| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 274, Issue 42, 29791-29795, October 15, 1999
From the Department of Cell Biology and Physiology, University of New Mexico Health Science Center, Albuquerque, New Mexico 87131
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
Following activation by ligand, most G
protein-coupled receptors undergo rapid phosphorylation. This is
accompanied by a drastic decrease in the efficacy of continued or
repeated stimulation, due to receptor uncoupling from G protein and
receptor internalization. Such processing steps have been shown to be
absolutely dependent on receptor phosphorylation in the case of the
N-formyl peptide receptor (FPR). In this study, we report
results that indicate that the mechanisms responsible for
desensitization and internalization are distinct. Using site-directed
mutagenesis of the serine and threonine residues of the FPR carboxyl
terminus, we have characterized regions that differentially regulate
these two processes. Whereas substitution of all 11 Ser/Thr residues in
the carboxyl terminus prevents both desensitization and
internalization, substitution of four Ser/Thr residues between 328-332
blocks desensitization but has no effect on internalization. Similarly,
substitution of four Ser/Thr residues between positions 334 and 339 results in a deficit in desensitization but again no decrease in
internalization, suggesting that phosphorylation at either site evokes
receptor internalization, whereas maximal desensitization requires
phosphorylation at both sites. These results also indicate that
receptor internalization is not involved in the process of
desensitization. Further analysis of the residues between 328-332
revealed that restoration either of Ser328 and
Thr329 or of Thr331 and Ser332 was
sufficient to restore desensitization, suggesting that phosphorylation within either of these two sites, in addition to sites between residues
334 and 339, is sufficient to produce desensitization. Taken together,
these results indicate that the mechanisms involved in FPR processing
(uncoupling from G proteins and internalization) are regulated
differentially by phosphorylation at distinct sites within the carboxyl
terminus of the FPR. The relevance of this paradigm to other G
protein-coupled receptors is discussed.
The human N-formyl peptide chemoattractant receptor
(FPR)1 is a member of the
seven-transmembrane receptor superfamily. Expressed predominantly on
leukocytes, the FPR, which binds ligands such as
N-formyl-Met-Leu-Phe (fMLF), couples to heterotrimeric G
proteins, activating numerous effectors including phospholipase C and
thereby initiating responses such as chemotaxis, superoxide production, and degranulation (1). The FPR is one of the better studied of the
chemoattractant/chemokine family of receptors, which is responsible, in
large part, for control of numerous immune functions (2). Much interest
has recently been devoted to this receptor family following the
discovery that a number of chemokine receptors are co-receptors for
human immunodeficiency virus (3). Despite the importance of this family
of receptors, relatively little is known regarding the molecular
mechanisms involved in activating and terminating receptor function.
Control and termination of effector functions, through a process termed
adaptation, is essential to cellular function and occurs in the
continued presence of activating ligand to prevent damage to the host.
The mechanisms responsible for these processes, although poorly
understood, are believed to consist of two essential components:
uncoupling of the receptor from G proteins (desensitization) and
removal of the receptor from the cell surface through receptor internalization (4, 5). Phosphorylation of the carboxyl terminus of the
FPR is proposed to play a major role in these processes for this
receptor (6). Members of the family of G protein-coupled receptor
kinases have been shown to phosphorylate serine and threonine residues
in the carboxyl terminus of the FPR (6). Substitution of all 11 serine
and threonine residues in the carboxyl terminus with alanine and
glycine residues produces a receptor incapable of undergoing either
functional desensitization (uncoupling from G proteins) or
ligand-induced internalization (7, 8). We have recently shown,
however, that receptor phosphorylation, desensitization, and
internalization are not obligatory steps for chemotaxis
(8).
Studies with glutathione S-transferase fusion proteins
containing the 47 amino acid carboxyl terminus of the FPR suggested that phosphorylation of the FPR occurs in a hierarchical manner within
a region in which 8 out of 12 continuous residues (between residues
328-339) are either serine or threonine (6). The presence of acidic
amino acids at the beginning and in the middle of this region is
consistent with the demonstrated preferential recognition sites for
phosphorylation by G protein-coupled receptor kinase 2 (9), suggesting
that either or both of the clusters of Ser and Thr residues following
an acidic residue may be sites of phosphorylation. Our experimental
results indicated that significant phosphorylation occurred within each
of these clusters of serine and threonine residues (7). However, the
functional consequences of phosphorylation at specific sites within the
carboxyl terminus are not known.
