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J. Biol. Chem., Vol. 275, Issue 32, 24590-24594, August 11, 2000
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From the Department of Cell Biology and Physiology,
University of New Mexico Health Science Center, Albuquerque, New
Mexico 87131
Received for publication, May 9, 2000
Following activation by ligand, the
N-formyl peptide receptor (FPR) undergoes processing events
initiated by phosphorylation that lead to receptor desensitization and
internalization. Our previous results have shown that FPR
internalization can occur in the absence of receptor desensitization,
suggesting that FPR desensitization and internalization are controlled
by distinct mechanisms. More recently, we have provided evidence that
internalization of the FPR occurs via a mechanism that is independent
of the actions of arrestin, dynamin, and clathrin. In the present
report, we demonstrate that stimulation of the FPR with agonist leads
to a significant translocation of arrestin-2 from the cytosol to the
membrane. Fluorescence microscopy revealed that the translocated arrestin-2 is highly colocalized with the ligand-bound FPR. A D71A
mutant FPR, which does not undergo activation or phosphorylation in
response to ligand, did not colocalize with arrestin-2. Surprisingly, an R123G mutant FPR, which does not bind G protein but does become phosphorylated and subsequently internalized, also did not bind arrestin. These results indicate that arrestin binding is not required
for FPR internalization and demonstrate for the first time that a
common motif, the conserved "DRY" domain of G protein-coupled receptors, is essential for phosphorylation-dependent arrestin binding, as well as G protein activation.
G protein-coupled receptors
(GPCRs)1 play essential roles
in most physiological responses. Of particular significance is their role in the regulation of the complex signaling pathways of the immune
system. Chemokine and chemoattractant receptors are largely responsible
for leukocyte trafficking and activation (1). The pathways responsible
for receptor activation are critical to the proper functioning of a
given system, and of equal importance are the pathways involved in
terminating or attenuating these responses. Termination of receptor
signaling has been shown to be dependent on receptor phosphorylation,
primarily by the family of G protein-coupled receptor kinases (GRKs)
(2). However, receptor phosphorylation alone is insufficient to
preclude G protein binding and activation. For this to occur, another
protein, a member of the arrestin family, must first bind the
phosphorylated receptor. The binding of arrestins to phosphorylated
receptors prevents G protein binding and results in an inactive
receptor (3, 4). For many G protein-coupled receptors, arrestin also acts as an adapter protein, mediating internalization through clathrin-coated pits (5). Furthermore, arrestin-mediated recruitment of
Src has been shown to be essential for the activation of
mitogen-activated protein kinases by certain G protein-coupled
receptors (6). More recently, it has been suggested that the ability of
arrestins to dissociate from an internalized or internalizing receptor
regulates the rate at which the receptor is resensitized and
re-expressed at the cell surface (7). It is thus clear that arrestins
can play multiple roles in receptor desensitization, internalization, signal transduction, and resensitization.
Mechanisms for the binding of arrestins to receptors have been
described based largely on biochemical, biophysical, structural, and
mutational studies of visual arrestin (8). This protein exhibits great
selectivity for binding to light-activated, phosphorylated rhodopsin,
with binding resulting in signal termination (9). With the recent x-ray
crystallographic determination of visual arrestin, a much more precise
understanding of the functional properties of arrestins has come to
light (8). The structure of visual arrestin consists of two domains
with a highly polar core comprised mostly of amino-terminal domain
residues. The extreme carboxyl terminus of the protein lies over the
cavity containing the polar core. Removal of the carboxyl terminus or
mutation of the polar core of the homologous arrestin-2 protein results
in an arrestin molecule that discriminates poorly between
phosphorylated, activated rhodopsin and non-phosphorylated, activated
rhodopsin, suggesting the phosphorylated carboxyl terminus of rhodopsin
binds to and disrupts this polar region (10). Disruption of the polar core is believed to result in a large conformational change within arrestin, leading to its activation (11). A key feature of arrestin, essential to its proper functioning, is its ability not only to recognize the phosphorylation state of a receptor but also its activation state, reflecting whether ligand is bound. This has been
shown for visual and non-visual arrestins and is elegantly demonstrated
by studies of a mutant form of arrestin, which disrupts the polar core
(partially pre-activating the protein), preventing the protein from
discriminating the phosphorylation state of the receptor (10). Despite
this deficit, the protein continues to display significantly higher
affinity for the light-activated form of rhodopsin compared with the
inactive form. These results indicate that arrestins recognize at least
two distinct features of G protein-coupled receptors.
