From the Immunology Division, Department of Pediatrics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada
Received for publication, December 3, 2002 , and in revised form, April 15, 2003.
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ABSTRACT |
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INTRODUCTION |
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Exposure of GPCRs to their agonists is usually followed by a rapid
desensitization of signaling
(1315).
A series of distinct events is known to participate in this mechanism,
including a functional uncoupling of the receptor from its cognate G protein,
sequestration of the receptors into intracellular compartments, and a net loss
of receptors (down-regulation). GPCR internalization has become the subject of
intensive investigation over the past several years, and a large volume of
data has accumulated regarding the mechanisms regulating their endocytosis.
The 2-adrenergic receptor and a large number of
GPCRshavebeendemonstratedtointernalizethroughadynamin-dependent mechanism
involving clathrin-coated pits
(16,
17). There are exceptions to
this generalization, however, because some receptors such the dopamine D2
receptor, the angiotensin 1A receptor, and the m2 muscarinic receptor can
internalize by an unidentified mechanism that shows an atypical sensitivity to
dynamin
(1821).
In addition, further analysis revealed that the N-formyl peptide
receptor and the C5a chemoattractant receptor are internalized via an
arrestin- and dynamin-independent pathway, which leads to questions about an
alternative mechanism involved in mediating internalization of these receptors
(22).
An increasing number of studies have shown that many GPCRs form homodimers
as well as heterodimers (23).
Subsequent reports demonstrated homodimerization among the
2-adrenergic receptor
(24), the
-opioid
receptor (25), the chemokine
receptors CCR2b, CCR4, and CCR5
(26,
27), the
Ca2+-sensing receptor
(28), and the metabotropic
glutamate receptor 5 (29).
Agonists have also been shown to stabilize the dimeric form of several
receptors including the
2-adrenergic receptor
(24) as well as the chemokine
receptors CCR2b, CCR4, and CCR5
(26,
27). This suggests that
homodimerization might play a role either directly in the activation mechanism
of receptors or in subsequent agonist-dependent internalization as shown for
the
-opioid receptor
(25).
We have demonstrated previously that coexpression of certain mutant hPAFR receptors with wild-type hPAFR modified specific characteristics of the native receptor, such as basal level of activity, affinity for the ligand, and cell surface expression (30). To determine whether artificially induced oligomerization of hPAFR could modulate the internalization process of the receptor, we applied the coumermycin-induced dimerization system (31), which is based on the binding of coumermycin to the amino-terminal 24-kDa subdomain of the B subunit of bacterial DNA gyrase (GyrB) (32). A hPAFR-GyrB fusion protein was thus generated, with the GyrB moiety fused to the COOH-terminal tail of hPAFR. We report here that coumermycin treatment on CHO cells stably expressing c-Myc-hPAFR-GyrB led to the formation of dimers/oligomers, which is sufficient to induce the sequestration process by an unknown endocytic machinery. This coumermycin-driven desensitization of hPAFR-GyrB is regulated by PKC as well as a tyrosine kinase- and phospholipase C-independent mechanism. More broadly, these data suggest that signals determined by secondary structure/conformation of the receptor involved in the conversion between the monomeric and the dimeric form of GPCRs may be implicated in the heterologous desensitization process, which may be correlated with the receptor susceptibility to phosphorylation by second messenger-dependent kinases.
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MATERIALS AND METHODS |
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Construction of Epitope-tagged PAFR-GyrBHuman PAF receptor was epitope tagged in the amino-terminal extracellular domain with either a c-Myc or a HA epitope, as described previously (12) and subcloned into the pcDNA3 vector. To construct PAFR-GyrB, NheI, NotI, and ApaI sites were introduced in the pcDNA3-c-Myc-hPAFR by digestion using the oligonucleotides 5'-CTGGCAATTCCCTCAAAAATGCTAGCTAGGCGGCCGCGGGC-3' corresponding to the last 23 nucleic acids of the hPAFR. The stop codon was then replaced at the NheI-NotI sites with the coding region of GyrB isolated from pcDNA3-Raf-GyrB at XbaI and NotI sites. Constructs were verified by restriction enzyme digestion.
