNle-Leu-Phe-Nle-Tyr-Lys (Sigma) were each
dissolved in anhydrous dimethyl sulfoxide (Me2SO) to
2 mM. Equal volumes were incubated with 100 mM
triethylamine at room temperature overnight, and the product was used
directly. All other chemicals were from Sigma unless otherwise indicated.
Cell Culture--
As described previously, stably transfected
U937 cells were grown in tissue culture medium at 37 °C in a
humidified 5% CO2 atmosphere (7). Cells were passaged from
near confluent cultures every 3-4 days by reseeding at 2 × 105 cells/ml, expanded for membrane preparations in sealed,
5% CO2-equilibrated, 1-liter, baffled spinner flasks
(Pyrex), and incubated at 37 °C.
Cell Stimulation and Membrane Preparation--
As described
previously, spinner flasks containing near confluent FPR-expressing
U937 cells were stimulated for 8 min at 37 °C with 10 µM fMLF, which results in ~90% maximal receptor
phosphorylation (7). Following stimulation, flasks were placed on ice,
and an equal volume of ice-cold PBS was added.
For membrane preparation, cells were harvested by centrifugation and
resuspended in cavitation buffer (10 mM HEPES, 100 mM KCl, 30 mM NaCl, 3.5 mM
MgCl2, 600 µg/ml ATP, pH 7.3) at 4 °C. The cell
suspension was then placed in a nitrogen bomb for 15-20 min at
500 p.s.i. Following cavitation, nuclei were separated by
centrifugation. The membrane fraction was resuspended in HEPES sucrose
buffer (200 mM sucrose, 25 mM HEPES, pH 7.0)
with protease inhibitor and phosphatase inhibitor cocktails
(Calbiochem) prior to flash freezing. Aliquots were stored until use at
Detergent Solubilization--
Membranes were thawed, diluted in
an intracellular binding buffer (30 mM HEPES, 100 mM KCl, 20 mM NaCl, 1 mM EGTA,
0.1% w/v bovine serum albumin, 0.5 mM MgCl2),
and isolated by centrifugation. The membrane pellet was resuspended in
binding buffer containing protease inhibitor mixture set I, phosphatase
inhibitor mixture, and 1% n-dodecyl
Receptor Reconstitution--
Detergent-solubilized FPR (8-12
µl of receptor preparation) was incubated with either a mixture of
bovine brain Gi/Go heterotrimer, purified
arrestins, or buffer alone. A fluorescent agonist,
N-formyl-Met-Leu-Phe-Lys-fluorescein 5-isothiocyanate (10 nM), was added, and the samples were gently mixed at
4 °C for up to 120 min. Blocked samples received a large excess of
an unlabeled peptide, N-formyl-Met-Leu-Phe-Phe (fMLFF), prior to fluorescent ligand addition.
Spectrofluorometric Analysis--
As described previously,
fluorescence was measured by an SLM 8000 spectrofluorometer
(Spectronics) using the photon counting mode in time acquisition mode
(7). Excitation was fixed at 490 nm with a 490-nm, 10-nm band pass
filter (Corion) and an excitation monochrometer. Emission was monitored
using a 520-nm, 10-nm band pass interference filter (Corion) and a
500-nm long pass filter (Kopp). Antibody and nucleotide additions were
made through a microinjection port above the sample holder.
Following protein reconstitution and ligand incubation at 4 °C,
samples were diluted with a room temperature binding buffer containing
0.1% n-dodecyl Confocal Microscopy--
U937 cells (8 × 106)
that stably express the wild type FPR were electroporated with 25 µg
of DNA for wild type or mutant arrestin-GFP (pEGFP-N1) constructs. The
wild type arrestin-GFP construct was a generous gift from Dr. Jeffrey
Benovic (Thomas Jefferson University). The truncated arrestin-GFP was
generated through PCR amplification of amino acids 1-382. After 24-48
h, cells were incubated with 10 nM
fNle-Leu-Phe-Nle-Tyr-Lys-Alexa546 for 10 min at either 0 °C or
37 °C. Stimulated samples were immediately fixed with ice-cold 2%
paraformaldehyde for 30 min. Cells were washed, resuspended in
Vectashield (Vector Laboratories), and mounted onto glass slides. Fluorescence images were acquired on a Zeiss LSM 510 confocal microscope to localize both the FPR (red) and arrestin-2
(green).