The N-formyl peptide receptor (FPR) is a chemoattractant
receptor found predominantly on leukocytes (1). We have demonstrated that following ligand stimulation, this receptor is rapidly
phosphorylated on its carboxyl terminus (12). This phosphorylation,
likely mediated by GRK2, is essential for the subsequent
desensitization and internalization of the receptor (13, 14). However,
studies of U937 cells expressing receptor mutants partially defective in phosphorylation have revealed that, unlike the paradigm outlined above, receptor internalization can occur in the absence of
desensitization (15). This suggested that FPR internalization and
desensitization are mediated by distinct mechanisms. Using a dominant
negative arrestin mutant (arrestin-2 319-418), which binds to clathrin but not activated receptors, we have subsequently confirmed in HEK
cells that internalization of the FPR occurs in an arrestin-independent manner.2 Furthermore,
co-expression of a dominant negative dynamin mutant or a dominant
negative clathrin mutant also had no effect on FPR internalization,
substantiating that neither clathrin nor caveolae are involved in this
process. Lastly, fluorescence microscopy revealed that the
In this study, we demonstrate that stimulation of the FPR does indeed
result in a translocation of arrestin-2 from the cytosol to the
membrane and furthermore that the translocated arrestin-2 colocalizes
with the FPR. Despite not being required for internalization, arrestin-2 can clearly be seen to be associated with endosomes containing internalized FPR. Activation of the FPR is required for this
association as demonstrated by the lack of arrestin colocalization with
an inactive D71A mutant form of the FPR. However, another mutant form
of the FPR, R123G, which does not bind G protein but does become
phosphorylated and undergoes internalization, was also shown not to
bind arrestin. These results suggest that the binding of arrestin to G
protein-coupled receptors may be regulated in part by the same receptor
activation signal utilized by G proteins, namely the highly conserved
"DRY" sequence, located at the interface between the third
transmembrane helix and the cytoplasm.
Materials--
The cDNA encoding the FPR was originally
obtained from a human HL-60 granulocyte library (17). The generation of
FPR mutants has been described previously (18). Anti-arrestin-2 rabbit
polyclonal antiserum was generously supplied by Dr. Jeffrey Benovic,
Thomas Jefferson University. Texas Red-conjugated goat anti-rabbit
antibody was from Vector Laboratories. fMLF, GTP Translocation Assay--
U937 cells (108) were
collected, washed once in PBS, and resuspended into 10 ml of PBS.
One-half of the sample was stimulated with 50 µM fMLF for
8 min at 37 °C. The reaction was stopped with the addition of 10 ml
of cold PBS. Stimulated and unstimulated cells were then pelleted,
resuspended in 750 µl of PBS plus protease inhibitor mixture
(Calbiochem), and sonicated. The membrane and cytosolic fractions were
separated by centrifugation at 30,000 × g for 30 min.
The membrane fraction was resuspended once and recentrifuged. Western
blot analysis was carried out to determine the relative arrestin
concentration in each fraction.
Western Blot Analysis--
Proteins were separated by SDS
polyacrylamide gel electrophoresis and transferred to polyvinylidene
difluoride membranes (Gelman) with a semi-dry transfer apparatus (Owl
Scientific). Membranes were blotted with antibody against the indicated
arrestin protein followed by an HRP secondary antibody. The blots were
developed using ECL Plus (Amersham Pharmacia Biotech) and imaged using
a PhosphorImager (Molecular Dynamics).
Ligand Affinity Measurements and G Protein
Interactions--
Membrane fractions were generated by nitrogen
cavitation as described previously (19) and stored until use at
Receptor Internalization--
Receptor internalization was
determined as the uptake of N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys
fluorescein. Cells were incubated with 10 nM
N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys fluorescein at 37 °C or
on ice for 8 min and rapidly chilled. Samples were washed extensively
with successive washes of ice-cold 50 mM glycine (pH 3.0),
1× PBS, and 1× PBS containing 10 µM
N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys to remove surface-bound
fluorescent ligand. Cells were then analyzed for residual fluorescent
intensity, representing internalized ligand, on a FACScan flow
cytometer (Becton Dickenson) with dead cells excluded by a gate on
forward and side scatter.