Cell Culture and TransfectionsCOS-7 and CHO cells were maintained at 37 °C in a 5% CO2 atmosphere in Dulbecco's modified Eagle's medium (high glucose) and Dulbecco's modified Eagle's medium F-12 (Ham's medium, high glucose), respectively, supplemented with 5% fetal bovine serum. Cells were grown in 100-mm dishes to 7080% confluence and transiently transfected the following day with 7 ml of a mixture of 100 µM chloroquine and 0.25 mg/ml DEAE-dextran containing 4 µg of plasmid DNA (pcDNA3-c-Myc-hPAFR, pcDNA3-HA-hPAFR, or pcDNA3-c-Myc-hPAFR-GyrB) in Dulbecco's modified Eagle's medium with 5% fetal bovine serum. After 2 h at 37 °C, the solution was removed, and the cells were treated for 1 min at room temperature with 10% dimethyl sulfoxide in phosphate-buffered saline (PBS), rinsed twice with PBS, and returned to the 37 °C incubator in growth medium supplemented with 5% fetal bovine serum. For confocal microscopy experiments, cells were seeded into 6-well plates (11.5 x 105 cells/ml) and transiently transfected with 1 µg of plasmid DNA/well by using the FuGENE 6 transfection reagent (Roche Applied Science) according to the manufacturer's instructions. Stable CHO transfectants expressing the pcDNA3-c-Myc-hPAFR were generated as described previously (33). CHO stably transfected with the pcDNA3-c-Myc-hPAFR-GyrB were established as above except that positive cells were selected with a FACS-Vantage cell sorter (BD Biosciences) after labeling with anti-c- Myc monoclonal antibody and fluorescein isothiocyanate-conjugated goat anti-mouse Ab.
Immunoprecipitation and Western BlottingCOS-7 were rinsed 48 h after transfection with PBS. Cells were then removed from 100-mm plates and collected by centrifugation in PBS. The cell pellet from each plate was disrupted in 500 µl of radioimmunoprecipitation assay buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% IGEPAL, 0.5% deoxycholic acid, 0.1% SDS) containing protease inhibitors (1 µg/ml soybean trypsin inhibitors, 1 µM leupeptin, 20 µg/ml aprotinin, 40 µg/ml prefabloc, and 40 µg/ml 1-chloro-3-tosylamido-7-amino-2-heptanone) and incubated 30 min on ice. Lysates were then precleared with 25 µg of protein A-Sepharose for 30 min at 4 °C and incubated with anti-HA or anti-c-Myc Ab overnight at 4 °C. The protein A-Sepharose was shaken gently in lysis buffer containing 1% BSA for 30 min at room temperature before use. Epitope-tagged hPAFRs were precipitated by incubation with 100 µg of protein A-Sepharose for 2 h at 4 °C. After washing three times in lysis buffer, complexes were dissolved in 1 x Laemmli sample buffer (34). Immunoprecipitated proteins were then separated by 10% Tris-glycine precast gels (Invitrogen) and transferred electrophoretically to nitrocellulose membranes. Phorbol 12-myristate 13-acetate (PMA)-stimulated cells (80 nM for 0, 30, 60, and 90 min) were lysed in radioimmunoprecipitation assay buffer, and immunoprecipitation was performed as described above. For coumarin experiments, CHO cells stably expressing c-Myc epitope-tagged hPAFR-GyrB and nontransfected cells were resuspended in ice-cold buffer containing 10 mM Tris-HCl, pH 8.0, and 1 mM EDTA. The cells were then disrupted by sonication and pelleted by centrifuging at 12,500 rpm for 30 min at 4 °C. The supernatants were collected as total cell lysates, and protein concentration was determined by the Bio-Rad procedure with BSA as standard. Samples containing 20 µg of protein were resolved using 6% Tris-glycine precast gels under nonreducing conditions. For Western blotting analysis, nitrocellulose membranes were blocked in PBS and 0.1% Tween containing 10% dried milk for 1 h and incubated with anti-HA or anti-Myc in PBS and 1% dried milk 1 h at room temperature. After washing with PBS-Tween and incubation with secondary Abs, a chemiluminescence detection system was used for protein detection (PerkinElmer Life Sciences).