Data Analysis--
Data were analyzed using GraphPad Prism
software. In general, data were plotted as normalized FITC intensity as
a function of time. Non-linear regression analyses were performed to
generate dose response curves and normalized to maximal values.
Effects of Truncated Arrestin-2 on the Phosphorylated, Wild Type
FPR--
Carboxyl-terminal truncation of arrestin-2 has been shown to
result in phosphorylation-independent binding to GPCRs as well as
confer enhanced affinity for an agonist (24). In the current report, we
examine the effects of carboxyl-terminal truncation of arrestin-2 on
full-length FPR assemblies, employing a non-cellular, spectrofluorimetric assay. The system is predicated on three facts. First, the anti-fluorescein antibody specifically quenches the fluorescence of unbound ligand (37). The assay therefore provides a
direct measurement of the FITC-ligand dissociation rate. Second, ligand
dissociation rates vary on the basis of receptor assemblies. Ternary
complexes of both arrestins and G proteins with the phosphorylated and
nonphosphorylated FPR, respectively, display high affinity for the
agonist (7, 24, 34, 35, 38). Finally, the addition of guanine
nucleotides causes the rapid dissociation of G proteins, but not
arrestins, from receptors (7, 24, 34). This dissociation, in turn,
results in the rapid dissociation of ligand, because the uncoupled
receptor exhibits low affinity for the agonist.
We initially sought to examine the effects of arrestin truncation on
the ligand dynamics of the phosphorylated, wild type FPR. As shown in
Fig. 1A, incubation of the
phosphorylated wild type receptor with 10 µM wild type
arrestin-2 and fluorescent ligand for 90 min leads to the formation of
a slowly dissociating complex, as described previously (7). Truncated
arrestin-2 reconstitution, in contrast, at an equivalent concentration
results in a high fraction of slowly dissociating species (Fig.
1A). Titration studies of truncated arrestin-2 with the
phosphorylated FPR demonstrate an EC50 of 220 ± 50 nM (Fig. 1B) in contrast to results with wild type arrestin-2, which display an EC50 of ~600
nM (7). The truncated arrestin-2 therefore exhibits higher
affinity for the phosphorylated FPR. Moreover, the magnitude of the
ligand affinity shift at a theoretical maximum
(Bmax) is greater in the case of the truncated
mutant (see Ref. 7). These effects were entirely dependent upon
incubation time with the ligand, because pre-incubation with arrestins
alone in the absence of ligand had no demonstrable effects on
reconstitution. The t1/2 for FPR-truncated
arrestin-2 complex formation was 5 ± 2 min (Fig. 1C),
which was similar to results with wild type arrestins
(t1/2, ~9 min). Thus, we see no evidence of
activation-independent reconstitution in the case of the soluble FPR,
in contrast to studies with other GPCRs (25).
Effects of Truncated Arrestin-2 on the Nonphosphorylated,
Wild Type FPR--
Our prior work has suggested that a truncated
protein of arrestin-3 can bind to the nonphosphorylated, full-length
FPR without inducing a ligand affinity change (34). However, we have
also shown that neither truncated arrestin-2 nor arrestin-3 exhibits specific binding to the nonphosphorylated FPR carboxyl terminus alone
(30). Together, these results support a role for additional receptor
domains in arrestin binding.
Incubation of the nonphosphorylated FPR with truncated arrestin-2 had
no discernible effects on ligand affinity, in contrast to results with
the phosphorylated FPR (Fig. 2,
cf. Fig. 1). Therefore, to discriminate between a lack of
binding and a lack of a ligand affinity shift, we undertook competition
studies between heterotrimeric G proteins and truncated arrestin-2.
Incubation of the nonphosphorylated FPR with G proteins results in the
formation of a high affinity, nucleotide-sensitive complex (7, 34). We
therefore incubated the receptor with high concentrations of truncated
arrestin-2 and 1.5 µM G protein to assess competition. As
shown in Fig. 2, partial inhibition of high agonist affinity complex
formation was observed in the presence of excess truncated arrestin-2.