In this study, we have examined a series of FPR mutants with
substitutions of carboxyl-terminal serine and threonine residues and
determined the role(s) of these residues in receptor desensitization and internalization. The results of our study indicate that FPR desensitization and internalization must occur via distinct mechanisms due to the particular properties of individual
phosphorylation-deficient mutants. Furthermore, our results demonstrate
that receptor internalization is not required for desensitization to
occur, further supporting the idea that these two processes occur via
distinct mechanisms.
Materials--
fMLF and fetal bovine serum were purchased from
Sigma. N-Formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys-fluorescein and
indo-1/AM were obtained from Molecular Probes, Inc. (Eugene, OR). RPMI
1640 was from Hyclone.
Construction and Expression of Site-directed Mutants in U937
Cells--
The FPR gene was mutagenized as described previously (10).
For transfection into U937 cells, 4 × 106 cells grown
in RPMI 1640 (supplemented with 2 mM
L-glutamine, 100 units/ml penicillin, 100 µg/ml
streptomycin, 10 mM HEPES, pH 7.4, and 10% fetal bovine
serum) were centrifuged and resuspended with 400 µl of RPMI 1640 containing 10 mM glucose and 0.1 mM
dithiothreitol. Linearized DNA (5-20 µg) was added, and the cells
were electroporated with a 240-V pulse from a 960-microfarad capacitor
(resulting in a pulse time constant of 30-35 ms) and returned to 5 ml
of growth medium (11). For selection, G418 was added to a final active
concentration of 1 mg/ml. Cells were cultured at 37 °C in a
humidified atmosphere of 6% CO2.
Flow Cytometry--
Cells (5 × 105) were
harvested by centrifugation, washed once with Tris-buffered saline, and
resuspended to 106 cells/ml in Tris-buffered saline.
Binding was carried out in 0.5 ml with
N-formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys-fluorescein at 10 nM. Cells were analyzed on a FACSCaliber flow cytometer
(Becton Dickinson) for fluorescent intensity and gated on forward and side scatter to exclude debris and dead cells. Nonspecific binding was
determined in the presence of 1 µM
N-formyl-Met-Leu-Phe and was almost identical to the level
of fluorescence observed with vector-transfected cells.
Ligand Binding Determinations--
Ligand binding parameters
were determined by flow cytometry performed as described above with the
following changes. To determine binding parameters
(Kd and Bmax) more
effectively, the higher affinity ligand
N-formyl-Nleu-Leu-Phe-Lys-fluorescein was used at eight
concentrations between 10 Measurement of [Ca2+]i--
Cells were
collected by centrifugation and resuspended at 5 × 106 cells/ml in Hanks' buffered saline solution (HBSS).
The cells were incubated with 5 µM indo-1/AM for 30 min
at 37 °C with gentle rocking, washed once with HBSS, and resuspended
to a concentration of approximately 106 cells/ml in HBSS
containing 1.5 mM EGTA, pH 8.0. The mobilization of
intracellular Ca2+ by various concentrations of fMLF was
monitored using an SLM 8000 photon-counting spectrofluorometer
(SLM-Aminco) detecting the ratio of fluorescence at 400 and 490 nm, as
described (13). The concentration of intracellular Ca2+ was
calculated as described (14).
In Vivo Phosphorylation--
Phosphorylation of the wild type
FPR and FPR mutants was determined as described (8). Briefly,
FPR-transfected U937 cells were harvested and resuspended in
phosphate-free RPMI 1640 containing 1 mCi of carrier-free, acid-free
[32P]orthophosphate. Cells were loaded for 3 h at
37 °C and then stimulated with 1 µM fMLF for 10 min at
37 °C. Cells were lysed with radioimmune precipitation buffer, and
insoluble debris was removed by centrifugation. The supernatant was
added to 10 mg of protein A-Sepharose coated with an anti-FPR antibody
and incubated for 1 h while rotating at 4 °C. The beads
were serially washed, and bound proteins were eluted with
Laemmli sample buffer. Samples were separated by
electrophoresis on a 12.5% SDS-polyacrylamide gel, and 32P
content was determined with a Molecular Dynamics PhosphorImager.