Flow Cytometry StudiesReceptor internalization was determined as the level of receptor loss from the cell surface. Stably transfected CHO cells were seeded on 6-well plates (4 x 105 cells/well) 1 day prior to the assay. Cells were then washed with PBS and incubated in Dulbecco's modified Eagle's medium F-12 with the indicated concentration of coumarins for a specific time period. Cells were rinsed once with PBS and treated with 1 µM PAF for the appropriate time in medium containing 0.2% BSA. Cells were then washed once with PBS containing 2% BSA and harvested in ice-cold PBS. Cells were first labeled with or without anti-Myc antibody on ice for 1 h. After washing twice with ice-cold PBS, cells were incubated for an additional 45 min with fluorescein isothiocyanate-conjugated goat anti-mouse Ab on ice and washed as described above. Antibody-labeled cells were analyzed for fluorescence intensity on a FACScan flow cytometer (BD Biosciences) with dead cells excluded by gating on forward and side scatter.
Ligand InternalizationThe ligand internalization kinetics had been evaluated in CHO cells stably transfected with the c-Myc-hPAFR-GyrB in 12-well plates (2 x 105 cells/well). After 24 h, cells were pretreated or not with coumermycin (15 µM for 10, 45, 80, 120, 180, and 210 min). Cells were then incubated at 37 °C with 2 nM [3H]hexadecyl-PAF in a buffer containing 150 mM choline chloride, 10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, and 0.2% lipid-free BSA for 45 min. After the incubation period, cells were washed twice with 1 ml of the same buffer but containing 2% BSA. Cells were then lysed in 0.1 N NaOH, and internalized radioactivity was measured by liquid scintillation.
Confocal MicroscopyCOS-7 cells were grown on 25-mm coverslips and transiently transfected with pcDNA3-c-Myc-hPAFR-GyrB and green fluorescent protein (GFP)-conjugated arrestin 2 or arrestin 3 (kind gifts from Dr. J. Benovic, Philadelphia) using FuGENE 6 and processed as described previously (35). Cells were incubated at 37 °C in the presence or absence of coumermycin or novobiocin (15 µM, 180 min) and then treated or not with 1 µM PAF for 80 min. Cells were fixed with 4% paraformaldehyde (15 min at room temperature) and permeabilized in 0.1% saponin. Cells were then incubated with anti-Myc Ab followed by rhodamine-conjugated goat anti-mouse Ab. Cells were examined, as described (35), with a scanning confocal microscope (NORAN Instruments, Inc., Middleton, WI) equipped with a krypton/argon laser and coupled to an inverted microscope with a 40 x oil immersion objective (Nikon). Optical sections were collected at 1-µm intervals with a 10-µm pinhole aperture. Digitized images were obtained with 256 x line averaging and enhanced with Intervision software (NORAN Instruments, Inc.) on a Silicon Graphics O2-work station.
Inositol Phosphate (IP) DeterminationStably transfected CHO cells expressing c-Myc-hPAFR-GyrB were plated on 6-well dishes (4 x 105 cells/well) and incubated at 37 °C in complete medium 12 h before the assay. Cells were then washed once in PBS and labeled for 1824 h with myo-[3H]inositol at 3 µCi/ml in Dulbecco's modified Eagle's medium (high glucose, without inositol). After labeling, cells were washed once in PBS, preincubated at 37 °C in prewarmed modified medium containing 20 mM LiCl for 10 min. Medium was then removed, and cells were pretreated, or not, with 15 µM coumermycin or 15 µM novobiocin for the indicated periods of time in modified medium containing 20 mM LiCl. Cells were washed with PBS and incubated in prewarmed medium containing 0.2% BSA and 20 mM LiCl for 10 min with 1 µM PAF. The reactions were terminated with the addition of perchloric acid followed by a 30-min incubation on ice. IPs were extracted as described previously (30) and separated on Dowex AG 1-X8 columns. Total labeled IPs were then counted by liquid scintillation.
