From the Département de Réponse et Dynamique Cellulaires/Biochimie et Biophysique des Systèmes Intégrés, (UMR 5092, Commissariat à l'Energie Atomique (CEA)/CNRS/Université Joseph Fourier), CEA/Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France
Received for publication, October 3, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The human complement 5a (C5a) anaphylatoxin
receptor (CD88) is a G protein-coupled receptor involved in innate host
defense and inflammation. Upon agonist binding, C5a receptor (C5aR)
undergoes rapid phosphorylation on the six serine residues present
in the C-terminal region followed by desensitization and
internalization. Using confocal immunofluorescence microscopy and green
fluorescent protein-tagged The complement C5a1
anaphylatoxin receptor (C5aR) is a member of the G-protein-coupled
receptor family (GPCR) (1, 2). It is abundantly expressed in myeloid
cells and to a lower level in a variety of non-myeloid cells (3). In
neutrophils, the activation of chemoattractant receptors including the
C5aR triggers a complex array of cellular functions that results in
directed cell migration and release of large amounts of proteolytic
enzymes and reactive oxygen species (4). These responses are highly regulated since, despite the persistent presence of chemoattractants, the intracellular signaling events are transient.
As for many other GPCRs, this attenuated responsiveness is thought to
result from receptor desensitization through their phosphorylation and
rapid internalization. The current concept for GPCR desensitization, largely extrapolated from studies with rhodopsin and the
It has been previously established that the activated C5aR is
phosphorylated on the 6 serine residues located in the C-terminal tail
at positions 314, 317, 327, 332, 334, and 338 (19). Mutants with
combined amino acid replacements exhibit different capacities to
incorporate phosphate on the remaining serine residues (20, 21).
Combined mutations at positions 332 and 334, at positions 334 and 338, or at all 3 positions yield phosphorylation-deficient mutants, whereas
mutants for which the serine pairs
Ser327/Ser338 or
Ser334/Ser338 are conserved retain the capacity
to be phosphorylated (21). This phosphorylation step plays a key role
in the attenuation of the cellular response since
phosphorylation-deficient mutants trigger sustained intracellular
signaling events that result in a significant increase in C5a-mediated
superoxide production by neutrophil-like differentiated HL-60 cells
(21). The phosphorylation of the first membrane proximal serine residue
is dispensable for receptor desensitization, and agonist sequestration
is apparently independent of desensitization (21).
In the present study, we used confocal microscopy and the green
fluorescent protein conjugate of Reagents--
Bovine serum albumin fraction V, human recombinant
C5a, 1,4-diazabicyclo(2.2.2)octane (DABCO), aprotinin, leupeptin, and
pepstatin were purchased from Sigma. Goat anti-rabbit Alexa
568-conjugate antibody was from Molecular Probes, Inc. (Eugene, OR).
Tissue culture media were from Invitrogen. Dithiobis(succinimidyl
propionate) was from Pierce. Dithiothreitol cycloheximide, okadaic
acid, and 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride were
from Roche Molecular Biochemicals. Puromycin and polyclonal anti-GFP antibodies were from Clontech. Blasticidine was
from Invitrogen. The rabbit polyclonal antibodies directed against the
last 10 amino acid residues of C5aR has been previously described (22). The dominant negative dynamin mutant (hemagglutinin-dynamin K44E) (11)
were supplied by Dr. S. L. Schmid (Department of Cell Biology, Scripps Research Institute, La Jolla, CA).
Cell Culture and Transfection--
The stable expression of
wild-type C5aR and mutant receptors C5aR-A314,317,332,334
and C5aR-A332,334,338 in the insulin-secreting cell line
RINm5F has been previously described (20). RINm5F cells that stably
expressed the mutant receptors C5aR-A314,317,327,332 and
C5aR-A334,338 were obtained by transfecting cells
with 1 µg of pPUR plasmid (Clontech)
and 20 µg of CDM8 mutant receptors as described previously (24).
DNA-mediated gene transfer into RINm5F cells was performed by
electroporation. Cells were cultured in RPMI 1640 medium/GlutaMax I
supplemented with 1 µg/ml puromycin and 10% heat-inactivated fetal
calf serum. Resistant clones were assayed for receptor expression by
testing their ability to bind 125I-labeled C5a as described
previously (25). RINm5F cells expressing either wild-type or mutant
C5aR were further transfected with 1 µg of pEF-Bsd plasmid
(Invitrogen) and 20 µg of
For transient transfection, HEK-293 cells were plated onto
6-well plates. Twenty-four hours after plating, cells were
co-transfected with C5aR and either a control vector, Subcellular Fractionation--
Cells incubated with or without
50 nM C5a were washed with ice-cold phosphate-buffered
saline and scraped into 0.25 M sucrose, 10 mM
Tris, pH 7.4, 1 mM EDTA, and 1 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, and disrupted
by Dounce homogenization. Nuclei and unbroken cells were removed by
centrifugation at 1000 × g for 10 min at 4 °C. A
crude plasma membrane fraction was prepared by centrifugation of the
supernatant at 3000 × g for 15 min. Then centrifugation of the resulting supernatant at 300,000 × g for 30 min at 4 °C gave rise to a supernatant
containing cytosol.
Indirect Immunofluorescence Staining and Fluorescence
Microscopy--
For all microscopic analyses, cells were seeded
on polylysine-coated coverslips 48 h before the experiments.
Protein synthesis was blocked by incubating cells in RPMI 1640 supplemented with 1% serum albumin fraction and 100 µg/ml
cycloheximide for 3 h before stimulation with C5a. Cells were
treated in the same medium with 50 nM C5a at 37 °C for
various time periods ranging from 0 to 30 min. Cells not treated with
the ligand were used as a control. Cells were then fixed with 4%
paraformaldehyde in phosphate-buffered saline supplemented with 1 mM MgCl2 and 1 mM CaCl2
for 30 min on ice, quenched with 50 mM NH4Cl in
RPMI 1640 for 15 min at 4 °C, permeabilized with 0.1% Nonidet P-40
for 10 min at room temperature, and incubated with blocking buffer (2%
serum albumin fraction in RPMI 1640). For indirect immunofluorescence
staining of C5aR, fixed and permeabilized cells were incubated with
affinity-purified rabbit polyclonal antibody to the C5aR C terminus for
1 h at room temperature. Cells were then washed 3 times and
incubated with red-fluorescent Alexa 568-conjugate goat anti-rabbit
antibody for 30 min at room temperature. After washing, cells were
embedded in 1,4-diazabicyclo(2.2.2)octane (DABCO). For detailed
microscopic analysis, we used a Leica TCS-SP2 confocal-scanning
microscope. EGFP was excited at 488 nm with an argon laser, and its
fluorescence was collected between 497 and 532 nm. Alexa 568 was
excited at 543 nm, and fluorescence emission was collected between 557 and 667 nm.