Desensitization--
Desensitization of calcium mobilization was
determined as follows. Cells (1 × 107) were loaded
with indo-1/AM as described above for calcium flux determinations and
divided into two parts. One was stimulated with 1 µM fMLF
for 10 min, whereas the other was treated with buffer only. The cells
were then washed three times with HBSS at room temperature to remove
surface-bound fMLF and resuspended for assay of calcium mobilization as
described above. The response of the treated and untreated cells to a
stimulation with 100 nM fMLF was determined.
Desensitization is expressed as the percentage decrease of the response
of the treated cells relative to the untreated cells.
Receptor Internalization--
Receptor internalization was
determined as the agonist-dependent loss of FPR from the
cell surface (15). FPR-transfected U937 cells were harvested, washed,
and resuspended in HBSS as above. Cells were then stimulated with 1 µM fMLF for 10 min (or the indicated time) at 37 °C
and washed three times with HBSS. Remaining cell surface receptors were
determined with 10 nM
N-formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys-fluorescein. Ligand-stained cells were then analyzed for fluorescent intensity on a
FACSCaliber flow cytometer with dead cells excluded by a gate on
forward and side scatter. Receptor internalization is expressed as the
percentage decrease of the cell surface receptors of the treated cells
relative to the untreated cells.
G protein-coupled receptors are known to undergo rapid
phosphorylation following agonist stimulation. Multiple kinases can be
involved in these phosphorylation reactions, including members of the G
protein-coupled receptor kinase family, which specifically recognize
agonist-activated receptors, and second messenger-activated kinases,
such as protein kinase A and C (16). Receptor phosphorylation by itself
does not appear to prevent coupling to G proteins. Desensitization most
likely requires the binding of an accessory molecule, such as arrestin,
which specifically binds phosphorylated GPCRs and sterically prevents G
protein association (17). Recently, arrestin has also been shown to
bind to clathrin, suggesting a mechanism for the concerted
desensitization and internalization of GPCRs (18). Whether receptor
desensitization and internalization are coupled or independent
processes remains largely unknown.
In this report, we characterize the ability of a series of mutant forms
of the FPR to undergo phosphorylation, desensitization, and
internalization. The mutant receptors used in this study represent variants of the FPR in which Ser and Thr residues have been
replaced by Ala or Gly residues (Fig.
1). Mutant
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
12 and 3.3 × 108 M (12). Cell density was decreased to
105/ml to prevent depletion of ligand at low ligand
concentrations. Nonspecific binding was determined in the presence of
100 µM N-formyl-Met-Leu-Phe. Binding
parameters were calculated using the program Prism (Graphpad). Absolute
Bmax values were determined by comparing
fluorescent intensity with reference beads containing known amounts of
fluorescein (Flow Cytometry Standards Corp.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ST(S3191A,T325G,S328A,T329A, T331A,S332G,T334G,
T336G,S338G,T339A,S342G) lacks all eleven potential phosphorylation sites within the carboxyl terminus and has
previously been shown not to undergo either
ligand-dependent internalization or desensitization.
Mutant A (S328A,T329A,T331A,S332G) and mutant B
(T334G,T336G,S338G,T339A) represent two clusters of Ser and Thr
residues that each follow acidic residues (Glu326 and
Asp327 in the case of mutant A and Asp333 in
the case of mutant B) and have previously been shown to be deficient
in desensitization. Mutants C (S328A,T329A) and D (T331A,S332G)
each alter two of the residues altered in mutant A. Last, mutant
3 (S3191A,T325G,S342G) replaces the three Ser and Thr not
collectively replaced in mutants A and B.
View larger version (11K):
[in a new window]
Fig. 1.
Sequence of the carboxyl terminus of the FPR
and the locations of site directed mutants. The carboxyl-terminal
34 amino acids of the FPR are shown. Amino acids that differ from the
wild type sequence are indicated for each of the mutants. Note the
acidic amino acid(s) preceding the Ser and Thr residues defined by
mutant A and B.
Mutant receptors were expressed in the promyelocytic cell line, U937,
which, in its undifferentiated state, does not express endogenous FPR
but is capable of processing the transfected wild type FPR in terms of
phosphorylation, desensitization, and internalization (7, 8, 11). Flow
cytometric analysis was used to determine the distribution of
receptor-expressing transfectants (Fig.