Radioligand Binding AssayCompetition binding curves were done on CHO cells expressing the c-Myc wild-type or GyrB receptor species, and binding reactions with 10 nM [3H]WEB2086 were done as described before (12). Internalization experiments were done on stably transfected CHO cells seeded onto 100-mm dishes 24 h prior to assay. Cells were washed once with PBS and pretreated or not with the indicated inhibitors (3 µM staurosporine, 20 min; 2 µM GF109203X, 30 min; and 10 µM tyrphostin 51, 25 min) or 80 nM PKC activator PMA, 30 min at 37 °C in Dulbecco's modified Eagle's medium F-12. Cells were then washed with PBS and incubated at 37 °C with 15 µM coumarins for the indicated periods of time. Binding reactions were done on ice as described above.
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RESULTS |
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Characterization of the hPAFR-GyrBTo study the effect of artificially induced dimerization of hPAFR on cells, we constructed a chimeric cDNA encoding hPAFR and GyrB as a fusion protein (hPAFR-GyrB) and generated a stable c-Myc-hPAFR-GyrB-expressing CHO cell line. Coumermycin acts as a natural dimerizer of GyrB because it binds GyrB with a stoichiometry of 1:2, whereas a related coumarin antibiotic, novobiocin, binds GyrB as a 1:1 complex and thus serves as a nondimerizing control (36). We next determined the effect of fusing GyrB on hPAFR properties by comparing CHO cells stably expressing the c-Myc-wild-type hPAFR with CHO cells stably transfected with c-Myc-hPAFR-GyrB. Flow cytometry studies showed that wild-type hPAFR and hPAFR-GyrB had similar cell surface expression levels (Fig. 2, A and B), indicating that GyrB had no significant effect on cellular trafficking and distribution of the receptor. Binding characteristics were determined using the PAF receptor antagonist, [3H]WEB2086, by competition with WEB2086. Competition binding experiments showed that the affinity of WEB2086 for hPAFR-GyrB was the same as for the wild-type receptor (Fig. 2C). To investigate the signaling capacity of the chimeric receptor, we examined the ability of hPAFR-GyrB to mediate the stimulation of phosphatidylinositol hydrolysis. Agonist-independent basal IP production and agonist-induced increased IP production were very similar in both c-Myc-hPAFR and c-Myc-hPAFR-GyrB transfected cells (Fig. 2D). Thus, the addition of the GyrB moiety did not lead to detectable changes in hPAFR coupling.
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Coumermycin Treatment Induces the Dimerization of hPAFR-GyrBTo verify that coumermycin modulates dimerization of hPAFR-GyrB as predicted by the system, CHO cells stably expressing c-Myc-hAPFR-GyrB were treated for various periods of time with coumarins (novobiocin or coumermycin) at 37 °C. Western blotting experiments were then performed on cell lysates using anti-c-Myc antibody. Immunoreactive bands hPAFR-GyrB monomeric form were detected at 83 kDa and around 165 kDa (Fig. 3A), respectively, corresponding to monomers as well as dimers of hPAFR-GyrB. Exposure of cells to coumermycin induced a rapid increase in the relative abundance of the dimeric form, up to a 5-fold increase of the dimer to monomer ratio after a 60-min treatment (Fig. 3B). As expected, no change was observed after addition of the monomeric coumarin antibiotic novobiocin.