The truncated mutant can therefore compete for the nonphosphorylated FPR with an estimated EC50 of 15-25 µM.
Moreover, FPR-arrestin complexes display low agonist affinity in
comparison to phosphoreceptor-arrestin assemblies.
Effects of Truncated Arrestin-2 on the Phosphorylated
Incubation of the phosphorylated
Because phosphorylation of the Effects of Truncated Arrestin-2 on the Phosphorylated In Vivo Co-localization of Arrestins and the FPR--
To confirm
the binding characteristics of the truncated arrestin with the FPR, we
electroporated U937 cells stably expressing FPR constructs with the DNA
for either arrestin-2-GFP or truncated arrestin-2-GFP. After 24 h
of recovery, cells were treated with an Alexa546-labeled formyl
hexapeptide and analyzed by confocal microscopy. We initially verified
the proper trafficking of the arrestin-GFPs using wild type arrestin-2
and the wild type FPR. As shown in Fig.
5, stimulation for 10 min at 37 °C
results in the co-localization of both wild type and truncated
arrestins with wild type receptors into punctate, internal structures,
which is consistent with prior results (31). Stimulation of cells on
ice did not result in either receptor clustering or arrestin co-localization. In agreement with in vitro results, both
the In this study, we initially demonstrated that carboxyl-terminal
truncation of arrestin-2 resulted in activation-dependent reconstitution with the wild type FPR. The truncated mutant displayed enhanced affinity for the phosphorylated receptor as well as an enhanced agonist affinity shift in comparison to wild type arrestins. In agreement with prior studies, the lack of receptor phosphorylation did not prohibit binding to the FPR (36), but such interactions were of
markedly lower affinity. Furthermore, receptor-arrestin assemblies of
nonphosphorylated receptors displayed low agonist affinity, in
contrast to results with phosphorylated receptors. Together, these
results suggest that truncation of arrestin-2 reduces its selectivity
for the phosphorylated FPR, which may permit its association with a
greater subset of a heterogeneously phosphorylated receptor population.
It also implies that the phosphorylation status of the receptor is a
critical determinant underlying the agonist affinity of
receptor-arrestin assemblies.
Because the truncated arrestin binds to both the phosphorylated and
unphosphorylated wild type FPR but only induces an affinity change for
the ligand in the former case, we investigated the individual
contributions of distinct phosphorylation sites. We have previously
determined that wild type arrestin binding to the FPR is contingent
upon phosphorylation in both the A and B domains (36). Binding of the
truncated arrestin to the phosphorylated The presence of two essential arrestin-binding motifs in the FPR is not
unique. Studies with the m2 muscarinic receptor have demonstrated the
importance of two distinct phosphorylation domains in the third
intracellular loop of the receptor (39, 40). It has been shown that
targeted mutation of either of these motifs disrupts stable arrestin
interactions. Furthermore, work on other GPCRs, such as the
neurotensin-1 receptor, oxytocin receptor, angiotensin II type 1A
receptor, and substance P receptor, has revealed the importance of
multiple phosphoamino acid clusters in regulating arrestin interactions
(27). However, the differential effects of distinct clusters or sites
of phosphorylation have not previously been described. Furthermore,
this is the first definitive identification of specific sites mediating
the high agonist affinity of arrestin-GPCR assemblies. This is an
entirely novel finding in that it has been widely assumed that the
regulation of receptor agonist affinity by arrestin was an all or
nothing phenomenon, dependent only on the actual binding of arrestin to the receptor and not the specific sites of phosphorylation (24). It
stands to reason, however, that a discrete subset of the
phosphorylation sites within the A and/or B domains of the FPR are
responsible for the phosphorylation-dependent differences.
We are therefore continuing studies to examine the role(s) of
individual phosphorylation sites in the regulation of arrestin and
receptor function.