2). In all cases, greater than 90% of
the cells expressed receptor. Quantitative binding analyses using the
high affinity FPR ligand N-formyl-Nleu-Leu-Phe-Lys-fluorescein revealed that there
were no significant differences in ligand binding affinity between the
mutants and the wild type receptor (Table
I). Receptor expression levels were found
to vary less than 2-fold from the highest (mutant A) to the lowest
(mutant ST). Receptor-mediated calcium mobilization was also not
altered in any of the mutants as compared with the wild type receptor,
with an approximately 2-fold range in EC50 values.
|
|
We next evaluated the ability of the site-directed mutants of the FPR
to undergo phosphorylation when treated with agonist. Previous results
and those obtained here indicate that the ST mutant does not undergo
any significant phosphorylation as compared with the wild type FPR
(Fig. 3). Mutants A and B each are
phosphorylated at a level corresponding to approximately 50% that of
the wild type receptor. Mutants C and D show still greater
increases in the level of phosphorylation as Ser and Thr residues are
restored to the receptor. Mutant 3, which contains all eight Ser and
Thr residues in the central portion of the FPR carboxyl terminus, displays phosphorylation similar to that of the wild type FPR. These
results suggest that receptor phosphorylation is approximately proportional to the content of potentially phorsphorylated residues within the central cluster of eight Ser and Thr residues.
|
FPR desensitization in response to treatment with fMLF was examined
next (Fig. 4, upper
panel). Cells were first treated for 10 min with a
saturating dose of fMLF, washed free of ligand, and then assayed for
calcium mobilization. The wild type receptor displays an approximately
80% reduction in peak calcium mobilization to the second challenge of
fMLF. On the contrary, mutant ST displays only a 20-25% reduction
in calcium mobilization under these assay conditions. Mutant A shows
a similar deficit to ST, whereas mutant B shows a less pronounced
effect. This is consistent with our previous finding that mutant B
produces an intermediate level of desensitization. However, mutants
C, D, and 3 demonstrated no defect in their ability to undergo
desensitization. The result that mutant 3 exhibited no defect in
desensitization indicates that phosphorylation solely within the
remaining eight Ser and Thr residues defined by mutants A and B
is sufficient to elicit desensitization. The much greater deficit
observed with mutant A as compared with mutant B indicates that
phosphorylation of residues Ser328, Thr329,
Thr331, and/or Ser332 plays a critical role in
the desensitization process, whereas phosphorylation of residues
Thr334, Thr336, Ser338, and/or
Thr339 plays a more minor or secondary role. We next
examined whether removal of all of the Ser and Thr residues within
mutant A was necessary to prevent desensitization. Mutants C and
D alter the first and second pair of residues altered in mutant
A, respectively. Analysis of these mutants revealed that they
desensitized normally, indistinguishably from the wild type receptor.
These results indicate that phosphorylation of either site
(Ser328/Thr329 or
Thr331/Ser332), in conjunction with the sites
defined by mutant B, is sufficient to promote desensitization of the
receptor, suggesting redundant sites for FPR desensitization.
Furthermore, these results suggest that in the wild type receptor,
phosphorylation probably occurs at minimally three sites, defined by
C, D, and B.
|
Receptor internalization has been suggested to represent a possible
mechanism for, or at least a contributing factor to, desensitization. In support of this, the mechanisms responsible for receptor
desensitization and internalization appear to be intimately associated
due to the pivotal role played by arrestin in both these processes. To examine this relationship in the FPR, we next tested the ability of
each mutant to be internalized (Fig. 4, lower
panel). In the case of the wild type receptor, approximately
80% of the receptor internalizes in 10 min. The ST mutant, on the
contrary, demonstrates no internalization, indicating an absolute
requirement for phosphorylation in this process. However, unlike the
profile seen with desensitization, all partially phosphorylated mutants
underwent complete internalization, indistinguishable from that of the
wild type receptor. To examine whether determining internalization at a
fixed time point might be inadequate to resolve differences in the
rates of internalization, we determined the time course of FPR
internalization for the wild type and mutant receptors. The results
indicate that there are no significant differences in the rates of
internalization, with half-times for internalization ranging from 3 to
4 min for all mutants including the wild type FPR (Fig.
5). Furthermore, the total levels of
internalization are also very similar, varying by no more than 10%.