Internalization Assay--
C5aR sequestration was performed as
previously described (21). Briefly, HEK-293 cells were seeded in
polyornithine-coated 6-well plates 48 h before co-transfection
with plasmids containing the C5aR cDNA and either the DynK44E
cDNA, Detection of C5aR and EGFP Conjugates by
Immunoblotting--
Cell monolayers were lysed in 2-fold Laemmli
sample buffer supplemented with 10 mM dithiothreitol and
briefly sonicated with a microtip. Proteins were then separated by
SDS-PAGE and transferred to a 0.2-µm Protran nitrocellulose filter
for immunoblotting. Immunodetection of C5aR and EGFP conjugates was
performed by affinity-purified rabbit anti-C5aR IgGs (1/200) and a
rabbit polyclonal anti-GFP antiserum (1/400) followed by a 1-h
incubation period time with 125I-labeled protein A. Immune
complexes on membranes were visualized by autoradiography.
Immunoprecipitation and Western
Blotting--
Immunoprecipitation experiments were performed by using
stably transfected RINm5F cells in 100-mm dishes. Cells were stimulated as described in the figure legends and solubilized in 1 ml of immunoprecipitation buffer containing 150 mM NaCl, 10 mM Tris, pH 7.5, 1% Triton X-100, 1 mM EDTA,
0.1% SDS, 10 mM NaF, 10 mM NaPi, 1 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 0.1 µM okadaic acid, 1 mM sodium orthovanadate,
and 1 µg/ml aprotinin, leupeptin, and pepstatin. To detect
C5aR-associated proteins, stimulated cells were subjected to covalent
protein cross-linking by using the permeable and cleavable cross-linker
dithiobis(succinimidyl propionate), as previously described (26).
Immunoprecipitation of C5aR was performed with affinity-purified
polyclonal anti-C5aR, as previously described (19). Immunoprecipitates
were resolved by SDS-PAGE and transferred to nitrocellulose membranes
for immunoblotting. Protein immunoblotting for full-length and
truncated Generation of Cell Lines Expressing C5aR and
Mutant receptors have been previously described and analyzed for their
ability to be phosphorylated after C5a binding when expressed in COS-7
or RINm5F cells (20, 21). With respect to the ability/inability to be
phosphorylated, wild-type C5aR, C5aR-A314,317,332,334, and
C5aR-A332,334,338 behave similarly that they are expressed
in COS-7 or RINm5F cells. Phosphorylated receptors, i.e.
wild-type C5aR and C5aR-A314,317,332,334, have a reduced
electrophoretic mobility on polyacrylamide gel in the presence of
sodium dodecyl sulfate, whereas the phosphorylation-deficient mutant
C5aR-A332,334,338 lacks this electrophoretic shift (20,
28). This hallmark of phosphorylation was used in the present study to
analyze two additional mutants, A314,317,332,334 and
C5aR-A334,338, when expressed in RINm5F cells (Fig.
1C). The absence of phosphorylation of the latter was
further confirmed by receptor immunoprecipitation after metabolic
labeling with radioactive orthophosphate (not shown).
Intracellular Trafficking of
To further examine the intracellular trafficking of C5aR and
Binding of Effect of
Because
To have a more precise measurement of the effect of Effect of Dominant Negative K44E Dynamin and
Number of reports have shown that agonist stimulation of GPCRs promotes
the formation of receptor-containing vesicles, which are pinched off
from the plasma membrane and translocated into endocytic compartments.
The pinching or sealing off of the vesicles from the plasma membrane is
dependent upon dynamin, a GTPase-containing molecule (11, 32). Because
Identification of Residues within the C5aR C Terminal Region That
Allow Interaction with
Confocal microscopy was used to assess the intracellular distribution
of the aforementioned mutant receptors and
Surprisingly, although cross-linking and immunoprecipitation
experiments did not demonstrate a C5a-mediated association of the
A332,334,338 mutant with any For most GPCRs, the primary effect of agonist binding is to bring
about a conformational change that triggers the coupling to
heterotrimeric G proteins and promotes the phosphorylation of the
receptor and its interaction with the Judging from the persistent association of In a previous study, we have shown that the phosphorylation of C5aR
occurs through a hierarchical process on serine residues located in the
C-terminal domain (21). The two most distal serine residues
(i.e. Ser334 and Ser338) serve as
primary phosphorylation sites. This step is strictly required for the
phosphorylation of the other residues. Partial phosphorylation on
Ser327/Ser338 as well as
Ser334/Ser338 is sufficient to confer a
wild-type phenotype of desensitization (21). Here, we show by confocal
microscopy that the phosphorylation of either two-serine pair is
sufficient to allow the co-trafficking of Of particular interest is the observation by confocal microscopy that
In addition to their role as clathrin adapters, In conclusion, we have demonstrated that C5a induces a marked
redistribution of -arrestins (
-arr 1- and
-arr
2-EGFP) we show a persistent complex between C5aR and
-arrestins to
endosomal compartments. Serine residues in the C5aR C terminus were
identified that control the intracellular trafficking of the
C5aR-arrestin complex in response to C5a. Two phosphorylation mutants
C5aR-A314,317,327,332 and
C5aR-A314,317,332,334, which are phosphorylated only on
Ser334/Ser338 and
Ser327/Ser338, respectively, recruited
-arr
1 and were internalized. In contrast, the phosphorylation-deficient
receptors C5aR-A334,338 and C5aR-A332,334,338
were not internalized even though observations by confocal microscopy indicated that
-arr 1-EGFP and/or
-arr 2-EGFP could be recruited to the plasma membrane. Altogether the results indicate that C5aR activation is able to promote a loose association with
-arrestins, but phosphorylation of either Ser334/Ser338 or
Ser327/Ser338 is necessary and sufficient for
the formation of a persistent complex. In addition, it was observed
that C5aR endocytosis was inhibited by the expression of the dominant
negative mutants of dynamin (K44E) and
-arrestin 1 (
-arr
1-(319-418)-EGFP). Thus, the results suggest that the C5aR is
internalized via a pathway dependent on
-arrestin, clathrin, and dynamin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptor, is that, once activated and
phosphorylated, the GPCRs form a stable complex with a family of
adapters known as arrestins and
-arrestins (5). There is growing
evidence indicating that the formation of this complex is a general
intermediate for endocytosis of most GPCRs. The
-arrestins interact
with clathrin and the adapter protein complex AP-2 (6-8) and target
the agonist-occupied receptors to pre-existing clathrin-coated pits for
internalization (9, 10). Expression of a GTPase-defective dynamin
mutant blocks the clathrin-dependent endocytic pathway in a
dominant-negative manner (11), and the agonist-mediated internalization
of most GPCRs is inhibited (12). However, some GPCRs appear to depart from this standard model and are capable of utilizing alternative pathways. For instance, the angiotensin II 1A and N-formyl
peptide receptors, although able to interact with
-arrestins, can be internalized through a process independent of
-arrestin and dynamin (13-16), whereas the 5-hydroxytryptamine 2A receptor is internalized via a pathway dependent on dynamin and independent of
-arrestin (17). The internalization of the M2 muscarinic acetylcholine receptor
appears to proceed through an atypical pathway that is independent of
clathrin (18).