One puzzling observation is the apparent inverse relationship between
arrestin affinity and agonist affinity in terms of the phosphorylation
status of the A and B clusters of the FPR (Table II). One possible explanation emerges
from the recent discovery of an additional phosphate-binding element in
arrestin. It has been suggested that lysines 14 and 15 in visual
arrestin (equivalent to lysines 10 and 11 in arrestin-2) act as a
phosphate-sensitive "guide" for GPCRs, presumably by optimally
orienting the receptors for polar core interactions (41). The
engagement of lysines 10 and 11 by GPCR phosphodomains most likely also
results in arrestin activation, due to disruption of a hydrophobic
anchor in the carboxyl terminus (Fig. 7).
This activation may be similar to what is achieved by truncation of the
carboxyl terminus of the protein. However, mutation of these residues
to neutral amino acids results in significantly diminished binding to
receptors, even in the face of additional activating arrestin
mutations, indicating that these residues are also critical in
establishing stable receptor-arrestin complexes (41).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C.
-D-maltoside (Calbiochem). Suspensions were solubilized
on a nutator (Clay Adams) for 90 min. The soluble fraction was
collected by centrifugation and placed on ice for immediate experimentation.
-D-maltoside and inhibitors.
Samples were placed into the spectrofluorometer with gentle stirring, and data were acquired for 70-100 s with a 1.0-s integration time. For
the first 10 s, equilibrium levels were obtained. At 10 s, excess anti-fluorescein antibodies, prepared as described previously, were added to the sample. The antibodies rapidly quenched the fluorescence of unbound, labeled ligand. In some assays, excess GTP
S
(guanosine 5'-3-O-(thio)triphosphate) (Sigma) was added at
40 s to assess receptor-G protein coupling.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effects of exogenous arrestins on the agonist
affinity characteristics of wild type, phosphorylated FPR.
A, phosphorylated, wild type FPRs were incubated with 10 nM FITC-fMLFK, a fluorescent agonist, on ice for 90 min
with binding buffer alone ( ), 10 µM exogenous wild
type (wt) arrestin-2 (
), or 10 µM truncated
arrestin-2 (
). B, to assess the relative affinity of the
truncated protein for the phosphorylated receptor, dose response curves
were generated by titration of the mutant arrestin protein from 35 nM to 9 µM, indicating an EC50 of
220 ± 50 nM. C, the time dependence of the
arrestin-mediated agonist affinity changes was assessed by incubating
10 µM truncated arrestin-2 with a phosphorylated receptor
and fluorescent ligand for 0 to 90 min, revealing a
t1/2 of 5 ± 1 min. In general, samples were
prepped immediately prior to spectrofluorimetric analysis by dilution
in room temperature binding buffer and transfer to glass cuvettes. At
10 s, 60 nM anti-fluorescein antibody was added
through a micro-injection port, which quenches the fluorescence of
unbound ligand. At 40 s, excess GTP
S was added, which disrupts
coupling of receptor and G proteins. Data are plotted as peptide
intensity versus time. Normalized means are representative
of at least three independent experiments conducted in duplicate.
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Fig. 2.
Competition studies of truncated arrestin-2
and heterotrimeric G proteins for the nonphosphorylated, wild type
FPR. Solubilized samples of the nonphosphorylated, wild type FPR
were incubated with fluorescent ligand (10 nM) and either
buffer alone, 10 µM wild type arrestin-2
(Arr-2), or 10 µM truncated arrestin-2
(Arr-2[1-382]) for 90 min on ice. Given the
lack of discernible reconstitution, competition studies were undertaken
between heterotrimeric G proteins and truncated arrestin-2 to assess
coupling. Nonphosphorylated FPR were reconstituted with FITC-ligand,
1.5 µM bovine brain G protein and either buffer alone or
10 µM truncated arrestin-2 for 120 min prior to analysis.
Addition of the truncated protein at high concentrations partially
inhibited the formation of the high agonist affinity,
nucleotide-sensitive G protein-coupled complex. Thus, similar to
results with uncoupled receptors, assemblies of the nonphosphorylated
receptor with arrestins display low agonist affinity. Normalized means
are representative of at least three independent experiments conducted
in duplicate.