The result that phosphorylation at site B (occurring in mutant A)
can support internalization while being inadequate to promote
densensitization suggests that distinct mechanisms mediate these two
processes. Furthermore, this result demonstrates that FPR
internalization cannot be responsible for its desensitization.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
In this paper, we have determined the ability of mutant forms of
the FPR to undergo desensitization and internalization. The surprising
result is that preventing phosphorylation at residues 328, 329, 331, and 332 (defined as site A) completely abolishes receptor
desensitization but has no effect on receptor internalization. Similar
results were observed by preventing phosphorylation at residues 334, 336, 338, and 339 (defined as site B), although the effect on
desensitization is not as great. Of further interest is the result that
restoration of either residues 328 and 329 or residues 331 and 332 within mutant A is sufficient to restore receptor desensitization.
Together, these results suggest that the processes of FPR
desensitization and internalization must occur through distinct mechanisms.
In support of the conclusion that desensitization and internalization
of the FPR occur by distinct mechanisms are results obtained with the
m2 muscarinic acetylcholine receptor (m2AChR). This GPCR contains a
large third intracellular loop of approximately 180 amino acids, which
is known to undergo agonist-mediated phosphorylation (19). In the
middle portion of this loop are two clusters of Ser and Thr residues
very similar to those in the carboxyl terminus of the FPR. Fig.
6 shows an alignment of the two
sequences. The two clusters are designated site A and site B to
represent the NH2- and COOH-terminal cluster, respectively.
The alignment demonstrates that there is remarkable homology between
these two sites between the FPR and the m2AChR. Within each site, three
of the four Ser and Thr residues can be aligned perfectly between the
FPR and the m2AChR, with variation in the position of the fourth
Ser/Thr residue. Each cluster is immediately preceded by one or two
acidic amino acid residues. The presence of N-terminal acidic residues has been shown to be stimulating factor in the activity of G
protein-coupled receptor kinase 2 toward peptide substrates (9).
Interestingly, whereas the two sites are directly juxtaposed in the
case of the FPR, they are separated by 13 amino acids in the case of
the m2AChR. Studies by Pals-Rylaarsdam and Hosey (19) have analyzed the effects of mutations at these homologous sites. Their results demonstrated that mutation of the Ser and Thr residues at site B
prevented desensitization, whereas mutation of the Ser and Thr residues
at site A desensitized normally. Furthermore, both of these mutants
internalized normally. The m2AChR results are similar to ours except
for the apparent transposition of sites A and B. In the case of the
FPR, mutation of site A prevents desensitization, whereas in the case
of the m2AChR, mutation of site B prevents desensitization. Mutation of
site B in the FPR produces a modest inhibition of desensitization,
indicating that for maximal FPR desensitization, phosphorylation must
take place within both sites A and B. With both mutant receptors,
phosphorylation at either site is completely sufficient for
internalization to occur.
|
These results lead to two important questions. First, what structural
and functional similarities are there between the FPR and the m2AChR?
Second, what are the mechanisms that distinguish between
phosphorylation at sites A and B in these receptors? With regard to the
first question, similar clusters of Ser and Thr residues exist in
numerous other receptors, including the chemoattractant receptors for
C5a (20) and interleukin-8 (21), which bear high overall homology to
the FPR, as well as receptor subtypes for serotonin (22), angiotensin
(23), and bombesin (24). It is also of interest that the location of
the Ser/Thr clusters does not appear to be critical, since they are
located either on the third intracellular loop (as in the case of the
m2AChR) or the carboxyl-terminal tail (as in the case of most GPCR
including the FPR). Many other receptors known to undergo
agonist-mediated phosphorylation do not possess clusters as distinct as
those observed for the FPR and m2AChR. In the case of the
2-adrenergic receptor, the mapped phosphorylation sites
span a large portion of the carboxyl-terminal tail (25). In the case of
the lutropin/choriogonadotropin receptor, phosphorylation of four Ser
residues spanning 17 amino acids has been demonstrated (26).
Interestingly, in this latter case mutation of one of these four Ser
residues impedes receptor internalization but has no effect on
desensitization, whereas mutation of either of two other Ser residues
delays the onset of desensitization with a lesser effect on
internalization. These results support the conclusion here that
distinct mechanisms may be involved in receptor desensitization and
internalization of GPCR.