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Ligand-independent Internalization of hPAFR-GyrB Mediated by Coumermycin-induced DimerizationInternalization of receptors in response to coumermycin was assayed by flow cytometry, measuring the level of depletion of epitope-tagged cell surface receptors. Internalization was defined as the fraction of total cell surface receptors that, after exposure to agonist, were removed from the plasma membrane and thus not accessible to antibodies. CHO cells stably expressing the c-Myc-tagged-hPAFR or -hPAFR-GyrB receptors were first pretreated with dimethyl sulfoxide (vehicle) for 100 min at 37 °C and exposed to PAF for 080 min. After an 80-min stimulation with the agonist, cell surface expression of hPAFR-GyrB sequestered to a maximum of 12 ± 2%, compared with 24 ± 0.4% observed for wild-type receptor (Fig. 4, A and B). A similar kinetic was observed after novobiocin pretreatment. However, coumermycin pretreatment resulted in a specific 3-fold increase of internalized hPAFR-GyrB, which has not been observed for wild-type receptor, suggesting that coumermycin-induced dimerization of hPAFR-GyrB may have stimulated the rate and extent of agonist-mediated internalization (Fig. 4, A and B). The loss of surface-bound receptors, corresponding to the sequestration process, was potentiated in the presence of less than 150 nM coumermycin, with a maximal response at 15 µM, yielding levels of hPAFR-GyrB internalization of 35 ± 0.2% (Fig. 4C). To examine the effect of coumermycin-induced dimerization on hPAFR-GyrB internalization further, CHO cells stably expressing c-Myc-hPAFR-GyrB were subjected to 15 µM coumermycin alone in the absence of agonist. As shown in Fig. 4D, a significant decrease in the level of cell surface receptors by 30 ± 1% was observed after the coumermycin treatment. The agonist-independent and coumermycin-mediated internalization was a very slow process that required more than 100 min compared with agonist-mediated sequestration, which was observed already at 20 min poststimulation (Fig. 4A). This indicates that coumermycin may activate an alternative endocytic mechanism(s) in addition to agonist-induced, clathrin-dependent endocytosis.
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Coumermycin-mediated Sequestration of the hPAFR-GyrB in CHO CellsTo demonstrate directly the effect of coumermycin-induced dimerization of hPAFR-GyrB on the internalization process, binding experiments using [3H]PAF were carried out. CHO cells stably expressing c-Myc-hPAFR-GyrB were thus exposed to coumermycin, for various times, at 37 °C, prior to the addition of 2 nM [3H]PAF for 40 min. Cells were then washed with 2% BSA to remove surface-bound [3H]PAF and lysed to allow the detection of internalized ligand. As shown in Fig. 5, coumermycin induced a time-dependent decrease in [3H]PAF uptake with a nadir of 61 ± 1% of the values obtained for untreated control cells, at 180 min of treatment. These results strongly suggest that the coumermycin-induced disappearance of cell surface receptors (Fig. 4A) is responsible for the loss of ligand binding sites in c-Myc-hPAFR-GyrB- expressing cells.
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Mechanisms of hPAFR-GyrB InternalizationIt is well established that nonvisual arrestins bind to agonist-activated GPCRs and serve as adaptor proteins during internalization of GPCRs through clathrin-coated pits. Arrestin (arr)2-GFP has been demonstrated to redistribute from the cytosol to the plasma membrane upon receptor stimulation (37) and colocalize with selective internalized GPCRs (38, 39). To evaluate the arrestin dependence of the coumermycin-mediated sequestration pathway, c-Myc-hPAFR-GyrB was cotransfected with the arr2-GFP or arr3-GFP fusion protein in COS-7 cells, and subcellular distribution was analyzed by confocal microscopy. In unstimulated cells, hPAFR-GyrB was found predominantly on the cellular membrane, and arr2-GFP was distributed evenly throughout the cytoplasm (data not shown). After an 80-min stimulation with PAF, a punctate pattern of arrestin fluorescence (green) at the plasma membrane was observed, in proximity to the receptor (red). Extensive colocalization (yellow) of hPAFR-GyrB and arr2-GFP fluorescence was also observed in endocytic vesicles (Fig. 6A). We next evaluated the internalization behavior of the receptor under PAF stimulation after coumermycin treatment. Under these conditions, arr2-GFP redistributed from a homogeneous cytosolic localization to a membrane-associated pattern, and hPAFR-GyrB appeared in endocytic vesicles distributed randomly throughout the cytoplasm (Fig. 6B). The overall distribution pattern of arr2-GFP appeared similar to the agonist-mediated internalization of hPAFR-GyrB observed above. In contrast, arr2-GFP fluorescence did not colocalize with hPAFR-GyrB immunofluorescence emanating from endocytic vesicles which were observed after the coumermycin treatment (Fig. 6C). Arrestins remained exclusively in the cytoplasm, suggesting an arrestin-independent internalization pathway. Novobiocin also failed to induce any change in the cellular distribution of hPAFR-GyrB and arr2-GFP, which remained on the cell surface and cytoplasm, respectively (data not shown). Similar results were found using arr3-GFP (data not illustrated). NH4Cl pretreatment did not inhibit the coumermycin-induced internalization, indicating that this process is also independent of clathrin-coated pits (data not shown).
