Trafficking, Ubiquitination, and Down-regulation of the Human Platelet-activating Factor Receptor*

Platelet-activating factor (PAF) is a potent phospholipid mediator involved in various disease states such as allergic asthma, atherosclerosis and psoriasis. The human PAF receptor (PAFR) is a member of the G protein-coupled receptor family. Following PAF stimulation, cells become rapidly desensitized; this refractory state can be maintained for hours and is dependent on PAFR phosphorylation, internalization, and down-regulation. In this report, we characterized ligand-induced, long term PAFR desensitization, and pathways leading to its degradation. Some GPCRs are known to be targeted to proteasomes for degradation while others traffic via the early/late endosomes toward lysosomes. Specific inhibitors of lysosomal proteases and inhibitors of the proteasome were effective in reducing the ligand-induced PAFR down-regulation by 40 and 25%, respectively, indicating the importance of receptor targeting to both lysosomes and proteasomes in long term cell desensitization to PAF. The effects of the proteasome and lysosomal protease inhibitors were additive and, together, completely blocked ligand-induced degradation of PAFR. Using dominant-negative Rab5 and 7 and colocalization of the PAFR with the early endosome autoantigen I (EEAI) or transferrin, we confirmed that ligand-induced PAFR down-regulation was Rab5/7-dependent and involved lysosomal degradation. In addition, we also demonstrated that PAFR was ubiquitinated in an agonist-independent manner. However, a dominant negative ubiquitin ligase (NCbl) reduced PAFR ubiquitination and inhibited ligand-induced but not basal receptor degradation. Our results indicate that PAFR degradation can occur via both the proteasome and lysosomal pathways and ligand-stimulated degradation is ubiquitin-dependent.

types (1). In humans, various diseases have been associated with PAF, such as allergic asthma, endotoxic shock, acute pancreatitis, and dermal inflammation such as psoriasis and pruritis (2). PAF structural requirements are highly specific for its biological actions, which are mediated through the binding and activation of a specific, high affinity PAF receptor (PAFR) on the target cell surface. cDNA cloning from various sources revealed that PAFR belongs to the G-protein coupled receptors family (GPCR) and its signaling is linked to various second messenger systems, including phospholipase A 2 , C, and D activation (3)(4)(5)(6)(7)(8)(9). This receptor also activates the mitogen-activated protein kinase cascade (8,10,11) and the Jak/STAT pathway (12). PAF is known to be involved in a variety of biological activities related to inflammatory and immune responses, respiratory and nervous system physiology as well as circulatory system disorders such as atherosclerosis (13). Transgenic mice, which overexpress PAFR spontaneously develop melanocyte tumors and a severe response to lipopolysaccharide-induced endotoxin shock. With their enhanced sensitivity to PAF, they also demonstrate bronchial hyperresponsiveness and have problems with fertilization (14). PAFR knockout mice, on the other hand, are resistant to endotoxic shock (15).
The attenuation of GPCR signaling, after stimulation, is known as desensitization and involves several distinct mechanisms. Within seconds after agonist binding, GPCRs become functionally uncoupled from G proteins and rapidly phosphorylated by different kinases. The receptors then undergo endocytosis into endosomes and, for some receptors such as the ␤ 2 AR, colocalize with the transferrin receptor and the Rasrelated rab5 GTPase (16). After endocytosis, a receptor can be recycled to the cell surface or targeted for degradation. The down-regulation of the total number of receptors after a prolonged treatment with an agonist is thought to mediate long term desensitization.
Recent studies have revealed distinct mechanisms implicated in GPCRs degradation. Several receptors are, at least in part, degraded via an endocytosis-independent mechanism (V2R (17), ␤ 2 AR (18)) or via a clathrin-mediated endocytosis pathway as described for the ␤ 2 AR or the kappa opioid (16,19,20). Studies with various receptors demonstrate that for the platelet-derived growth factor receptor, Met tyrosine kinase receptor, ␤ 2 AR, and the ␦Ϫopioid receptors (21)(22)(23)(24) degradation can occur via the proteasome whereas the vast majority of receptors are degraded by lysosomal proteases (19,(25)(26)(27)(28). Although pathways of degradation for some GPCRs are known, the trafficking of the majority of receptors toward degradation is not well described. Following internalization in clathrincoated vesicles, GPCRs are targeted to the early endosome compartments where Rab4 and Rab5 are implicated in recycling or regulation of the early endocytic pathway, respectively (29). Rab7 is known to regulate trafficking from early to late endosomes and might be linked to trafficking toward lysosomes (30). The ␤ 2 AR can accumulate in perinuclear Rab-11 dependent compartments (16) while Rab5-and Rab7-dependent trafficking is implicated in -opioid receptor trafficking toward degradation (20). Moreover, monoubiquitination on cytoplasmic lysine residues of the ␤ 2 AR (23) and CXCR4 (31) promotes their targeting to lysosomes.
In the present study, we investigated whether PAF-induced receptor endocytosis is a prerequisite for PAFR down-regulation. Using markers such as labeled transferrin, EEA1, rab5, and rab7 and specific inhibitors, we also examined the trafficking pathways involved in receptor down-regulation and evaluated the role of proteasomes and lysosomes in this phenomenon. The possible agonist-dependent or -independent mono-ubiquitination of PAFR was also assessed since ubiquitination is known to be linked to degradation by both lysosomes and proteasomes. and the mixture was incubated for 1 h. After extensive washing with RIPA buffer, the immunoprecipitated proteins were eluted from beads with 50 l of SDS sample buffer, resolved by SDS-PAGE, and dried gels were exposed to Hyperfilm MP.
Confocal Microscopy Analysis of EEA1-Confocal microscopy analysis was performed as previously described, with some modifications (10). The cells were grown on coverslips (22 mm), 40 h post-transfection, incubated with serum-free DMEM containing PAF (10 Ϫ7 M), anti-cMyc antibodies and 0.1% BSA at 4°C for 1 h, then at 37°C for indicated times. The coverslips were fixed with 3% paraformaldehyde for 20 min at room temperature, then placed in 0.1% saponin in PBS for 20 min, and sequentially incubated with 5% casein and 0.01 M glycine at room temperature for 20 min, each. The cells were then incubated with goat anti-EEA1 antibodies at room temperature for 1 h, followed by biotinconjugated donkey anti-goat antibodies for 1 h, then with a mixture of rhodamine-conjugated goat anti-mouse IgG antibodies and streptavidin-FITC for 1 h. After washing, the coverslips were mounted on slides. The cells were analyzed on a Molecular Dynamics (Sunnyvale, CA) Multi-Probe 2001 confocal argon laser scanning system equipped with a Nikon Diaphot epifluorescence inverted microscope. Scanned images were transferred to a Silicon Graphics Indy 4000 workstation equipped with Molecular Dynamics Imagespace analysis software.
Confocal Microscopy Analysis of Transferrin-The cells were grown on coverslips (22 mm), 40 h post-transfection, incubated with serumfree DMEM containing PAF 10 Ϫ7 M, anti-cMyc antibodies, 50 g/ml transferrin-Alexa568 and 0.1% BSA at 4°C for 1 h, then at 37°C for indicated times, and fixed with 3% paraformaldehyde for 20 min at room temperature. The coverslips were then placed in 0.1% saponin in PBS for 20 min, and then sequentially incubated with 5% dry milk and 0.01 M glycine at room temperature for 20 min, each. The cells were then incubated with rhodamine-conjugated goat anti-mouse IgG antibodies for 1 h. After washing, the coverslips were mounted on slides for image acquisition and analysis.
Receptor Sequestration-The evaluation of receptor sequestration was done on COS-7 cells transiently expressing wild-type receptor and indicated coexpressed proteins, as described previously (32). Cells were exposed to medium or PAF in the presence of [ 3 H]PAF at room temperature for 30 min in following buffer (150 mM choline chloride, 10 mM MgCl 2 , 10 mM Tris, pH 7.4, in presence of 0.25% lipid-free BSA) (33). Cells were then washed twice with the same buffer ϩ 2% BSA and harvested in 0.1 M NaOH. [ 3 H]PAF uptake was then measured by liquid scintillation.
Statistics-Student's t test was performed to determine statistical significance.