With regard to the second question, the mechanisms responsible for
receptor desensitization and internalization are only partly understood. In the case of the 2-adrenergic receptor,
the protein arrestin is known to be crucial in the desensitization and
internalization of the receptor. However, in the case of the m2AChR, it
appears that arrestin may play a role in desensitization but not
internalization (27). Yet another scenario is described by the rat
follitropin receptor, which appears to be phosphorylated on multiple
intracellular loops yet can be internalized in a
phosphorylation-independent, although arrestin-dependent,
pathway (28). We have recently demonstrated that G protein activation
by the FPR is not necessary to elicit agonist-mediated internalization
of the receptor (15). An FPR mutant, R123G, located in the highly
conserved DRY motif at the beginning of the second intracellular loop,
is unable to bind or activate G protein yet is competent in undergoing
agonist-mediated phosphorylation and internalization, consistent with
the requirement for receptor phosphorylation in desensitization and internalization.
In conclusion, we have demonstrated the existence of a highly conserved
sequence motif, consisting of two clusters of Ser and Thr residues
preceded by acidic residues, involved in directing the desensitization
and internalization of GPCRs. Phosphorylation of either of these two
clusters is sufficient to mediate internalization, whereas disruption
of one cluster can completely inhibit desensitization. These
distinctions clearly indicate that these two events, which both require
receptor phosphorylation, proceed by different mechanisms. The
existence of similar sequences in other GPCR suggests that the results
described here may be much more widespread throughout the GPCR superfamily.
| |
FOOTNOTES |
|---|
* This research was supported by National Institutes of Health Grants AI36357 and AI40115, a grant-in-aid from the American Heart Association (National Center), and the University of New Mexico Cancer Center, 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.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: FPR, N-formyl peptide receptor; fMLF, N-formyl-methionyl-leucyl-phenylalanine; G protein, guanine nucleotide-binding regulatory protein; HBSS, Hanks' balanced salt solution; m2AChR, m2 muscarinic acetylcholine receptor; GPCR, G protein-coupled receptor; Nleu, norleucinyl.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Ye, R. D., and Boulay, F. (1997) Adv. Pharmacol. 39, 221-289 |
| 2. | Prossnitz, E. R., and Ye, R. D. (1997) Pharmacol. Ther. 74, 73-102[CrossRef][Medline] [Order article via Infotrieve] |
| 3. | Choe, H. (1998) Arch. Pharmacal. Res. 21, 634-639[Medline] [Order article via Infotrieve] |
| 4. | Zhang, J., Ferguson, S. S., Barak, L. S., Aber, M. J., Giros, B., Lefkowitz, R. J., and Caron, M. G. (1997) Receptors Channels 5, 193-199[Medline] [Order article via Infotrieve] |
| 5. |
Lefkowitz, R. J.
(1998)
J. Biol. Chem.
273,
18677-18680 |
| 6. |
Prossnitz, E. R.,
Kim, C. M.,
Benovic, J. L.,
and Ye, R. D.
(1995)
J. Biol. Chem.
270,
1130-1137 |
| 7. |
Prossnitz, E. R.
(1997)
J. Biol. Chem.
272,
15213-15219 |
| 8. |
Hsu, M. H.,
Chiang, S. C.,
Ye, R. D.,
and Prossnitz, E. R.
(1997)
J. Biol. Chem.
272,
29426-29429 |
| 9. | Onorato, J. J., Palczewski, K., Regan, J. W., Caron, M. G., Lefkowitz, R. J., and Benovic, J. L. (1991) Biochemistry 30, 5118-5125[CrossRef][Medline] [Order article via Infotrieve] |
| 10. | Prossnitz, E. R., Quehenberger, O., Cochrane, C. G., and Ye, R. D. (1993) Biochem. J. 294, 581-587 |
| 11. | Kew, R. R., Peng, T., DiMartino, S. J., Madhavan, D., Weinman, S. J., Cheng, D., and Prossnitz, E. R. (1997) J. Leukocyte Biol. 61, 329-337[Abstract] |
| 12. | Vilven, J. C., Domalewski, M., Prossnitz, E. R., Ye, R. D., Muthukumaraswamy, N., Harris, R. B., Freer, R. J., and Sklar, L. A. (1998) J. Recept. Signal Transduct. Res. 18, 187-221[Medline] [Order article via Infotrieve] |
| 13. | Prossnitz, E. R., Quehenberger, O., Cochrane, C. G., and Ye, R. D. (1991) Biochem. Biophys. Res. Commun. 179, 471-476[CrossRef][Medline] [Order article via Infotrieve] |
| 14. | Cobbold, P. H., and Rink, T. J. (1987) Biochem. J. 248, 313-328[Medline] [Order article via Infotrieve] |
| 15. | Prossnitz, E. R., Gilbert, T. L., Chiang, S., Campbell, J. J., Qin, S., Newman, W., Sklar, L. A., and Ye, R. D. (1999) Biochemistry 38, 2240-2247[CrossRef][Medline] [Order article via Infotrieve] |
| 16. | Lefkowitz, R. J. (1996) Nat. Biotechnol. 14, 283-286[CrossRef][Medline] [Order article via Infotrieve] |
| 17. |
Lohse, M. J.,
Benovic, J. L.,
Codina, J.,
Caron, M. G.,
and Lefkowitz, R. J.