-arrestin 1,
-arrestin 2, and
the dominant negative mutant
-arrestin 1-(319-418) to examine the
cellular trafficking of a series of C5aR mutants in response to C5a.
Our results demonstrate that a minimal level of phosphorylation is
required for internalization through a dynamin-, arrestin-, and
clathrin-dependent internalization pathway. Moreover,
-arrestin 1 and
-arrestin 2 were found to have differential abilities to associate with the activated receptor. In contrast to
-arrestin 1,
-arrestin 2 redistributed to the plasma membrane regardless of whether C5aR was phosphorylated provided it was activated.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Arrestin-EGFP Fusion Proteins and C5aR Phosphorylation
Mutants--
To construct a
-arr 1-EGFP fusion, a
HindIII-XbaI fragment encoding human
-arrestin
1 (
-arr 1) was isolated from plasmid pBJI-neo (supplied by Dr.
DeBlasi Mario Negri, Instituto di Richerche Farmacologiche, Santa Maria
Imbaro, Milan, Italy) and subcloned into pcDNA3.1
(Invitrogen). A HindIII-KpnI fragment encoding
residues 1-410 of
-arrestin 1 and a KpnI-SalI
synthetic fragment encoding the last eight residues of
-arrestin 1 were then ligated into pEGFP-N3 (Clontech). The
-arr 1-(319-418)-EGFP fusion was prepared as follows; a 300-bp
fragment encoding the last 100 residues of
-arrestin 1 was amplified
by PCR from plasmid
-arr 1-EGFP with the sense primer
5'-CCCAAGCTTACCATGGTTTCCTACAAAGTGAAAGTG-3' and the reverse
primer 5'-GTCGCCGTCCAGCTCGACCAG-3'. After cleavage with HindIII and BamHI, the PCR fragment was
cloned into pEGFP-N3. The nucleotide sequence of the amplified fragment
was verified by sequencing. To construct a
-arr 2-EGFP fusion, a
SacI-PstI fragment and a
PstI-BamHI fragment encoding rat
-arr 2 were
isolated from plasmid
-arr 2-GFP pS65-T (23) and cloned into
pEGFP-N3.
-arr-EGFP plasmids (
-arr 1-,
-arr
2-, and
-arr 1-(319-418)-EGFP). Selection of
-arr-EGFP-expressing clones was performed by culturing cells in the
presence of 10 µg/ml blasticidine. Resistant clones were screened for
-arr-EGFP expression by epifluorescence with an inverted microscope.
-arr
1-(319-418)-EGFP, or hemagglutinin-dynamin K44E using a standard
calcium phosphate co-precipitation protocol. After 60-72 h, cells were
assayed for their ability to internalize C5a labeled with radioactive
iodine as previously described (25).
-arr 1-(319-418)-EGFP cDNA, or the empty vector
alone. Surface-expressed receptors were saturated with
125I-labeled C5a (100 nM) in Dulbecco's
modified Eagle's medium, 1% serum albumin fraction, 20 mM
Hepes, pH 7.5, for 60 min, at 4 °C. Control cells were incubated
with an excess of unlabeled C5a to determine nonspecific binding.
Internalization was initiated by diluting cells in 5 volumes of
Dulbecco's modified Eagle's medium at 37 °C. At the indicated
times, cells were washed and treated for 10 min with ice-cold buffer
containing 0.15 M NaCl and 0.2 M acetic acid at
pH 2.5 to remove 125I-labeled C5a bound to cell surface
receptors, thus giving a measure of C5a internalization/sequestration.
The kinetics of C5a internalization are expressed as the percentage of
specifically bound 125I-labeled C5a that is internalized as
follows: (cpm resistant to acid wash after warming minus cpm resistant
to acid wash before warming)/(cpm specifically bound at 4 °C under
saturating conditions).
-arrestin was performed by using rabbit polyclonal
anti-GFP antiserum. Immune complexes on nitrocellulose membrane were
visualized by 125I-labeled protein A and autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Arrestins--
To
examine whether the C5aR interacts with the
-arrestins and to what
extent the phosphorylation of the C-terminal region of C5aR influences
this interaction, we established RINm5F cell lines that expressed
either wild-type or mutant C5aRs (see Fig. 1A) together with either
-arr 1-EGFP,
-arr 2-EGFP, or a dominant negative form of
-arrestin 1 referred to as 1
-arr 1-(319-418)-EGFP (27). RINm5F
cells are adherent cells of rat insulinoma origin that have been
previously shown to rapidly and efficiently internalize the C5aR in
response to C5a binding (20). The expression of each
-arrestin
variant was assessed by Western blotting of whole cell lysates using
anti-GFP polyclonal antibodies. As illustrated in Fig. 1B,
single protein species of around 75, 40, and 70 kDa were immunodetected
that corresponded to
-arr 1-EGFP,
-arr 1-(319-418)-EGFP, and
-arr 2-EGFP, respectively.
View larger version (47K):
[in a new window]
Fig. 1.
