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Department of Cell Biology and Physiology, University of New Mexico Health Science Center, Albuquerque, NM 87131
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ST, which is incapable of undergoing
ligand-stimulated phosphorylation. We observed that stimulation of
wild-type FPR resulted in very low levels of degranulation compared
with that mediated by cross-linking of the Fc
RI receptor.
Stimulation of the ST mutant, however, resulted in levels of
degranulation comparable to those of the FcRI receptor,
demonstrating that neither receptor phosphorylation nor arrestin
binding was necessary to initiate FPR-mediated degranulation.
Degranulation initiated by the ST mutant was proportional to the
level of active cell surface receptor, suggesting that either receptor
internalization or desensitization may be responsible for terminating
degranulation of the wild-type FPR. To distinguish between these
possibilities, we used a partially phosphorylation-deficient mutant of
the FPR that can undergo internalization, but not desensitization.
Degranulation by this mutant FPR was indistinguishable from that of the
ST mutant, indicating that FPR phosphorylation or binding of
arrestin but not internalization terminates the degranulation
response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit of a
heterotrimeric G protein. In turn, the 
subunit of the
heterotrimeric G protein is released, and the downstream signaling
events are initiated (2). Because of the potentially
harmful effects of excessive superoxide production and degranulation, a
fine balance between receptor activation, desensitization, and
resensitization must be maintained, while allowing the cell to carry
out its designated functions. Failure to do so can have deleterious
effects, resulting in conditions such as reperfusion injury,
atherosclerosis, and arthritis. The regulation of signaling events induced by ligand binding of a G protein-coupled receptor (GPCR) is mediated by phosphorylation of the receptor, which, in turn, leads to the binding of accessory proteins to the phosphorylated intracellular domain. One group of such accessory proteins is the arrestins. Arrestins are cytoplasmic proteins that were originally shown to be involved in the desensitization of numerous GPCRs, presumably through steric interference with G protein binding (2, 3). It was subsequently demonstrated that arrestins could bind directly to clathrin and AP-2, thereby mediating internalization of certain GPCRs (4, 5). Most recently, arrestins have been shown to serve as scaffolds for a number of kinases, including Src (and related kinases), mitogen-activated protein kinases, and Disheveled (6, 7, 8). In the case of FPR, however, arrestin binding has been shown not to be required for receptor internalization, with recent in vitro work suggesting that receptor phosphorylation alone may substantially inhibit G protein interactions (9, 10, 11).
Overall, the role of arrestin in GPCR-mediated signaling remains ambiguous and seems to vary depending upon the GPCR. For example, arrestins induce degranulation via the IL-8R, CXCR1 (12), but are not required to mediate degranulation via the C3a receptor (13). Downstream cellular events, triggered by activation of the C3a receptor, however, such as monocyte chemoattractant protein 1 (MCP-1) production, are dependent on arrestin binding to the receptor (13). This interaction seems to be dependent upon phosphorylation of the C3a receptor and probably activation of arrestin, since stimulation of a phosphorylation-deficient mutant fails to induce MCP-1 production (13). Whether receptor phosphorylation or arrestin binding has a role in regulating the signaling events initiated by binding of agonist to the FPR remains unclear (9, 11). Although internalization of the FPR is an arrestin-independent process (10), arrestins colocalize with the FPR upon stimulation with fMLP. The pathways that result in FPR desensitization, however, are distinct from those involved in receptor internalization (14).
The rat basophil leukemia cell line (RBL-2H3) provides a useful model to examine the regulation of signaling pathways that originate at the FPR. Stimulation of the FPR in RBL-2H3 cells transfected with the FPR has provided ambiguous results with respect to degranulation (15, 16). In addition, recent studies have reported conflicting data regarding the roles of receptor phosphorylation and accessory proteins in mediating degranulation initiated by binding of ligands to their cognate receptors (13, 17). To examine the role of receptor phosphorylation and arrestin in the regulation of signaling events that mediate the degranulation of RBL-2H3 cells upon stimulation of the FPR, we used a mutant of the FPR that lacks phosphorylation sites in the C terminus. In addition, using a second mutant that fails to bind arrestin, but is capable of internalization, we examined the consequence of receptor internalization on FPR-mediated signaling events in the absence of arrestin binding. We report that while degranulation of RBL cells in response to stimulation with the formyl peptide can be initiated in the absence of receptor phosphorylation and arrestin binding, receptor phosphorylation and/or arrestin binding lead to termination of FPR-mediated degranulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FMLP was purchased from Sigma-Aldrich (St. Louis, MO). Hexapeptide (N-fNleLFNleYK), hexapeptide-FITC, and Alexa-633 were purchased from Sigma-Aldrich and Molecular Probes (Eugene, OR). RPMI 1640 medium was obtained from BioWhittaker (Walkersville, MD), and MEM was obtained from Invitrogen (San Diego, CA). DNP-specific IgE was a gift from Dr. J. Rivera (National Institutes of Health, Bethesda, MD). Anti-arrestin rabbit polyclonal serum that reacts with both arrestin-2 and arrestin-3 was provided by Dr. J. Benovic (Thomas Jefferson University, Philadelphia, PA). Texas Red-conjugated goat anti-rabbit was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).