(1990)
Science
248,
1547-1550 |
| 18. | Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450[CrossRef][Medline] [Order article via Infotrieve] |
| 19. |
Pals-Rylaarsdam, R.,
and Hosey, M. M.
(1997)
J. Biol. Chem.
272,
14152-14158 |
| 20. | Boulay, F., Mery, L., Tardif, M., Brouchon, L., and Vignais, P. (1991) Biochemistry 30, 2993-2999[CrossRef][Medline] [Order article via Infotrieve] |
| 21. |
Thomas, K. M.,
Taylor, L.,
and Navarro, J.
(1991)
J. Biol. Chem.
266,
14839-14841 |
| 22. | Hartig, P. R., Adham, N., Zgombick, J., Macchi, M., Kao, H. T., Schechter, L., Branchek, T., and Weinshank, R. (1993) Psychopharmacol. Ser. (NY) 10, 15-25 |
| 23. | Nawata, H., Takayanagi, R., Ohnaka, K., Sakai, Y., Imasaki, K., Yanase, T., Ikuyama, S., Tanaka, S., and Ohe, K. (1995) Steroids 60, 28-34[CrossRef][Medline] [Order article via Infotrieve] |
| 24. |
Battey, J. F.,
Way, J. M.,
Corjay, M. H.,
Shapira, H.,
Kusano, K.,
Harkins, R.,
Wu, J. M.,
Slattery, T.,
Mann, E.,
and Feldman, R. I.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
395-399 |
| 25. |
Fredericks, Z. L.,
Pitcher, J. A.,
and Lefkowitz, R. J.
(1996)
J. Biol. Chem.
271,
13796-13803 |
| 26. |
Lazari, M. de F. M.,
Bertrand, J. E.,
Nakamura, K.,
Liu, X.,
Krupnick, J. G.,
Benovic, J. L.,
and Ascoli, M.
(1998)
J. Biol. Chem.
273,
18316-18324 |
| 27. |
Pals-Rylaarsdam, R.,
Gurevich, V. V.,
Lee, K. B.,
Ptasienski, J. A.,
Benovic, J. L.,
and Hosey, M. M.
(1997)
J. Biol. Chem.
272,
23682-23689 |
| 28. |
Nakamura, K.,
Krupnick, J. G.,
Benovic, J. L.,
and Ascoli, M.
(1998)