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Functional Characterization of the Coumermycin-induced Internalization of hPAFR-GyrB in CHO CellsTo determine whether the coumermycin-mediated internalization of hPAFR-GyrB was associated with receptor signaling, phospholipase C activation was assessed in intact, stably transfected CHO cells by measuring IP accumulation. As shown in Fig. 7, no IPs were produced by hPAFR-GyrB-transfected cells over the entire duration of stimulation with coumermycin. In contrast, however, a further 10-min incubation with PAF at 40, 100, or 120 min did result in significant IP accumulation, showing that coumermycin treatment did not impair a functional response of the receptor to its ligand. These data suggested that phospholipase C activation does not participate in the coumermycin-induced internalization of hPAFR-GyrB and that coumermycin stimulates the internalization of a nonsignaling subset of receptors, in the absence of ligand.
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Agonist-independent and PKC-dependent Desensitization of the hPAFR-GyrBTo examine the possible role of coumermycin-driven dimerization in the desensitization process, CHO cells stably expressing the c-Myc-epitope-tagged hPAFR or hPAFR-GyrB receptors were treated with coumarins for the indicated times, and the number of antagonist binding sites on the cell surface was measured using a saturating concentration of the membrane-impermeant ligand [3H]WEB2086. Surprisingly, exposure to coumermycin alone initiated a rapid, time-dependent loss of receptor binding sites which reached 37 ± 2% of controls (Fig. 8A). The phenomenon occurred within 5 min after stimulation and reached a plateau after 1 h. The coumermycin-induced desensitization of hPAFR-GyrB, which is independent of the agonist, did not prevent the further loss of binding sites induced by PAF, the two effects being additive (data not shown). In addition to agonist-specific receptor desensitization, functions of GPCRs can be regulated by second messenger-dependent protein kinases, leading to heterologous desensitization (14). To investigate further the molecular mechanism mediating the coumermycin-induced ligand-independent desensitization of hPAFR-GyrB, we determined whether the effect of coumermycin occurred through activation of PKC. We preincubated CHO cells stably expressing c-Myc-hPAFR-GyrB in the presence or absence of the PKC inhibitors staurosporine and GF109203X prior to a 40-min incubation with coumermycin at 37 °C (Fig. 8B). Under these conditions, staurosporine and GF109203X inhibited the coumermycin-induced loss of [3H]WEB2086 binding sites by 75 and 50%, respectively. On the other hand, preincubation with the PKC activator PMA or the tyrosine kinase inhibitor tyrphostin 51 had no significant effect on desensitization of hPAFR-GyrB. Exogenous PKC activation by PMA did, however, significantly increase multimerization of hPAFR-GyrB after 60 min of incubation (Fig. 8, C and D). These data suggest that PKC plays a role in coumermycin-mediated desensitization of hPAFR-GyrB, possibly by enhancing receptor multimerization.
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DISCUSSION |
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Coumermycin-induced Internalization of hPAFR-GyrBReceptor
dimerization has been linked to GPCR sequestration in a number of instances.
Thus, the coexpression of an endocytosis-defective mutant of the yeast
-factor receptor with the wild-type receptor leads to efficient
endocytosis of the mutant receptor, indicating that these receptors are
internalized as dimers/oligomers
(42). Dimerization, however,
is not necessarily associated with internalization in all GPCRs. In the case
of the
-opioid receptor, it was shown that the reduction in the level
of dimers was faster than internalization, suggesting that monomerization
precedes internalization (25).