Cell lines and cDNA
The cDNA encoding the FPR was obtained from a human HL-60
granulocyte library (18). The ST mutant in which all 11
of the serine and threonine residues have been replaced (S319A, T325G,
S328A, T329A, T331A, S332G, T334G, T336G, S338G, T339A, S342G) and the
A mutant (S328A, T329A, T331A, S332G) have been described previously
(19). RBL-2H3 cells were a gift from Dr. B. Wilson
(University of New Mexico, Albuquerque, NM). Plasmid DNA was
transfected into RBL-2H3 cells by a lipid-based (Effectene; Qiagen,
Valencia, CA) method. Briefly, cells were plated overnight, transfected
with 1 µg of plasmid DNA, and then selected for 8–12 days in G418.
Surviving cells were pooled, and the expression levels were analyzed by
flow cytometry of 10 nM N-fNleLFNleYK-FITC. RBL cell lines
expressing three different levels of ST receptor were isolated by
flow cytometry: RBL-ST (high; 
200,000 receptors/cell), RBL-ST
(mid; 100,000 receptors/cell), and RBL-ST (low; 10,000
receptors/cell). Receptor numbers were quantitated using Simply
Cellular calibration beads (Bangs Laboratories, Fiskers, IN). RBL cell
lines were maintained at 37°C in 5% CO2 in
MEM-Earle’s medium with 15% heat-inactivated FBS, 2 mM
L-glutamine, 100 U/ml penicillin, 0.1 mg/ml
streptomycin, and 1 mg/ml G418.
Degranulation measurements of -hexosaminidase
All degranulation assays were conducted in 1x HBSS containing
10 mM HEPES, 0.05% BSA, and 15 mM NaHCO3. Three
days before assays, G418 was removed from the medium. For FcR1
activation cells were incubated for at least 14 h with 1 µg/ml
of DNP-specific IgE (anti-DNP IgE) to bind FcR1, then rinsed,
and Ag (100 ng/ml of DNP-BSA) was added to cross-link the IgE-FcR1
complexes. FPR was activated with 100 nM fMLP. Degranulation assays
were conducted at 37°C for 30 min. Supernatants containing
-hexosaminidase were incubated with
p-nitrophenyl-N-acetic-3-D-glucosamide
(Sigma-Aldrich) for 1 h at 37°C, neutralized with 200 mM glycine
(pH 10.7), diluted in water, and read on a spectrofluorometer at
405 nm. Degranulation values were normalized to the degranulation
values measured upon stimulation of the IgE receptor.
Colocalization studies
Transfected RBL-2H3 cells were seeded onto glass coverslips and stimulated for 20 min with 10 nM fMLP at 37°C. The cells were fixed in 2% paraformaldehyde solution and permeabilized in 0.02% saponin in PBS. Cells were stained indirectly with anti-arrestin-2 rabbit polyclonal Ab. Confocal microscopy was conducted on a confocal microscope (Zeiss, New York, NY) using a 40x oil immersion objective.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To understand the role of arrestin in the FPR-mediated signaling
events that induce degranulation of RBL-2H3 cells, stable transfectants
of wild type (RBL-FPR) or a phosphorylation-deficient mutant of the
FPR, ST (RBL-ST), was generated. The ST mutant, which lacks
all the potential phosphorylation sites found in the C terminus (serine
and threonine residues) when expressed in U937 pro-monocytic cells, has
previously been shown to mediate ligand-induced calcium fluxes, but not
to undergo ligand-induced internalization or desensitization (14, 19). The expression of the receptors on the cell surface was
confirmed by FACS detection of N-fNleLFNleYK-FITC, a
fluorescent ligand that binds to the FPR. To facilitate comparisons
between these cell lines, RBL-FPR and RBL-ST stable cell lines were
sorted for expression levels of 100,000 receptors/cell (Fig. 1
A).