J. Biol. Chem.
273,
24346-24354 |
This article has been cited by other articles:
|
|
 |
|
  M. Xue, G. Hsieh, M. A. Raymond-Stintz, J. Pfeiffer, D. Roberts, S. L. Steinberg, J. M. Oliver, E. R. Prossnitz, D. S. Lidke, and B. S. Wilson Activated N-Formyl Peptide Receptor and High-Affinity IgE Receptor Occupy Common Domains for Signaling and Internalization Mol. Biol. Cell, April 1, 2007; 18(4): 1410 - 1420. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Potter, D. C. Maestas, D. F. Cimino, and E. R. Prossnitz Regulation of N-Formyl Peptide Receptor Signaling and Trafficking by Individual Carboxyl-Terminal Serine and Threonine Residues J. Immunol., May 1, 2006; 176(9): 5418 - 5425. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Revankar, C. M. Vines, D. F. Cimino, and E. R. Prossnitz Arrestins Block G Protein-coupled Receptor-mediated Apoptosis J. Biol. Chem., June 4, 2004; 279(23): 24578 - 24584. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Vines, C. M. Revankar, D. C. Maestas, L. L. LaRusch, D. F. Cimino, T. A. Kohout, R. J. Lefkowitz, and E. R. Prossnitz N-Formyl Peptide Receptors Internalize but Do Not Recycle in the Absence of Arrestins J. Biol. Chem., October 24, 2003; 278(43): 41581 - 41584. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Bylund, A. Bjorstad, D. Granfeldt, A. Karlsson, C. Woschnagg, and C. Dahlgren Reactivation of Formyl Peptide Receptors Triggers the Neutrophil NADPH-oxidase but Not a Transient Rise in Intracellular Calcium J. Biol. Chem., August 15, 2003; 278(33): 30578 - 30586. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Key, T. D. Foutz, V. V. Gurevich, L. A. Sklar, and E. R. Prossnitz N-Formyl Peptide Receptor Phosphorylation Domains Differentially Regulate Arrestin and Agonist Affinity J. Biol. Chem., January 31, 2003; 278(6): 4041 - 4047. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Vines, M. Xue, D. C. Maestas, D. F. Cimino, and E. R. Prossnitz Regulation of N-Formyl Peptide-Mediated Degranulation by Receptor Phosphorylation J. Immunol., December 15, 2002; 169(12): 6760 - 6766. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Dumont, S. Dremier, I. Pirson, and C. Maenhaut Cross signaling, cell specificity, and physiology Am J Physiol Cell Physiol, July 1, 2002; 283(1): C2 - C28. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Willets, R. A. J. Challiss, and S. R. Nahorski Endogenous G Protein-coupled Receptor Kinase 6 Regulates M3 Muscarinic Acetylcholine Receptor Phosphorylation and Desensitization in Human SH-SY5Y Neuroblastoma Cells J. Biol. Chem., May 3, 2002; 277(18): 15523 - 15529. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Potter, T. A. Key, V. V. Gurevich, L. A. Sklar, and E. R. Prossnitz Arrestin Variants Display Differential Binding Characteristics for the Phosphorylated N-Formyl Peptide Receptor Carboxyl Terminus J. Biol. Chem., March 8, 2002; 277(11): 8970 - 8978. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Lamey, M. Thompson, G. Varghese, H. Chi, M. Sawzdargo, S. R. George, and B. F. O'Dowd Distinct Residues in the Carboxyl Tail Mediate Agonist-induced Desensitization and Internalization of the Human Dopamine D1 Receptor J. Biol. Chem., March 8, 2002; 277(11): 9415 - 9421. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. A. W. Tawfeek, F. Qian, and A. B. Abou-Samra Phosphorylation of the Receptor for PTH and PTHrP Is Required for Internalization and Regulates Receptor Signaling Mol. Endocrinol., January 1, 2002; 16(1): 1 - 13. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Desai, H. April, C. Nwaneshiudu, and B. Ashby Comparison of Agonist-Induced Internalization of the Human EP2 and EP4 Prostaglandin Receptors: Role of the Carboxyl Terminus in EP4 Receptor Sequestration Mol. Pharmacol., April 13, 2001; 58(6): 1279 - 1286. [Abstract] [Full Text]
|
|
|
|
|
|
T. A. Bennett, D. C. Maestas, and E. R. Prossnitz Arrestin Binding to the G Protein-coupled N-Formyl Peptide Receptor Is Regulated by the Conserved "DRY" Sequence J. Biol. Chem., August 4, 2000; 275(32): 24590 - 24594. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Bennett, T. D. Foutz, V. V. Gurevich, L. A. Sklar, and E. R. Prossnitz Partial Phosphorylation of the N-Formyl Peptide Receptor Inhibits G Protein Association Independent of Arrestin Binding J. Biol. Chem., December 21, 2001; 276(52): 49195 - 49203. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Blaukat, A. Pizard, A. Breit, C. Wernstedt, F. Alhenc-Gelas, W. Muller-Esterl, and I. Dikic Determination of Bradykinin B2 Receptor in Vivo Phosphorylation Sites and Their Role in Receptor Function J. Biol. Chem., October 26, 2001; 276(44): 40431 - 40440. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Key, T. A. Bennett, T. D. Foutz, V. V. Gurevich, L. A. Sklar, and E. R. Prossnitz Regulation of Formyl Peptide Receptor Agonist Affinity by Reconstitution with Arrestins and Heterotrimeric G Proteins J. Biol. Chem., December 21, 2001; 276(52): 49204 - 49212. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||
, wild type FPR; 
, mutant
, mutant 
, mutant 
, mutant 
,
mutant 