Fluorescence Microscopy--
Control cells were incubated with
10 nM N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys
fluorescein for 8 min at 0 °C. Receptor internalization was
stimulated by incubating cells with 10 nM
N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys fluorescein for 8 min at
37 °C. Each of the samples were then fixed with 2% paraformaldehyde
for 30 min and permeabilized with 0.02% saponin. Arrestin-2 was
detected by incubating cells with anti-arrestin antibody followed by a
goat anti-rabbit secondary antibody conjugated to Texas Red. After
three washes, cells were resuspended in Vectashield (Vector
Laboratories) and placed on a slide. Fluorescence images were acquired
on a Zeiss Axioplan 2 equipped with a digital camera (Orca, Hammamatsu)
to localize both the FPR (green) and arrestin proteins (red).
The role of arrestins in the desensitization, and more recently
internalization, of numerous G protein-coupled receptors has been
thoroughly documented (21, 22). However, our recent work in the
characterization of phosphorylation-deficient FPR mutants has suggested
that desensitization and internalization of this receptor occur through
distinct mechanisms (15). Subsequently, we have demonstrated that FPR
internalization proceeds via a pathway that is not only independent of
arrestin but also independent of dynamin and clathrin.2 The
mediators of this mode of internalization have yet to be described.
Despite this, our results have led to questions regarding the existence
of an interaction between arrestins and the FPR and the role, if any,
of arrestins in FPR function.
To investigate the potential interaction between the FPR and arrestins,
we utilized a model promonocytic cell line, U937, stably transfected
with the wild type FPR (23). Analysis of whole cell lysates by Western
blot revealed that this cell line predominantly expresses arrestin-2
(data not shown). To determine the cellular location of the arrestin-2,
cells were incubated for 10 min either in the presence or absence of
agonist, disrupted by sonication, and separated into cytosolic and
membrane fractions by centrifugation. In the nonstimulated cells,
approximately 15% of the total arrestin-2 was associated with the
membrane fraction (Fig. 1). Following a
10-min treatment with agonist, a significant translocation occurred
with approximately 50% of the arrestin-2 now detected in the membrane
fraction. Because arrestin is known to reside predominantly in the
cytosol in unstimulated cells (24), this result is consistent with the
activated FPR being able to recruit arrestin to the membrane.
Given that activation of the FPR induced arrestin-2 translocation to
the plasma membrane, we next examined cells utilizing fluorescence
microscopy techniques to investigate whether the translocating arrestin
was being directed to sites containing the FPR. Cells were stimulated
with a fluorescent agonist to identify and track the FPR as it was
processed. The cells were then immediately fixed and permeabilized, and
arrestin-2 was detected with an anti-arrestin antibody followed by a
secondary antibody conjugated to Texas Red. As demonstrated in Fig.
2, both arrestin-2 and the FPR have a
diffuse appearance in a cell treated with the fluorescent ligand but
kept at 0 °C. Following an 8-min incubation with the fluorescent agonist at 37 °C, the FPR can be seen to be localized in punctate structures, presumably largely endosomes at this point. Translocation of arrestin into punctate structures is also apparent. The arrestin-2 colocalized with the FPR as seen in endosomes that are close to the
cell surface, as well as in structures that have migrated further into
the interior of the cell. The arrestin-2 seems to remain associated
with the receptor throughout the internalization process. This is in
contrast to the
Arrestin Binding to the G Protein-coupled N-Formyl
Peptide Receptor Is Regulated by the Conserved "DRY" Sequence*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2-adrenergic receptor, which does internalize via
clathrin-coated pits, does not colocalize with the FPR during simultaneous internalization of both receptors. Together, these results
provide clear evidence that the FPR need not bind arrestins to be
processed for internalization.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
S, and
HRP-conjugated goat anti-rabbit antibodies were purchased from Sigma.
N-Formyl-Nle-Leu-Phe-Nle-Tyr-Lys fluorescein was from
Molecular Probes.