Moreover, dimerization of the m3 receptor and the calcium-sensing receptor has
been shown to be agonist-insensitive
(43,
44), indicating that agonist
stimulation neither promotes nor destabilizes dimer formation. It thus remains
to be clarified whether the presence of receptor dimers or oligomers in the
absence of ligand is sufficient to drive internalization, or whether this
process requires ligand-occupied receptors. Here we investigated this question
for the hPAFR, using the coumermycin-GyrB dimerization strategy. As for other
GPCRs, we quantified cell surface epitopes by flow cytometry, which is a
nonradioactive method of monitoring receptor sequestration
(4547).
We found that the pattern and overall level of expression as well as
agonist-mediated internalization of hPAFR-GyrB were almost indistinguishable
from the wild-type receptor, indicating that the pharmacological properties of
the receptor were not affected by the GyrB. We first demonstrated that
coumermycin pretreatment substantially increases the rate and extent of
agonist-mediated internalization of c-Myc-tagged-hPAFR-GyrB by flow
cytometry. We also found that exposure to coumermycin alone results in a
concentration-dependent internalization of hPAFR-GyrB, indicating that this
process is dimerization-dependent. Similarly, binding experiments using
[3H]PAF demonstrated a time-dependent coumermycin-driven
attenuation in radioligand uptake, resulting from the disappearance of cell
surface receptors. Because novobiocin or dimethyl sulfoxide did not induce any
change in the surface expression level of receptors, these results suggest
that by inducing dimerization of hPAFR-GyrB, coumermycin specifically induced
a PAF-independent conformational change leading to the sequestration process.
These data thus demonstrate that dimerization of hPAFR-GyrB is sufficient to
induce its sequestration.
Using a membrane-impermeant antagonist radioligand to estimate changes in
the binding sites on the cell surface, we then observed that coumermycin
induced a rapid loss of [3H]WEB2086 binding sites in CHO cells
stably expressing the hPAFR-GyrB. Coumermycin-mediated dimerization of
hPAFR-GyrB might thus induce a conformational change of the receptor which
leads initially to a loss of ligand binding capacity and later to a
disappearance of the unoccupied hPAFR-GyrB from the cell surface.
Coumermycin-induced internalization was not preceded by receptor activation
because coumermycin treatment did not increase phosphoinositide hydrolysis, to
which the hPAFR is coupled via the G protein Gq. Both the
rapid desensitization and the long term sequestration may explain the reduced
intracellular accumulation of receptor-bound [3H]PAF in
coumermycin-pretreated cells compared with cells treated only with
agonist.
Dimerization-induced Heterologous DesensitizationPrevious
results suggest that GPCR kinases are the primary protein kinases involved in
agonist-induced phosphorylation of hPAFR, but it has also been shown that
desensitization of the PAFR is accompanied by PKC-dependent phosphorylation as
well (10). Here, we found that
the two PKC inhibitors, staurosporine and GF109203X, greatly attenuated the
agonist-independent binding site-depleting actions of coumermycin, whereas the
tyrosine kinase inhibitor tyrphostin 51 had no effect. In contrast to receptor
phosphorylation by GPCR kinases, phosphorylation by second messenger-activated
kinases does not require receptor occupancy by agonist. This kind of
desensitization, termed heterologous desensitization, has been implicated in
the regulation of a number of GPCRs
(48,
49). Hence, the induction of
-opioid receptor internalization has been shown to be one of the
functional consequences of agonist-independent phosphorylation of the receptor
after activation of PKC via a heterologous signaling pathway
(50). In the case of the
-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate-type glutamate
receptor, it has been demonstrated that heterologous desensitization by PKC
involves the interaction of kinase and receptor with an auxiliary protein.
Originally described as protein interacting with PKC, PICK1 binds via its PDZ
domain to the extreme COOH terminus of the
-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate-R subunit GluR2,
resulting in the clustering of receptors into intracellular membrane-bound
compartments (51). Thus, the
enzymatic pathway involved in agonist-independent, PKC-dependent hPAFR-GyrB
desensitization/internalization mediated by coumermycin may involve either
direct phosphorylation of the receptor by PKC or prior interaction with other
signaling molecules. Our findings also indicate that PKC activation can
enhance receptor multimerization, which may also contribute to hPAFR-GyrB
desensitization/internalization.