Construction of cell lines co-expressing
wild-type or mutant receptors and
-arrestins. A, primary amino acid
sequence of the C-terminal region of C5aR. It has been previously
established that the C5aR can be phosphorylated on serine residues at
positions 314, 317, 327, 332, 334, and 338 (19). Alanine substitution
mutants used in this study are shown. B, analysis of total
protein extracts from RINm5F cells co-expressing C5aR and
-arr
1-EGFP,
-arr 1-(319-418)-EGFP, or
-arr 2-EGFP. Immunoblotting
was performed with a GFP-specific polyclonal antibody. C,
detection of wild-type (wt) and mutant C5aR by
immunoblotting with anti-C5aR rabbit serum. RINm5F cells were directly
lysed in Laemmli sample buffer under reducing conditions at 37 °C
before (
) and after (+) 10 min of stimulation with C5a. Based on
previous studies (20), the retarded bands correspond to the
phosphorylated form of C5aR.
-Arrestins upon Activation of
C5aR--
The cellular localization of
-arrestin 1 in cells
co-expressing C5aR and
-arr 1-EGFP was first examined by subcellular
fractionation of cells that were stimulated or not with C5a. Consistent
with previous observations indicating that
-arrestin 1 resides in the cytosol of unstimulated cells (29),
-arr 1-EGFP was mainly associated with the cytosolic fraction of RINm5F cells in the absence
of C5a stimulation (Fig. 2A).
When cells transfected with both wild-type C5aR and
-arr 1-EGFP were
stimulated with C5a, a significant amount of
-arr 1-EGFP was
recruited to the plasma membrane (Fig. 2A). Under the same
conditions of stimulation there was no translocation of
-arr
1-(319-418)-EGFP (Fig. 2B). The translocation of
-arr
1-EGFP to the plasma membrane of cells expressing the
phosphorylation-deficient mutant C5aR-A332,334,338 receptor
was not significantly detectable after C5a stimulation (Fig.
2C). Thus, activated C5aR recruits
-arrestins to the
plasma membrane. The redistribution of
-arrestin 1-EGFP appears to
be linked to the ability of C5aR to be phosphorylated and is unlikely to result from C5a-mediated signaling since the
phosphorylation-deficient A332,334,338 mutant, which
supports sustained signaling (21), fails to induce a significant
translocation of
-arrestin 1-EGFP to the plasma membrane.
View larger version (13K):
[in a new window]
Fig. 2.
Translocation of
-arrestin 1 to the membrane after agonist
stimulation. RINm5F cells stably co-expressing wild-type C5aR
(wt) and
-arr 1-EGFP (A) or
-arr
1-(319-418)-EGFP (B) or co-expressing the
phosphorylation-deficient mutant C5aR-A332,334,338 and
-arr 1-EGFP (C) were treated without (
) or with (+) 50 nM C5a for 5 min at 37 °C. After harvesting, cells were
subjected to subcellular fractionation as described under
"Experimental Procedures." Membrane fractions were dissolved in
lysis buffer and subjected to SDS-PAGE and Western blot analysis using
a GFP-specific polyclonal antibody. PM, plasma
membrane.
-arrestins at the cell level and to explore whether
-arrestin 1 and
-arrestin 2 were targeted to sites containing the C5aR, we used
confocal microscopy to detect
-arrestin-EGFP chimera and
immuno-decorated C5aR in fixed and permeabilized cells. In resting
cells, the C5aR was mainly recovered in the plasma membrane, as
indicated by a thin red fluorescent staining at the cell periphery (Fig. 3, A and B,
top panels). Both
-arr 1-EGFP and
-arr 2-EGFP were
evenly distributed throughout the cytoplasm of C5aR-expressing cells.
Consistent with previous reports (30),
-arr 1-EGFP was also
recovered in nucleus, whereas
-arr 2-EGFP was completely excluded
from this region. Merging of the red and green signals indicated a
complete absence of co-localization of C5aR and fluorescent
-arrestins. The addition of C5a rapidly led to the movement of both
EGFP-tagged
-arrestins from the cytosol to the plasma membrane. By 2 min, red and green fluorescence signals were still co-localized at the
cell periphery. At further time points, distinct punctate foci of
-arrestin-EGFP began to appear at sites below the plasma membrane
(not shown), and by 10 min, the green fluorescence of
-arrestin-EGFP
chimera was seen as bright spots that localized in the perinuclear
region. These fluorescent spots had the same size, shape, and location
as those containing C5aR, as indicated by superimposition of confocal
images where a yellow color denotes co-localization. As illustrated in
Fig. 3, A and B (lower panels), this
pattern was maintained over the longest time points monitored (30 min).
Thus, C5a induces translocation of both
-arrestins from the cytosol
to the plasma membrane, where they colocalize with the C5aR, followed
by the endocytosis of the C5aR and the association of
-arrestins to
C5aR-containing vesicles.
View larger version (34K):
[in a new window]
Fig. 3.
Agonist-stimulated colocalization of C5aR
with -arrestin-EGFP.
Representative laser-scanning micrographs showing the distribution of
wild-type C5aR (left panels) in RINm5F cells expressing
either
-arr 1-EGFP (A, middle panels) or
-arr 2-EGFP (B, middle panels) after treatment
for various periods of time with 50 nM of C5a. After
stimulation, cells were fixed, permeabilized, and stained as described
under "Experimental Procedures." Colocalization of C5aR and
-arrestin-EGFP is shown in the overlay images (right
panels). Confocal images are representative of more than four
independent experiments. Bar, 8 µm.
-Arrestins to Wild-type and Mutant C5aRs--
The
ability of
-arrestin-EGFP to interact with the C5a-occupied
receptors was further assessed. Cells expressing C5aR and either
-arr 1-EGFP or
-arr 2-EGFP were treated with C5a for 5 min and
subjected to reversible cross-linking with dithiobis(succinimidyl propionate). C5aR was immunoprecipitated with an antibody directed against its C terminus. After SDS-PAGE,
-arrestin-EGFP that was associated with immunoprecipitated receptors was detected by
immunoblotting. The treatment of cells with C5a strikingly increased
the interaction of C5aR with either
-arrestin 1 or
-arrestin 2, as shown in Fig. 4, A and
B (lanes 1 and 2), by the apparition
of an additional band migrating below two contaminating species that
were detected whether C5aR was expressed or not (not shown). No
interaction could be detected either between wild-type C5aR and
-arr
1-(319-418)-EGFP (Fig. 4A, lanes 3 and
4) or between the phosphorylation-deficient mutant
C5aR-A332,334,338 and the two
-arrestins (Fig. 4,
A, lanes 5 and 6, and B,
lanes 3 and 4, respectively). Altogether the
results indicate that there is a stable physical association between
-arrestins and C5aR provided that C5aR is activated and
phosphorylated.