80 °C. Lysed membranes were thawed and washed in binding buffer
(30 mM HEPES, 100 mM KCl, 20 mM
NaCl, 1 mM EGTA, 1 mg/ml bovine serum albumin, 0.5 mM MgCl2, pH 7.4). Membranes were centrifuged
at 135,000 × g and resuspended to 5 × 108 cell equivalents/ml of binding buffer containing a
protease inhibitor mixture (Calbiochem). Membrane extract (10 µl) was
incubated with 10 nM formyl-Met-Leu-Phe-Lys fluorescein
(Peninsula Laboratories) in a final volume of 20 µl for 2 h at
4 °C with agitation. Samples were analyzed by the
spectrofluorometric approach described previously (19). Briefly,
following preparation at 4 °C, samples were diluted to 200 µl and
equilibrated to 22 °C. They were then placed into the
spectrofluorimeter (SLM 8000, Spectromic Instruments), and fluorescence
was measured using the photon counting mode. Total fluorescence was
obtained for 20 s followed by the addition of an anti-fluorescein
Ab. The Ab binds formyl-Met-Leu-Phe-Lys fluorescein with high affinity
and results in essentially the complete quenching of fluorescence of
free ligand but does not recognize ligand bound to receptor. Thus, the
remaining fluorescence indicates ligand bound to receptor. At 120 s GTPS (100 µM) was added. Guanine nucleotide
sensitivity was used to assess the coupling between receptors and G
proteins based on characteristic ligand dissociation rates. Data were
analyzed and graphed using Prism software (GraphPad Software, Inc.).
Kinetic binding data was normalized to peak fluorescence with the mean
base line subtracted. Ligand dissociation rates were determined by
using a non-linear curve fit to either a single or a double exponential
decay as described previously (20).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (43K):
[in a new window]
Fig. 1.
Translocation of arrestin-2 from the cytosol
to the membrane following fMLF stimulation of FPR-transfected
cells. A, FPR-transfected U937 cells were treated with
50 µM fMLF for 8 min at 37 °C. Cells were then
sonicated to generate cytosolic and membrane fractions. Subsequent
Western analysis of arrestin-2 in either the cytosolic (Cyt)
or membrane (Mem) fractions in the absence or presence of
agonist was performed. B, quantitation of arrestin-2
associtated with the membrane fraction as a percentage of total
arrestin-2 in either untreated (unstim) or treated
(+fMLF) cells. Shown are the means ± S.E. of three
independent experiments.
2 adrenergic receptor, which has been
reported to dissociate from arrestin at the membrane prior to or
concomitant with release from the membrane (25). Thus, although the FPR
does not require arrestin binding for internalization, arrestin does
bind to the wild type FPR upon stimulation and appears to
co-internalize with the receptor.
View larger version (112K):
[in a new window]
Fig. 2.
Arrestin-2 colocalizes with the FPR following
exposure to ligand. U937 cells stably transfected to express wild
type FPR were incubated for 8 min at either 0 or 37 °C with
N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys fluorescein. The cells were
then fixed with 2% paraformaldehyde and permeabilized with saponin.
Arrestin-2 was visualized by staining with anti-arrestin antibodies
followed by a secondary antibody conjugated to Texas Red. Shown are
representative microscopic images displaying the cellular localization
of FPR and arrestin-2. Three independent experiments were performed
with identical results.
To characterize this interaction further, we employed two mutant forms of the FPR, R123G and D71A, that we have described previously (26). The R123G mutation is at the cytoplasmic boundary of the third transmembrane domain, and the D71A is located in the second transmembrane segment. The Arg-123 site is part of the highly conserved DRY consensus sequence that is conserved in all identified G protein-coupled receptors (27). Although the Asp and Tyr can permit a small number of substitutions, the Arg is unalterable. The Asp-71 residue is also highly conserved in the great majority of G protein-coupled receptors, with only a small number exhibiting substitutions. In our previous work we have demonstrated that neither the R123G nor the D71A mutant receptor was able to mediate a ligand-induced calcium response as seen with the wild type FPR. This suggested a defect in the ability of these mutant receptors to either bind or activate G protein (26). Although we initially hypothesized that both mutants were unable to attain the active receptor conformation, differences in receptor phosphorylation and internalization suggested otherwise (15).