A FPR
Mutant--
We have previously demonstrated that two
phosphorylation-deficient mutants of the FPR cannot bind to wild type
arrestins either in vivo or in vitro despite full
competency in terms of signaling and internalization (33, 36). These
A and
B receptors contain targeted substitutions of groups of
serines and threonines in the carboxyl terminus of the FPR with
non-phosphorylatable alanines and glycines (Table
I). Because the truncated arrestin
displayed binding toward both the fully phosphorylated and
unphosphorylated forms of the FPR, we sought to investigate the binding
properties and effects of reconstitution on the ligand dynamics of the
phosphorylated
A and
B receptors.
Carboxyl-terminal sequences of FPR
mutants
A receptor with 10 µM
truncated arrestin-2 results in the formation of a high affinity
complex, in contrast to an equivalent concentration of wild type
arrestin-2 (Fig. 3A). This
result demonstrates at a minimum that the truncated arrestin-2 acts on
a greater subset of the receptor population than wild type arrestins
and not simply with greater effect on the same receptors, because wild
type arrestins display no detectable binding to this
phosphorylation-deficient receptor. Titration studies reveal that the
affinity of the truncated arrestin-2 for the phosphorylated
A is
significantly lower than for the phosphorylated, wild type receptor,
with an EC50 of 2.2 ± 0.3 µM (Fig. 3,
A and C). However, the magnitude of the affinity
shift at a maximum is approximately equivalent to that seen with the
phosphorylated, wild type FPR (data not shown). Thus, the truncated
arrestin is able to induce a ligand affinity change in a submaximally
phosphorylated receptor population to the same extent as with a fully
phosphorylated form of the FPR, despite its weaker binding affinity to
the
A receptor. These results demonstrate that phosphorylation of
the A site is critical in determining the affinity of the receptor for
arrestin, but it is not responsible for arrestin-dependent agonist affinity changes.
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Fig. 3.
Reconstitution of exogenous arrestins and
heterotrimeric G proteins with the phosphorylated
A FPR mutant. A, solubilized,
phosphorylated preparations of the phosphorylation-deficient
A
mutant were incubated with FITC-ligand and either buffer (
), 10 µM wild type (wt) arrestin-2 (
), or 10 µM truncated arrestin-2 (
) for 90 min on ice. The
phosphorylated
A receptor reconstituted with the truncated, but not
wild type, arrestin-2 to form a high agonist affinity complex.
B, because the phosphorylated
A receptor can reconstitute
with heterotrimeric G proteins (bbG), competition with
truncated arrestin-2 was assessed. Incubation of phosphorylated
A
with both 10 µM truncated arrestin-2 and 1 µM heterotrimeric G proteins (
) resulted in complete
loss of nucleotide sensitivity in comparison to samples receiving G
proteins alone (
). Injections of both anti-fluorescein antibody and
GTP
S were made at 10 and 40 s respectively. Normalized means
are representative of at least three independent experiments conducted
in duplicate. C, dose response curves demonstrate an
EC50 of 2.2 ± 0.3 µM, suggesting that
the A sites principally mediate the affinity of receptors for
arrestins.
A receptor does not prohibit G
protein coupling, we could also demonstrate the successful competition
of heterotrimeric G protein binding with the truncated arrestin-2, as
suggested by the loss of nucleotide-sensitivity (Fig. 3B).
The EC50 for competition of G protein binding (used at its
Kd of ~1 µM) was ~1.7 ± 0.5 µM (Fig. 3C), similar to that observed for the
ligand affinity shift dose response data (i.e. 2.2 µM; see Fig. 3, A and C).
B
FPR--
The
B receptor contains targeted substitution of a cluster
of the four serines and threonines between amino acids 334-339 of the
FPR. Incubation of the phosphorylated
B receptor with truncated
arrestin-2 had no discernible effects on agonist affinity (Fig.