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70% of the wild-type receptors
were internalized, the ST mutant failed to internalize (Fig. 1
B). These results confirmed that while the receptor levels
were equivalent, the loss of phosphorylation sites in the C terminus
resulted in a receptor that failed to internalize upon exposure to the
agonist. FPR-induced degranulation of RBL-2H3 cells is not mediated by receptor phosphorylation and arrestin binding
Using the RBL-FPR and RBL-ST cell lines we examined whether the
FPR-mediated degranulation of RBL-2H3 cells was activated by
phosphorylation of FPR and subsequent arrestin binding. To this end we
compared degranulation of the RBL-FPR cells to RBL-ST cells (Fig. 2A). In response to a 30-min
stimulation with 100 nM fMLP, RBL-FPR cells released 11.8 ± 2%
of their available granules compared with IgE-mediated degranulation
(Fig. 2A). On the other hand, RBL-ST cells that lack
phosphorylation sites in the C terminus and are incapable of
interacting with arrestin released of 83.8 ± 19.4% of their
granules.
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ST cells, we examined the kinetics of the response (Fig. 2B). The cells were incubated in the presence of 100 nM
formyl peptide for the indicated times, and the supernatant was removed
and assayed for the release of -hexosaminidase. The
t1/2 for RBL-FPR degranulation was 15 s. It
is important to note that the attenuation of the degranulation response
closely parallels the rate of FPR phosphorylation in RBL cells, which
has a t1/2 of 1.5 min following FPR
stimulation (16). In contrast, the
t1/2 for RBL-ST was 1 min, with the
release of granules approaching the maximum level of granules available
in the cell by 5–10 min (Fig. 2B). These results were
unexpected, since it has been shown that RBL-2H3 degranulation mediated
by the CXCR1 receptor requires arrestin activation by the receptor
(17). In addition, the phosphorylation-deficient mutant of
the C3a receptor exhibited an identical degranulation potential as the
wild-type form of C3a (13). It was unclear, however,
whether arrestin might be mobilized/activated in the ST mutant in
response to stimulation with fMLP.
To examine the behavior of arrestin upon stimulation of the FPR and
ST receptors, two additional cell lines were generated,
RBL-FPR-green fluorescent protein (GFP) and RBL-ST-GFP. In these
cell lines the FPR was fused at the C terminus to GFP (20)
and was used to determine whether activation of the receptor resulted
in clustering of the arrestin. Stimulation of the RBL-FPR-GFP cell line
with hexapeptide resulted in activation of the cells, as determined by
spreading of the cells and generation of ruffles in the membrane.
Although the FPR is phosphorylated in RBL cells within the first 2.5
min following receptor stimulation (16), clusters of FPR
on the endosomes were difficult to detect at 2.5 min, and
colocalization of arrestin with the GFP-FPR was only weakly visible at
this time point (Fig. 3A). The
lack of a strong colocalization of arrestin with FPR at 2.5 min was not
surprising, since arrestin is not required for internalization of the
formyl peptide receptor and may only accumulate with the receptor on
the endosomes following internalization. Strong colocalization of
arrestin with FPR was observed at 20 min (Fig. 3A). In
contrast, while stimulation of the ST-GFP mutant with hexapeptide
resulted in flattening and activation of the cells, as assessed by
membrane ruffling and the mobilization of calcium, the arrestin failed
to cluster. This observation suggested that while receptor-mediated
activation of the cells induces degranulation, binding of arrestin is
not required to generate the fMLP-initiated degranulation response in
RBL-2H3 cells.
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ST cells were capable of mobilizing calcium in response to
150 nm fMLP (Fig. 3B) confirming that both cell lines were
activated in response to stimulation with formylated peptide. Because
the degranulation response of RBL-FPR cells, in which FPR is
phosphorylated and can bind arrestin, is significantly different from
that of RBL-ST cells, we reasoned that receptor phosphorylation and
possibly arrestin binding may be involved in terminating the signaling
events that result in degranulation.