To demonstrate the functional capabilities of the R123G and D71A
mutants, we used membrane preparations to investigate receptor activity
with respect to G protein coupling. Utilizing spectrofluorometric techniques that detect ligand dissociation from receptors, it can be
determined whether a given receptor is capable of interacting with G
proteins. Two distinct rates of ligand dissociation can be observed
dependent on whether a G protein is bound to the receptor. The
receptor-G protein complex has a higher affinity for ligand than
receptor alone. This can also be viewed in spectrofluorometric experiments as the ligand dissociation rate being sensitive to guanine
nucleotide (GTPS). An initial slow dissociation rate is present that
is followed by a fast dissociation rate after the addition of GTPS
with the wild type receptor (Fig.
3A). Neither the Arg-123 nor
the Asp-71 mutants display any nucleotide sensitivity, and there is no
initial slow dissociation rate (Fig. 3A). Ligand dissociates
at a single rate rather than two distinct rates as observed with the
wild type receptors, indicating that 70% of the wild type FPR is
initially coupled to G protein (Fig. 3B). Interestingly,
although both mutant receptors exhibit single rate dissociation
kinetics, the R123G rate is slower than the D71A, suggesting that the
Arg-123 receptor may be able to undergo a conformational change in the
presence of ligand, possibly to a higher affinity, activated
state.
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To determine the extent to which the receptors internalize under the conditions used in microscopy experiments, cells expressing either wild type, R123G, or D71A FPR were treated with fluorescent agonist for 8 min and washed extensively to remove fluorescent ligand from the exterior of the cell. Samples were then assayed by flow cytometry where detected fluorescence represents fluorescein that has been internalized with receptors into the cell (Fig. 3C). Approximately 70% of the total wild type cell surface receptors have been internalized under these conditions, and approximately 50% of the cell surface R123G mutant FPR was internalized. Under the same conditions, there is essentially no internalization of the D71A receptor. This is consistent with the rates and extents of internalization demonstrated previously for other agonists (28).
We next examined the ability of both of the mutant receptors to
redistribute arrestins following stimulation. Fluorescence microscopy
experiments were performed to determine whether either the R123G or
D71A mutant receptor was able to colocalize with arrestin-2. Both
receptors, like the wild type receptor, initially appeared diffuse,
spread evenly over the cell surface (data not shown). After incubation
with fluorescent agonist, the R123G receptor appears punctate, similar
to wild type receptor, with a significant fraction of the receptor
appearing in endosomes (Fig. 4). The D71A
receptor, however, remains diffuse over the cell surface reacting very
differently to agonist treatment. Stimulation of the cells has no
effect on the appearance of arrestin-2, and there is no apparent
colocalization with either mutant receptor. The result for the R123G
mutant FPR was particularly surprising, because it has been shown to
undergo ligand-dependent phosphorylation (28). To support
these findings, arrestin-2 translocation experiments were performed
using the R123G and D71A cells lines. For both cell lines, there is no
change in the fraction of arrestin that is membrane-associated upon
ligand stimulation, with <10% of the total arrestin being associated
with the membrane fraction in either the agonist-treated or
unstimulated cells (data not shown). These results indicate that
receptor activation is required for receptor processing and arrestin
binding as demonstrated by the D71A FPR mutant but that receptor
phosphorylation alone, in the presence of ligand, is insufficient for
arrestin binding as demonstrated by the R123G FPR mutant, which
undergoes phosphorylation and internalization but does not activate G
proteins.
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The results of our experiments are summarized in Table I. After stimulation with agonist, the wild type FPR assumes a fully active conformation, activates G protein, localizes to discrete membrane sites, recruits arrestin, and internalizes. The D71A mutant FPR appears totally inactive, exhibiting none of the features of the wild type receptor with the exception that it is able to bind ligand, be it in the low affinity state. The R123G mutant FPR represents an interesting intermediate. It binds ligand only with low affinity, indicating it does not couple to G protein. This has been confirmed previously in calcium mobilization experiments (26). Although we had at first believed this mutant was inactive like the D71A mutant, we were surprised to discover that it became phosphorylated and internalized almost as well as the wild type receptor. If receptor internalization required arrestin binding, then this mutant would also have to bind arrestin. However, our recent studies in HEK cells suggested that the FPR could internalize in an arrestin-independent manner.2 In the current study, when we examined the ability of the R123G mutant FPR to colocalize with arrestin in U937 cells, we found that no such interaction occurred, demonstrating that in a native myeloid cell line, FPR internalization occurs in the absence of arrestin binding.