View larger version (26K):
[in a new window]
Fig. 4.
Cross-linking of
-arrestin to C5a-stimulated C5aR. RINm5F cells
that stably co-expressed either wild-type C5aR (wt) or
A332,334,338 mutant receptor and either
-arr 1-EGFP,
-arr 1-(319-418)-EGFP (A), or
-arr 2-EGFP (B) were
incubated without (
) or with (+) 50 nM C5a for 15 min at
37 °C.
-Arrestin-receptor complexes were stabilized by covalent
cross-linking with dithiobis(succinimidyl propionate). After
cross-linking, wild-type C5aR or C5aR-A332,334,338 mutant
was immunoprecipitated with an affinity-purified rabbit polyclonal
antibody directed to the C-terminal end of C5aR. The presence of
-arrestin in the immunoprecipitates was further detected by Western
blot with anti-GFP antibodies and is indicated by an arrow.
Results are representative of three independent experiments.
-arr 1-(319-418)-EGFP Expression on C5aR
Internalization--
To determine whether clathrin is required for
agonist-induced endocytosis of C5aR, we analyzed C5aR internalization
in RINm5F-C5aR cells stably transfected with
-arr 1-(319-418)-EGFP.
This fragment, which constitutively binds to clathrin but is unable to
interact with phosphorylated GPCRs, acts as a dominant negative mutant that inhibits agonist-stimulated endocytosis of GPCRs via the classical
clathrin-dependent internalization pathway (31). Our observation that the stimulation of cells with C5a did not induce translocation of
-arr 1-(319-418)-EGFP from the cytosol to the plasma membrane fraction (Fig. 2B) nor its association with
the activated receptor (Fig. 4A) was further confirmed by
confocal microscopy as shown in Fig. 5.
The dominant negative mutant
-arr 1-(319-418)-EGFP was distributed
throughout the cell in superficial and perinuclear vesicles. Although a
punctate distribution of green fluorescence was observable at the cell
periphery, there was no clear overlap with C5aR regardless of whether
cells were stimulated with C5a.
View larger version (32K):
[in a new window]
Fig. 5.
Effect of dominant negative
-arr 1-(319-418)-EGFP on C5aR endocytosis.
RINm5F cells stably expressing wild-type C5aR together with
-arr
1-(319-418)-EGFP were treated with 50 nM C5a for 0, 5, 10, and 30 min at 37 °C. After stimulation, cells were fixed,
permeabilized, and stained as described under "Experimental
Procedures." Confocal microscopic images in the right
panels are formed by superimposition of images from the two other
panels in the same row. Bar, 8 µm.
-arr 1-(319-418)-EGFP constitutively interacts with
clathrin but not with GPCR (31), the expression of this truncated mutant is expected to inhibit the agonist-induced internalization of
C5aR if a clathrin-dependent pathway is the
major endocytic pathway involved in C5aR-internalization.
In control RINm5F-C5aR cells, i.e. not transfected with
-arrestin-EGFP, C5aR is clearly recovered in vesicles that
accumulate in the perinuclear region upon C5a addition (20). In cells
co-expressing
-arr 1-(319-418)-EGFP, C5aR largely remained at the
plasma membrane of the majority of cells, even 30 min after C5a
application (Fig. 5). These results indicate that the expression of
-arr 1-(319-418)-EGFP strongly inhibits C5aR endocytosis and
provide strong evidence that C5aR is mainly internalized via the
classical clathrin-dependent pathway.
-arrestin on the
internalization process, we assayed the capacity of RINm5F-C5aR cells
expressing or not
-arr 1-(319-418)-EGFP to internalize radiolabeled
C5a (see "Experimental Procedures"). Paradoxically, although the
expression of
-arr 1-(319-418)-EGFP has a marked inhibitory effect
on the cellular trafficking of C5aR, no dramatic change in the amount
of 125I-labeled C5a that was sequestered (i.e.
resistant to acid treatment) could be observed compared with control
cells (data not shown). Thus, it appears that even though it is not
routed to deep endosomal compartments, C5aR is still able to sequester
radiolabeled C5a in
-arr 1-(319-418)-EGFP-expressing RINm5F cells.
In these cells, agonist sequestration may be due to a relative
abundance of endogenous receptor-interacting proteins that stabilize
the agonist-receptor complex in a high affinity state insensitive to
mild acid treatment.
-arr
1-(319-418)-EGFP on C5aR Internalization in HEK-293 Cells--
We
further examined the internalization of 125I-labeled C5a in
HEK-293 cells when the C5aR was transiently overexpressed in the absence or the presence of
-arr 1-(319-418)-EGFP. As shown in Fig.
6, when C5aR was expressed alone,
~45-50% of the radiolabeled C5a bound at 4 °C was resistant to
acid treatment after warming at 37 °C, whereas little (less than
10%) radiolabeled C5a was internalized or sequestered when
-arr
1-(319-418)-EGFP was co-expressed with C5aR. This result is consistent
with the confocal microscopy experiments and suggests a critical role
of clathrin in C5aR internalization.
View larger version (21K):
[in a new window]
Fig. 6.
Sequestration of C5aR is inhibited by
expression of -arr 1-(319-418) and
DynK44E. HEK-293 cells transiently co-transfected with C5aR and
either a control vector (empty vector),
-arr 1-(319-418)-EGFP, or
dominant negative dynamin (DynK44E) were allowed to bind
125I-labeled C5a at 4 °C, and the capacity of cells to
internalize surface bound ligand was assessed as described under
"Experimental Procedures." After different periods of incubation at
37 °C, internalization was stopped by transferring cells into a
chilled low pH buffer to remove bound ligand that had not been
internalized. Results are presented as the mean percentage ± S.E.
(n = 3) of saturably bound 125I-labeled C5a
at 4 °C that is internalized in the different transfection
conditions.
-arrestins are thought to act as scaffolding proteins in coupling
GPCRs to clathrin-coated vesicles (6, 23, 33), we investigated the role
of dynamin in C5aR internalization by transiently co-expressing in
HEK-293 cells both C5aR and dynamin K44E, a mutant previously shown to
inhibit the endocytosis via clathrin-coated pits in a dominant negative manner (11). As illustrated in Fig. 6, expression of dynamin K44E
markedly impaired the ability of C5aR to internalize radiolabeled C5a.
Thus, altogether the results suggest that internalization of C5aR, like
most GPCRs, is an arrestin-, clathrin-, and
dynamin-dependent process.