Differences in receptor phosphorylation/arrestin binding account
for the difference in degranulation between FPR and ST
As mentioned, the wild-type FPR is internalized, and the ST
mutant, which fails to become phosphorylated or bind to arrestin,
remains on the surface of the RBL-2H3 cells following stimulation of
the receptor. Therefore, it was unclear whether the number of active
receptors remaining on the cell surface or the ability of the receptor
to desensitize controlled the extent of the degranulation response. To
determine whether the extent of the degranulation response could be
controlled by the level of receptor on the surface of the cells, we
generated two additional cell populations by FACS-based cell
sorting, RBL-ST high (200,000 receptors/cell) and RBL-ST low
(10,000 receptors/cell). Examination of these cell lines revealed
that the degranulation response in RBL-2H3 cells generated by
stimulating the nondesensitizing, noninternalizing FPR is related to
the number of active receptors on the cell surface (Fig. 4). Based on this observation, we
predicted that the cells expressing the wild-type FPR at 100,000
receptors/cell should yield a minimal degranulation response of 25%
based on the level of internalization observed at 30 min. The actual
response should be even higher, since the initial signaling occurs at a
level of 100,000 receptors/cell and only decreases over time as
receptors are removed from the cell surface. It remained unclear,
however, whether receptor internalization or inactivation attenuated
the degranulation response of the RBL-FPR cells. To determine whether
loss of cell surface receptors or a lack of receptor desensitization
was responsible, we used a third cell line that is incapable of binding
arrestin, but is capable of undergoing internalization.
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A (S328A, T329A, T331A, S332G) mutant lacks four of the 11
potential phosphorylation sites at the C terminus. This receptor, when
expressed in the U937 pro-monocytic cell line, was able to internalize,
but was only phosphorylated at 50% the level of the wild-type FPR
(14). As a result, the A receptor does not bind
arrestin (19). In addition, using calcium mobilization
assays, it was determined that the A mutant, like the ST mutant,
fails to desensitize in response to stimulation with the formyl peptide
(14). We hypothesized that if phosphorylation of the C
terminus and the subsequent association of arrestin attenuate
FPR-mediated signaling events that lead to degranulation, then the A
mutant, which does not bind arrestin upon stimulation with fMLP, should
degranulate to a similar extent as the ST mutant. If, however, the
number of active receptors on the surface of the cell controls the
extent of the degranulation response, then the response of the A
mutant should reflect the number of receptors on the cell surface that
were not internalized, similar to the wild-type FPR response.
RBL-A (233,000 receptors/cell) and RBL-ST (100,000
receptors/cell) expression levels and internalization rates were
compared (Fig. 5). The rate of
internalization of the A mutant was the same as that of the
wild-type FPR, although the overall extent of internalization of the
A mutant was 40% less than that of the FPR (Fig. 5B).
We predicted that if the number of active receptors on the cell surface
controlled the level of degranulation, then the extent of degranulation
exhibited by the A mutant should be greater than the level
exhibited by RBL-FPR. In contrast, since at the end of the signaling
event, the A mutant retains 120,000 receptors on the surface,
the degranulation levels for the A mutant should be equivalent
to the level expected in the presence of 100,000 ST receptors. To
determine whether the A mutant was capable of activating the RBL-2H3
cells we examined the behavior of the cells in response to stimulation
with fMLP (Fig. 6). All three cell lines
were stimulated in the presence of 100 nM fMLP. Although the A
mutant clustered and internalized in the presence of fMLP, the receptor
failed to colocalize with arrestin (Fig. 6). Activation of the A
receptor was also able to induce mobilization of
Ca2+ (data not shown). The level of degranulation
of the RBL-A mutant was significantly greater than the level induced
by stimulation of the FPR (Fig. 6), but was not different from that of
the ST mutant. From these observations we find that the lack of
phosphorylation of the C terminus results in sustained FPR-mediated
degranulation of RBL cells. We conclude that signals that are
transduced from the wild-type FPR that result in degranulation are
attenuated via phosphorylation of the receptor and possibly binding of
arrestin to the FPR C terminus.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A mutant to demonstrate that a loss of
receptors from the cell surface was insufficient to attenuate
degranulation. Instead we show that phosphorylation of the C terminus
and subsequent binding of arrestin in response to stimulation of FPR
result in an attenuated degranulation response.
Previous studies examining the degranulation response of stable RBL-FPR
demonstrated 22.5% (16) release of -hexosaminidase
and little to no release of preloaded
[3H]serotonin, (15). These RBL-2H3
cell lines used in these studies stably expressed between 90,000 and
200,000 receptors. The difference in the extent of degranulation
between these studies and ours may have been due to a change in the
degranulation potential over time. We have noticed that culturing
RBL-2H3 cells for >8–10 passages results in a decrease in
degranulation potential (C. Vines and E. Prossnitz, unpublished
observations). To maintain consistency between our data sets, the
stable cell lines were discarded from culture after 1 mo of
propagation, and we normalized our degranulation values to levels that
could be generated by stimulation of the IgE receptor.