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Our data further indicate the importance of the Arg-123 site not only in the interaction with G protein, but with arrestin as well. It is interesting to note that the R123G mutant is recognized by the kinase, as we have demonstrated previously, resulting in substantial ligand-dependent phosphorylation. This suggests that the R123G mutant may be able to form an overall active-like conformation state that can be recognized by the kinase but not by G protein or arrestin. This is supported by Fig. 3B, in which the ligand dissociation rate from the R123G mutant differs from that of the D71A mutant. If the D71A mutant is incapable of forming an active conformation and wild type can form a fully active conformation, the R123G mutant may represent a new intermediate state of the receptor. An alternative interpretation could propose that in addition to being an important point of contact for G protein, the Arg-123 site governs the phosphorylation pattern produced by the kinase, altering it in such a way that arrestin can no longer bind, even though phosphorylation-dependent internalization can take place. Even in this situation, a crucial role for Arg-123 is indicated in the regulation of receptor processing.
The role of the DRY sequence in two other GPCRs, the
gonadotropin-releasing hormone receptor (29) and the
1b-adrenergic receptor (16), has been extensively
modelled. Work with both receptors suggests that the arginine in the
DRY sequence plays a key structural role in receptor activation. Scheer
et al. (16) speculate that for the
1b-adrenergic receptor, mutations of the arginine
residue can induce different states of the receptor. Ballesteros
et al. (29) suggest that a highly conserved aspartate, which
is on helix 2 in most GPCRs and on helix 7 in the
gonadotropin-releasing hormone receptor, forms a salt bridge with the
arginine of the DRY sequence in the active state of the GPCR. This
conflicts with the view by the Scheer et al. (16), which
argues that the arginine is not engaged in a salt bridge interaction.
Molecular modelling by the latter group suggests that the Arg of the
DRY sequence is embedded within the receptor in the inactive state, and
upon ligand-mediated receptor activation, the ensuing conformational change results in the movement of the arginine side chain to the cytoplasmic surface, where it becomes solvent-exposed. This suggests that the exposure of the arginine side chain is a crucial triggering event in G protein binding and/or activation. This model supports our
conclusion that this same site is equally important in triggering the
binding of arrestin, consistent with the ability of arrestin to
distinguish between active, phosphorylated and inactive,
phosphorylated receptors.
In conclusion, we describe a novel function of a recognized G
protein-coupled receptor domain. The DRY motif of G protein-coupled receptors, known to be involved in G protein activation, is shown here
to be critical to arrestin recognition and binding, demonstrating the
use of a conserved receptor signal to signify the activation state of
the receptor to multiple binding partners. This represents the first
report, to our knowledge, of the mapping of a site used by arrestins to
assess the activation state of a receptor, independent of the
phosphorylation state of the receptor. It remains to be determined
whether this paradigm will extend to other G protein-coupled receptors.
In addition, we directly demonstrate that internalization of the FPR
occurs in the absence of arrestin binding, suggesting the existence of
alternative adapter proteins that recognize phosphorylated receptors
and mediate their internalization.
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ACKNOWLEDGEMENTS |
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We thank Dr. David Bear for use of microscopy facilities.
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FOOTNOTES |
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* This research was supported by Grants AI36357 and AI43932 from the National Institutes of Health and a grant-in-aid from the American Heart Association (National Center), and the University of New Mexico Cancer Center was supported by the New Mexico State Cigarette Tax.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 and reprint requests should be addressed.
Tel.: 505-272-5647; Fax: 505-272-1448; E-mail:
eprossnitz@salud.unm. edu.
Published, JBC Papers in Press, May 22, 2000, DOI 10.1074/jbc.C000314200
2 T. L. Gilbert, T. A. Bennett, D. C. Maestas, D. F. Cimino, and E. R. Prossnitz, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are:
GPCR(s), G
protein-coupled receptor(s);
G protein, guanine nucleotide-binding
regulatory protein;
FPR, N-formyl peptide receptor;
fMLF, N-formyl-methionyl-leucyl-phenylalanine;
Nle, norleucine;
GRK(s), G protein-coupled receptor kinase;
HEK, human embryonic kidney;
GTPS, guanosine 5'-3-O-(thio)triphosphate;
HRP, horseradish peroxidase;
PBS, phosphate-buffered saline;
Ab, antibody.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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