-Arrestin--
Previous studies show that
C5aR is phosphorylated in a hierarchical manner on the six serine
residues located in the C-terminal cytoplasmic portion (19, 21). By
substituting alanine for serine (see Fig. 1B), we have
previously shown that the internalization of the non-phosphorylable
mutant C5aR-A332,334,338 is severely impaired compared with
wild-type receptor, whereas the mutant
C5aR-A314,317,332,334 that is phosphorylated on serine 327 and/or serine 338 showed a rate of internalization in between that of
wild-type and C5aR-A332,334,338 receptor (20). To pinpoint
serine residues that stabilize receptor/
-arrestin interaction, we
further analyzed RINm5F cells that stably expressed two additional C5aR
mutants, C5aR-A314,317,327,332 and
C5aR-A334,338, that are phosphorylated and
non-phosphorylated, respectively.
-arr 1-EGFP at several
time points after the addition of C5a. As shown in Fig.
7, A and B,
substitution of alanine for serine either at positions 314, 317, 327, and 332 or at positions 314, 317, 332, and 334 affected neither the
C5a-mediated internalization of these two mutant receptors nor their
ability to recruit
-arr 1-EGFP. The complex was found to traffic
into the same endosomal vesicles, as indicated by merging confocal
images. In marked contrast, mutations either at positions 334 and 338 or at positions 332, 334, and 338 completely abolished the movement of
these two mutant receptors from the cell periphery to the vesicular
compartment in the perinuclear region (Fig.
8, A and B).
Interestingly, upon C5a application,
-arr 1-EGFP clearly
translocated to the plasma membrane of cells expressing the
A334,338 mutant receptor, whereas very little, if any,
translocation occurred with cells expressing the
A332,334,338 mutant. In the latter case,
-arr 1-EGFP
remained restricted to the cytoplasm even after a prolonged exposure to
C5a (Fig. 8B).
View larger version (37K):
[in a new window]
Fig. 7.
Trafficking of -arr
1-EGFP with C5aR into endocytic vesicles is mediated by specific serine
residues in the receptor C-terminal tail. RINm5F cells were stably
co-transfected either with
-arr 1-EGFP and either
C5aR-A314,317,327,332 (A) or
C5aR-A314,317,332,334 (B). Cells were treated
with 50 nM of C5a for 0, 2, 5, 10, and 30 min at 37 °C.
Cells were fixed, permeabilized, and stained as described under
"Experimental Procedures." Colocalization of C5aR and
-arrestin-EGFP is shown in the overlay images. Confocal images are
representative of at least three independent experiments.
Bar, 8 µm.
View larger version (29K):
[in a new window]
Fig. 8.
Differential translocation
of -arr 1-EGFP and
-arr 2-EGFP upon phosphorylation-deficient mutant
C5aR activation. RINm5F cells that stably co-expressed either
-arr 1-EGFP plus A334,338 C5aR (A),
A332,334,338 C5aR (B), or
-arr 2-EGFP plus
C5aR-A332,334,338 C5aR (C) were treated with 50 nM of C5a for 0, 10, and 30 min at 37 °C. Cells were
fixed, permeabilized, and stained as described under "Experimental
Procedures." Colocalization of C5aR and
-arrestin-EGFP is shown in
the overlay images. Confocal images are representative of at least
three independent experiments. Bar, 8 µm.
-arrestin isoform (Fig. 4,
A and B), we did observe a change in the
intracellular distribution pattern of
-arr 2-EGFP in response to C5a
(Fig. 8C). By 2 min, an increase in the level of green
fluorescence was observable at the cell periphery that also contained
the mutant receptor (not shown). This pattern of fluorescence remained
stable with time. By 30 min, we could only observe the presence of few
receptor- and
-arrestin 2-containing vesicles in a minority of
cells, indicating a slow rate of internalization of this
phosphorylation-deficient mutant. Thus, in the absence of
phosphorylation, the binding of C5a brings about a conformational
change that is apparently sufficient to mediate a loose interaction
with
-arrestin 2 but not with
-arrestin 1. Phosphorylation of
only two serine residues either at positions 327/338 or at positions
334/338 is sufficient to trigger a firm association with
-arrestins
and the subsequent endocytosis of C5aR.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin scaffolding proteins. In the present work, by using green fluorescent
protein-tagged
-arrestin 1 and 2, we find that C5a stimulated a
striking and rapid redistribution of
-arrestin 1 and 2 from the
cytosol to the plasma membrane followed by the formation of a
long-lasting complex between wild-type C5aR and both
-arrestins as
evidenced by the co-localization of C5aR and
-arrestins on the same
endosomal compartments. Similar agonist-mediated translocation and
association to endosomes have been previously described with a variety
of GPCRs (6, 23). The co-trafficking of
-arrestins and C5aR to
perinuclear vesicles was consistently best observed with
-arrestin 2, suggesting that this isoform has a higher affinity for the activated
wild-type receptor. The persistent association of
-arrestins with
intracellular vesicular compartments that contain internalized GPCRs is
not a general rule. The stability of
-arrestin-receptor interactions
seems to differ from receptor to receptor. For instance, although
"class B" receptors, such as the angiotensin II type 1A and the
neurokinin 1 receptors, colocalize with
-arrestin 2 on endosomal
compartments, "class A" receptors including the
2-adrenergic, dopamine D1A, and endothelin type A
receptors rapidly dissociate from
-arrestins, which remain confined
to the plasma membrane (14, 30, 34). In this respect, C5aR behaves as a
"class B" receptor. The significance of this prolonged
colocalization is not known, but it is likely to regulate the
dephosphorylation and recycling of C5aR.