4A). We therefore sought to
discriminate between binding without an accompanying agonist affinity
shift and a complete lack of binding by carrying out competition
studies with heterotrimeric G proteins. Reconstitution of the
phosphorylated
B receptor with 1 µM exogenous G
proteins results in the formation of a high agonist affinity,
nucleotide-insensitive complex (36). The addition of truncated
arrestin-2 results in the conversion of the receptor from a high
affinity to low agonist affinity state, as expected from the lack of a
ligand affinity change in the direct binding assay above. This
competition occurred with an EC50 of 340 ± 60 nM, a similar affinity to the interaction of wild type
arrestin-2 with the phosphorylated, wild type FPR. Thus, although
phosphorylation in the A site is not sufficient for
arrestin-dependent agonist affinity changes, it can provide
for a high affinity interaction of arrestin with the FPR.
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Fig. 4.
Reconstitution of exogenous arrestins and
heterotrimeric G proteins with the phosphorylated
B FPR mutant. A, solubilized,
phosphorylated preparations of the phosphorylation-deficient
B
mutant were incubated with FITC-ligand and either buffer (
), 10 µM wild type (wt) arrestin-2 (
), or 10 µM truncated arrestin-2 (
) for 90 min on ice.
Incubation of the phosphorylated
B receptor with both truncated and
wild type arrestin-2 had no measurable effect on agonist affinity.
B, competition studies with heterotrimeric G proteins
(bbG) were undertaken to assess coupling of the receptor
with truncated arrestin-2. Incubation of phosphorylated
B with 1 µM heterotrimeric G proteins and 10 µM
truncated arrestin-2 (
) resulted in a loss of nucleotide sensitivity
in comparison to samples receiving G proteins alone (
).
C, in contrast to the phosphorylated
A FPR, however, such
complexes displayed low affinity. Titration of truncated arrestin-2
demonstrates a Ki of 340 ± 60 nM.
Injections of both anti-fluorescein antibody and GTP
S were made at
10 and 40 s, respectively, during spectrofluorimetric analysis.
Normalized means are representative of at least three independent
experiments conducted in duplicate.
A and
B receptors co-localized with the truncated arrestin-2 into punctate structures after 10 min of stimulation (Fig.
6), unlike results with wild type
arrestin-2. These results confirm the interaction of the truncated
arrestin mutant with the phosphorylation-deficient receptors in a
native environment.
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Fig. 5.
Co-localization of GFP-arrestins with
Alexa546-labeled wild type FPR. Wild type (wt)
FPR-expressing U937 cells were electroporated with the DNA for either
arrestin-2-GFP or truncated arrestin-2-GFP. After 24-48 h of
recovery, cells were treated with an Alexa546-labeled formyl
hexapeptide for 10 min, fixed with paraformaldehyde, and analyzed by
confocal microscopy. The trafficking of wild type arrestins after
stimulation on ice (top row), at 37 °C (middle
row), and truncated arrestins at 37 °C (bottom row)
was assessed by the imaging of ligand-receptor complexes
(red) and arrestins (green). Co-localization
(yellow) of arrestins and receptors after 37 °C
stimulation confirms the stimulation-dependent trafficking
of the proteins into punctate, endosomal structures.
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Fig. 6.
Co-localization of GFP-arrestins with
Alexa546-labeled phosphorylation-deficient FPRs. A and
B
FPR-expressing U937 cells were electroporated with the DNA for either
arrestin-2-GFP or truncated arrestin-2-GFP. After recovery, cells were
treated with an Alexa546-labeled formyl hexapeptide, fixed, and
analyzed by confocal microscopy. The lack of trafficking of wild type
(wt) arrestins (green) with liganded receptors
(red) after stimulation at 37 °C was confirmed for both
the
A (top row) and
B (third row from top)
mutants. Co-localization (yellow) of truncated arrestin-GFP
proteins with
A (second row from top) and
B
(bottom row) mutants after stimulation into punctate,
internalized structures further suggests the interactions of the
proteins.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A FPR mutant resulted in
the formation of a high agonist affinity complex, although with an
order of magnitude decrease in the arrestin binding constant. This
suggests that phosphorylation of the A sites plays a pivotal role in
the regulation of the affinity of arrestins for the receptor but not in
the formation of a high agonist affinity complex. In contrast, the
phosphorylated
B receptor was determined to bind to truncated
arrestin with a similar affinity as that of the wild type receptor.