Our results are in contrast with those reported by Barlic et al.
(17), which suggested that arrestin is a critical
signaling component that is required to initiate degranulation in
RBL-2H3 cells via the CXCR1 receptor. In this study stable
transfectants of the CXCR1 receptor were used, and it was demonstrated
that upon stimulation of the CXCR1 receptor, the Src family kinases Hck
and Fgr are stimulated to bind to arrestin. This interaction was
thought to induce the activation and translocation of these Src family
members and their subsequent association with -arrestin 1.
Expression of a dominant-negative form of -arrestin 1 (P91G-P121E),
which expressed a mutated polyproline-rich SH3 domain binding region
and was shown to be defective in binding to Src (6),
inhibited degranulation of these cells. In addition, Barlic et al.
(17) demonstrated that expression of a CXCR1 mutant that
lacked all phosphorylation sites in the tail, 8STmCXCR1, failed to
degranulate. It is important to note that internalization of CXCR1 is
an arrestin-dependent event (12), while internalization of
the FPR is not (10). That arrestin has a different role in
the internalization of CXCR1 than it does in the internalization of the
FPR may indicate that arrestin has a unique function in response to
activation by either the FPR or the CXCR1.
Recent studies with the C3a receptor have found that a lack of
phosphorylatable serines or threonines in the C-terminal region
(ST-C3a) had no effect on the ability of transiently transfected
RBL-2H3 cells to degranulate, as measured by the release of
-hexosaminidase (13). Like the FPR, even in the absence
of a phosphorylatable C terminus, the C3a receptor could still induce
ERK activation (10). Interestingly, while the inability to
phosphorylate the C terminus did not positively or negatively affect
the extent of C3a-mediated degranulation in RBL-2H3 cells, it did
affect the production of MCP-1 in these cells. The former result could
be a result of the very high levels of receptor expression per cell in
transiently transfected cells. These authors conclude that the ability
to terminate degranulation was requiredfor the production of MCP-1.
They raise the possibility that Src family kinases may be involved in
the C3a-mediated degranulation process, since MCP-1 production was
substantially inhibited in the presence of a Src family kinase
inhibitor. Experiments to examine the role of Src family kinases
in the FPR-mediated degranulation of RBL-2H3 cells are currently
underway (C. M. Vines et al., manuscript in preparation).
In summary, we have found that lack of phosphorylation of the C-terminal domain and the commensurate lack of arrestin binding to the FPR do not adversely affect the initiation and propagation of degranulation. On the contrary, only in the absence of receptor phosphorylation is a significant level of degranulation observed, with the extent of FPR-mediated degranulation being dependent upon the number of active receptors on the cell surface. While RBL cells have been used as a model in this study, these cells may not accurately reflect the signaling events that take place in primary cells. Indeed, fMLP has been shown to be a strong degranulating agent in neutrophils. Therefore, the signaling pathways involved in the degranulation of these primary cells could be different (22, 23). This does not preclude the observation that in the case of wild-type FPR, receptor phosphorylation severely curtails the degranulation response. Thus, phosphorylation of the FPR yields a novel mechanism of regulation of degranulation not previously observed for other GPCRs.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Eric R. Prossnitz, Department of Cell Biology and Physiology, University of New Mexico Health Science Center, Albuquerque, NM 87131. E-mail address: eprossnitz{at}salud.unm.edu
3 Abbreviations used in this paper: FPR, formyl peptide receptor; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; MCP-1, monocyte chemoattractant protein 1.
Received for publication April 22, 2002. Accepted for publication October 18, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Arrestin2, a novel member of the arrestin/-arrestin gene family. J. Biol. Chem. 267:17882.2-adrenergic receptor/betaarrestin complex recruits the clathrin adaptor AP-2 during endocytosis. Proc. Natl. Acad. Sci. USA 96:3712.-Arrestin-dependent formation of 2 adrenergic receptor-Src protein kinase complexes. Science 283:655.2-adrenergic receptor chimera: pharmacological properties of a unique G protein-linked receptor. Naunyn Schmiedebergs Arch Pharmacol 365:341.[Medline]
-Arrestins regulate interleukin-8-induced CXCR1 internalization. J. Biol. Chem. 274:16287.R1 and formyl peptide receptor signaling pathways in RBL(FPR) cells: the contribution of receptor-specific Ca2+ mobilization responses. Biochim. Biophys. Acta 235:812.
-arrestin by CXCRI. Nat. Immunol. 1:227.[Medline]
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