Dimerization-induced -Arrestin-and Clathrin-independent
Internalization of hPAFR-GyrB
-Arrestins have been shown to
be involved in the internalization and signaling of many GPCRs
(14). After agonist-promoted
recruitment to the plasma membrane, they serve as clathrin adaptors, which
help to target agonist-occupied GPCRs to clathrin-coated pits for
internalization. Indeed, agonist activation of overexpressed wild-type hPAFR
receptors has been shown to trigger the redistribution of arrestins from
intracytoplasmic pools to the plasma membrane where they colocalize with
internalized receptors to discrete intracytoplasmic vesicles
(11). To characterize whether
coumermycin-driven hPAFR-GyrB endocytosis follows the same pathway as
agonist-induced internalization, we investigated the cellular trafficking of
GFP-conjugated arrestin-2 in COS-7 cells. However, activation with coumermycin
failed to induce a translocation of arrestins from the cytosol to the
membrane, suggesting a
-arrestin-independent mechanism. Moreover, the
coumermycin-induced desensitization/internalization occurred in a
clathrin-independent manner because NH4Cl-induced acidification did
not inhibit the process (52).
Thus, these results shows that coumermycin-induced dimerization occurs via a
mechanism distinct from PAF-induced hPAFR-GyrB endocytosis. Recent studies
have suggested that in addition to the clathrin-mediated sequestration pathway
utilized by a large number of GPCRs, alternative mechanisms of GPCR
internalization exist (13).
Indeed, m2 muscarinic and angiotensin 1A receptors have been shown to
internalize in an arrestin- and clathrin-independent manner via an unknown
machinery (19,
21). The potential involvement
of phospholipase C activation in the internalization process was considered
after the report that the AP-2 complex could bind to phosphatidylinositol
4,5-bisphosphate (53) and
inositol 1,4,5-trisphosphate
(54). However, the
agonist-independent sequestration induced by coumermycin did not induce
accumulation of IP, suggesting that phospholipase C activation does not
participate in this alternative sequestration pathway. Considering that some
PKC isoforms and GPCRs can associate with caveolae
(55), it is conceivable that
the coumermycin-driven internalization of hPAFR-GyrB occurred through this
mechanism, as observed for the bradykinin 2 receptor
(56). Alternatively, a novel
mechanism of GPCR sequestration would have to be postulated.
In conclusion, we have shown that coumermycin-induced dimerization of hPAFR-GyrB is sufficient to induce the desensitization/internalization of hPAFR-GyrB via a mechanism that is independent of arrestins, clathrin, or phospholipase C activation, but requires PKC activity. These data represent the first demonstration that dimerization of a GPCR can induce receptor desensitization and internalization without agonist stimulation. They may, therefore, provide insight into a novel mechanism governing cellular responsiveness to stimuli targeting GPCRs. If agonist-independent GPCR dimerization occurs in vivo, it is conceivable that potency and efficacy of an agonist of a given receptor may be shifted through dimerization-dependent desensitization, providing as well as concerns for drug development.
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FOOTNOTES |
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Present address: Montreal Neurological Institute, McGill University,
Montreal, Quebec H3A 2B4, Canada.
To whom correspondence should be addressed: Immunology Division, Dept. of
Pediatrics, Faculty of Medicine, Université de Sherbrooke, 3001 N. 12th
Ave., Sherbrooke, QC J1H 5N4, Canada. Tel.: 819-346-1110 (ext. 14851); Fax:
819-564-5215; E-mail:
mrolaple{at}courrier.usherb.ca.
1 The abbreviations used are: PAF, platelet-activating factor; Ab(s),
antibody(ies); arr, arrestin; BSA, bovine serum albumin; CHO, Chinese hamster
ovary; GFP, green fluorescent protein; GPCR(s), G protein-coupled receptor(s);
GyrB, bacterial DNA gyrase B; h, human; HA, hemagglutinin; IP, inositol
phosphate(s); PAFR, PAF receptor; PBS, phosphate-buffered saline; PKC, protein
kinase C; PMA, phorbol 12-myristate, 13-acetate.
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ACKNOWLEDGMENTS |
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REFERENCES |
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