-arrestins to
agonist-occupied C5aR and from the observation that co-expression of
-arrestin 1 increases by 1.4-fold the rate of C5aR
internalization,2 it is
likely that
-arrestins play an important role in C5aR internalization. In the present study, we provide strong evidence that
-arrestins target the activated C5aR to clathrin-coated pits in a
dynamin-dependent manner. First, confocal microscopy experiments with RINm5F cells clearly show that activated C5aR remains
at the periphery of cells expressing the clathrin-interacting domain of
-arrestin 1. Second, the co-expression of C5aR with either the
dominant negative mutant K44E of dynamin or
-arr 1-(319-418)-EGFP results in a severe reduction of internalization/sequestration of
radiolabeled C5a in HEK-293 cells. Thus, similarly to the interleukin 8 receptor (35), the C5aR appears to be internalized via clathrin-coated pits in a dynamin-dependent manner. The role of
-arrestins and dynamin in the endocytosis of GPCRs is not a general
rule since it has been reported that the internalization of several
GPCRs, including the M2 muscarinic and angiotensin II type A receptors, is independent of
-arrestin and dynamin (13, 36, 37). As C5aR, the
leukocyte chemoattractant receptors N-formyl peptide receptor (FPR) and formyl peptide receptor-like 1 (FPRL1) appear to
behave as "class B" receptors since they also form a persistent complex with
-arrestin 1.2 Our conclusions strikingly
contrast with recent studies suggesting that C5aR and FPR are
internalized through a
-arrestin-, clathrin-, and
dynamin-independent pathway (15, 16). These divergent results might
possibly be due to differences in the methods used to assess receptor
internalization. In the study by Bennett et al. (16), C5aR
internalization was detected by flow cytometry with an antibody
directed to the receptor N-terminal domain, whereas our conclusions are
based on confocal microscopy and the uptake of radiolabeled C5a. Our
results do not, however, exclude the possibility that, under certain
experimental conditions, the internalization of C5aR could proceed
through an alternative pathway.
-arrestins with the
receptor to intracellular vesicles. Whether
-arrestin binding is
required to inhibit the association of C5aR with the G protein is
presently not known. In the case of the FPR it has been shown by an
elegant in vitro reconstitution assay that a partial
phosphorylation is sufficient to inhibit FPR-G protein interactions
independently of arrestin binding (38).
-arrestins are recruited to the plasma membrane by phosphorylation-deficient mutant. The agonist-dependent
recruitment of
-arr 2-EGFP but not
-arr 1-EGFP by
C5aR-A332,334,338 provides additional support to the notion
that
-arrestin 2 is the preferred isoform for targeting wild-type
C5aR to clathrin-coated pits. Although the affinity of
-arrestins
for these mutant receptors is most likely low, it may be sufficient to
impose structural constraints that result in the sequestration of C5a
in its binding site. This could explain previous results where
radiolabeled C5a has been found to be significantly sequestered by
phosphorylation-deficient mutant receptors (20, 21).
-arrestins function
as scaffolding proteins for several signaling pathways. Although
-arrestin 1 has been shown to recruit and activate c-Src kinase to
the plasma membrane, thereby allowing the activation of extracellular
signal-regulated kinases (Erk1 and Erk2) (26),
-arrestin 2 forms a
complex with and target c-Jun N-terminal kinase 3 (JNK3) to specific
subcellular compartments and/or specific substrates (39). Recently,
-arrestin 2 has been shown to be essential, presumably through its
binding to phosphorylated receptors, for the directed migration of
mouse lymphocyte in a gradient of stromal-derived factor 1 (40). Our
data indicating that
-arrestins can be recruited to the plasma
membrane in the absence of receptor phosphorylation suggest that
-arrestin-mediated signaling is still possible in the absence of
receptor internalization. With other chemoattractant receptors, the
agonist-dependent recruitment of
-arrestin 2 by
phosphorylation-deficient mutant receptors may also occur to variable
levels, depending on the receptor and the sets of residues that are
mutated. This could explain why chemotaxis and the activation of MAP
kinase mediated by phosphorylation-deficient mutants of chemoattractant
receptors are variably affected (41-43).
-arrestins to the plasma membrane, where they
participate in clathrin-mediated endocytosis of C5aR. Moreover, we
further establish that phosphorylation of two serine pairs, namely
Ser327/Ser338 or
Ser332/Ser338, is sufficient to stabilize the
interaction between
-arrestins and C5aR. In these serine pairs, the
phosphorylation of Ser338 is likely to play a key role
since its replacement by an alanine yields a mutant with a better
ability to transduce signal (21), suggesting a weaker interaction with
-arrestins and, thereby, a slightly prolonged interaction with the G protein.
![]() |
FOOTNOTES |
---|
* This study was supported by grants from the Commissariat à l'Energie Atomique, CNRS, and the University Joseph Fourier.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.
Recipient of a postdoctoral fellowship from the Association pour
la Recherche sur le Cancer. Present address: Plasticité et
Expression des Génomes Microbiens (FRE 2383, CNRS/Université Joseph Fourier), 460 rue de la Piscine, 38041 Grenoble Cedex 9, France.
§ Present address: Euroscreen s.a. 47, Rue Adrienne Bolland, 6041 Gosselies Belgium.
¶ To whom correspondence should be addressed. Tel.: 33-438-78-31-38; Fax: 33-438-78-51-85; E-mail: fboulay@cea.fr.
Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M210120200
2 L. Braun and F. Boulay, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
C5a, complement
5a;
C5aR, C5a receptor;
Bsd, blasticidine;
GPCR, G-protein-coupled receptor;
GFP, green fluorescent protein;
EGFP, enhanced GFP;
-arr,
-arrestin;
FPR, N-formyl peptide
receptor.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Boulay, F., Mery, L., Tardif, M., Brouchon, L., and Vignais, P. (1991) Biochemistry 30, 2993-2999[Medline] [Order article via Infotrieve] |
2. | Gerard, N. P., and Gerard, C. (1991) Nature 349, 614-617[CrossRef][Medline] [Order article via Infotrieve] |
3. | Zwirner, J., Fayyazi, A., and Gotze, O. (1999) Mol. Immunol. 36, 877-884[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Bokoch, G. M.
(1995)
Blood
86,
1649-1660 |
5. | McDonald, P. H., and Lefkowitz, R. J. (2001) Cell. Signal. 13, 683-689[CrossRef][Medline] [Order article via Infotrieve] |
6. | Goodman, O. B., Krupnick, J. G. J., 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] |
7. | Ferguson, S. S. G., Downey, W. E., III, Colapietro, A.-M., Barak, L. S., Ménard, L., and Caron, M. G. (1996) Science 271, 363-366[Abstract] |
8. |
Laporte, S. A.,
Oakley, R. H.,
Zhang, J.,
Holt, J. A.,
Ferguson, S. S.,
Caron, M. G.,
and Barak, L. S.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3712-3717 |
9. |
Scott, M. G.,
Benmerah, A.,
Muntaner, O.,
and Marullo, S.
(2002)
J. Biol. Chem.
277,
3552-3559 |
10. |
Santini, F.,
Gaidarov, I.,
and Keen, J. H.