However, analogous to results with the nonphosphorylated receptor, such
complexes displayed low agonist affinity. This implies that the
phosphorylation status of the B sites is a critical determinant in the
formation of a high agonist affinity complex. Furthermore, in
comparison to the A sites, the B sites appear to play a less
significant role in determining the affinity of arrestins for receptors.
Affinity characteristics of arrestin ternary complexes
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Fig. 7.
Model of FPR ternary complex formation with
arrestin. Ternary complex assembly with arrestins involves the
stepwise interactions of multiple phosphorylation-dependent
and -independent domains in both arrestin and the FPR. Phosphorylated,
wild type receptor binds to wild type arrestin in an activation- and
phosphorylation-dependent manner to yield a high agonist
affinity complex. Carboxyl-terminal truncation of arrestin obviates the
need for arrestin tail release and allows for binding to
phosphorylation-deficient receptors. Following interactions between the
phosphoreceptor and the polar core of arrestin, the latter induces
and/or stabilizes a high agonist affinity state of the receptor.
Our results suggest that there are likely four functional sites of
interaction between GPCRs and arrestins (Fig. 7). The important and
regulatable sites on the FPR include the ligand binding site, an
activation-dependent binding site for G proteins and
arrestins (consisting in part of the "DRY" sequence of the second
intracellular loop), and at least two distinct sites of phosphorylation
that we define here as regulators of arrestin binding affinity and receptor affinity for agonist. Arrestins possess at least three primary sites of interaction with activated, phosphorylated GPCRs. The
first site recognizes the activation state of the receptor, likely
through interactions with the second intracellular loop of the
receptor. Two additional sites, represented by lysines 10/11 and the
polar core, interact with phosphate moieties on the receptor. Initial
interactions with lysine 10/11 (Fig. 7, Step 1)
result in the partial activation of arrestin, leading to alterations of
the carboxyl terminus of arrestin and exposure of the polar core (Fig.
7, Step 2). In the case of the truncated form of arrestin
this activation step is not required, and the truncated arrestin can
bind to forms of the FPR with lower levels of phosphorylation (mutants
A and
B). Following binding of phosphoresidues to the polar core
(Fig. 7, Step 3), the final interaction between arrestins and GPCRs results in the stabilization of a receptor conformation with a high affinity for the ligand (Fig. 7,
Step 4).
In conclusion, this work describes completely unanticipated results
that are essential in unraveling the complex interactions between an
activated phosphorylated GPCR and arrestins. Because arrestins bind to
virtually all GPCRs and yet elicit receptor-specific effects including
important differences in desensitization, internalization, and G
protein-independent kinase activation, it is likely that the pattern of
receptor phosphorylation specifies to a large extent the subsequent
functions of the bound arrestin molecule. Additional work with the FPR
and other GPCRs will be required to resolve these complexities.
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FOOTNOTES |
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* This work was supported by funds from the New Mexico Cancer Research Fund (to L. A. S.) and National Institutes of Health Grants AI36357 and AI43932 (to E. R. P.), GM60799, EB00265, and RR01315 (to L. A. S.), and EY11500 and GM63097 (to V. V. G). Confocal images and flow cytometry data in this paper were generated in the Fluorescence Microscopy and Flow Cytometry Data facilities at the University of New Mexico Health Sciences Center, which received support from National Center for Research Resources Grants 1 S10 RR14668 and P20 RR11830, National Science Foundation Grant MCB9982161, National Cancer Institute Grant R24 CA88339, the University of New Mexico Health Sciences Center, and the University of New Mexico Cancer Center.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. Tel.: 505-272-5647; Fax: 505-272-1421; E-mail: eprossnitz@salud.unm.edu.
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M204687200
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ABBREVIATIONS |
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The abbreviations used are:
GPCR, G
protein-coupled receptor;
FPR, N-formyl peptide
receptor;
GTPS, guanosine 5'-3-O-(thio)triphosphate;
GFP, green fluorescent protein;
FITC, fluorescein 5-isothiocyanate;
fMLF, N-formyl-Met-Leu-Phe;
fMLFF, N-formyl-Met-Leu-Phe-Phe;
fMLFK, N-formyl-Met-Leu-Phe-Lys.
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REFERENCES |
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