(2002)
J. Cell Biol.
156,
665-676 |
11. | Damke, H., Baba, T., Warnock, D. E., and Schmid, S. L. (1994) J. Cell Biol. 127, 915-934[Abstract] |
12. | Pierce, K. L., and Lefkowitz, R. J. (2001) Nat. Rev. Neurosci. 2, 727-733[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Zhang, J.,
Stephen, S. G.,
Barak, L. S.,
Ménard, L.,
and Caron, M. G.
(1996)
J. Biol. Chem.
271,
18302-18305 |
14. |
Zhang, J.,
Barak, L. S.,
Anborgh, P. H.,
Laporte, S. A.,
Caron, M. G.,
and Ferguson, S. S.
(1999)
J. Biol. Chem.
274,
10999-11006 |
15. | Gilbert, T. L., Bennett, T. A., Maestas, D. C., Cimino, D. F., and Prossnitz, E. R. (2001) Biochemistry 40, 3467-3475[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Bennett, T. A.,
Maestas, D. C.,
and Prossnitz, E. R.
(2000)
J. Biol. Chem.
275,
24590-24594 |
17. |
Bhatnagar, A.,
Willins, D. L.,
Gray, J. A.,
Woods, J.,
Benovic, J. L.,
and Roth, B. L.
(2001)
J. Biol. Chem.
276,
8269-8277 |
18. |
Roseberry, A. G.,
and Hosey, M. M.
(2001)
J. Cell Sci.
114,
739-746 |
19. |
Giannini, E.,
Brouchon, L.,
and Boulay, F.
(1995)
J. Biol. Chem.
270,
19166-19172 |
20. |
Naik, N.,
Giannini, E.,
Brouchon, L.,
and Boulay, F.
(1997)
J. Cell Sci.
110,
2381-2390 |
21. |
Christophe, T.,
Rabiet, M.-J.,
Tardif, M.,
Milcent, M.-D.,
and Boulay, F.
(2000)
J. Biol. Chem.
275,
1656-1664 |
22. |
Tardif, M.,
Mery, L.,
Brouchon, L.,
and Boulay, F.
(1993)
J. Immunol.
150,
3534-3545 |
23. |
Barak, L. S.,
Ferguson, S. S. G.,
Zhang, J.,
and Caron, M. G.
(1997)
J. Biol. Chem.
272,
27497-27500 |
24. | Lang, J., Boulay, F., Li, G., and Wollheim, C. B. (1993) EMBO J. 12, 2671-2679[Abstract] |
25. |
Mery, L.,
and Boulay, F.
(1994)
J. Biol. Chem.
269,
3457-3463 |
26. |
Luttrell, L. M.,
Ferguson, S. S. G.,
Daaka, Y.,
Miller, W. E.,
Maudsley, S.,
Della Rocca, G. J.,
Lin, F.-T.,
Kawakatsu, H.,
Owada, K.,
Luttrell, D. K.,
Caron, M. G.,
and Lefkowitz, R. J.
(1999)
Science
283,
655-661 |
27. |
Dery, O.,
Thoma, M. S.,
Wong, H.,
Grady, E. F.,
and Bunnett, N. W.
(1999)
J. Biol. Chem.
274,
18524-18535 |
28. | Langkabel, P., Zwirner, J., and Oppermann, M. (1999) Eur. J. Immunol. 29, 3035-3046[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Lin, F. T.,
Krueger, K. M.,
Kendall, H. E.,
Daaka, Y.,
Fredericks, Z. L.,
Pitcher, J. A.,
and Lefkowitz, R. J.
(1997)
J. Biol. Chem.
272,
31051-31057 |
30. |
Oakley, R. H.,
Laporte, S. A.,
Holt, J. A.,
Caron, M. G.,
and Barak, L. S.
(2000)
J. Biol. Chem.
275,
17201-17210 |
31. |
Krupnick, J. G.,
Santini, F.,
Gagnon, A. W.,
Keen, J. H.,
and Benovic, J. L.
(1997)
J. Biol. Chem.
272,
32507-32512 |
32. | Damke, H. (1996) FEBS Lett. 389, 48-51[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Zhang, J.,
Barak, L. S.,
Winkler, K. E.,
Caron, M. G.,
and Ferguson, S. S. G.
(1997)
J. Biol. Chem.
272,
27005-27014 |
34. |
Schmidlin, F.,
Dery, O.,
Bunnett, N. W.,
and Grady, E. F.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
3324-3329 |
35. |
Barlic, J.,
Khandaker, M. H.,
Mahon, E.,
Andrews, J.,
DeVries, M. E.,
Mitchell, G. B.,
Rahimpour, R.,
Tan, C. M.,
Ferguson, S. S.,
and Kelvin, D. J.
(1999)
J. Biol. Chem.
274,
16287-16294 |
36. |
Pals-Rylaarsdam, R.,
Gurevich, V. V.,
Lee, K. B.,
Ptasienski, J.,
Benovic, J. L.,
and Hosey, M. M.
(1997)
J. Biol. Chem.
272,
23682-23689 |
37. |
Claing, A.,
Perry, S. J.,
Achiriloaie, M.,
Walker, J. K.,
Albanesi, J. P.,
Lefkowitz, R. J.,
and Premont, R. T.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
1119-1124 |
38. |
Bennett, T. A.,
Foutz, T. D.,
Gurevich, V. V.,
Sklar, L. A.,
and Prossnitz, E. R.
(2001)
J. Biol. Chem.
276,
49195-49203 |
39. |
McDonald, P. H.,
Chow, C. W.,
Miller, W. E.,
Laporte, S. A.,
Field, M. E.,
Lin, F. T.,
Davis, R. J.,
and Lefkowitz, R. J.
(2000)
Science
290,
1574-1577 |
40. |
Fong, A. M.,
Premont, R. T.,
Richardson, R. M., Yu, Y. R.,
Lefkowitz, R. J.,
and Patel, D. D.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
7478-7483 |
41. |
Arai, H.,
Monteclaro, F. S.,
Tsou, C. L.,
Franci, C.,
and Charo, I. F.
(1997)
J. Biol. Chem.
272,
25037-25042 |
42. |
Richardson, R. M.,
Ali, H.,
Pridgen, B. C.,
Haribabu, B.,
and Snyderman, R.
(1998)
J. Biol. Chem.
273,
10690-10695 |
43. |
Kraft, K.,
Olbrich, H.,
Majoul, I.,
Mack, M.,
Proudfoot, A.,
and Oppermann, M.
(2001)
J. Biol. Chem.
276,
34408-34418 |