Down-regulation of PAFR Expression-Twenty-four hours
post-transfection, COS-7 cells were labeled with [ 35 S]methionine for 2 h, washed, and then incubated in new medium for indicated times. PAFR was then immunoprecipitated, proteins were separated by electrophoresis, and bands were quantified by densitometric analysis after gels were exposed to film. An 18-h stimulation with a non-hydrolyzable PAF analog, methylcarbamyl-PAF (10 Ϫ6 M), was performed to evaluate the maximum level of receptor degradation. In Fig. 1A, a representative autoradiogram indicates a decreased concentration of receptors after stimulation. Fig. 1B shows the densitometric representation of several experiments (n ϭ 4) and indicates that 42 Ϯ 6.5% receptors disappear in reference to unstimulated cells. To further characterize the down-regulation of PAFR, a time-dependent degradation curve was plotted (Fig. 1C). Basal constitutive degradation could be observed and corresponds to ϳ50% of ligand-induced degradation. Approximately 25% more recep-tors were degraded after 5 h of ligand exposure, and the degradation reached a plateau by 8 h of stimulation.
Dynamin-dependent Down-regulation of PAFR-Previous studies showed that PAFR internalization occurs through clathrin-coated pits and is dependent on dynamin (32,34,35). In order to determine whether internalization was necessary for down-regulation, we analyzed the role of dynamin in the methylcarbamyl-PAF-induced degradation of the receptor. Interestingly, the expression of wild-type dynamin (Dyn1A) increased receptor expression, compared with cells not expressing the construct. An 8-h stimulation with methylcarbamyl-PAF induced receptor down-regulation comparable to control cells (ϳ40%). However, PAF-stimulated receptor down-regulation was completely inhibited by the dominant-negative dy-namin (Dyn1A[K44A]), without a significant change in basal degradation ( Fig. 2A). Binding studies indicated that the numbers of receptors on the cell surface did not change significantly in cells expressing wild-type or dominant-negative dynamin (Fig. 2B), but PAF internalization was blocked in cells expressing Dyn1A[K44A] (Fig. 2C). These results indicate that PAFstimulated PAFR degradation is dependent on internalization.
Transferrin Receptor and Early Endosomal Localization of Internalized PAFR-In Fig. 3, we assessed whether PAFR followed the well-established internalization pathway of the transferrin receptor (TfnR). To visualize internalization of PAFR and TfnR simultaneously, cells were prelabeled with transferrin (Tfn) conjugated to Alexa-568, rinsed, and incubated with PAF. Fig. 3, A and B show the basal expression of both TfnR and PAFR, respectively, with no colocalization of transferrin and PAFR (Fig. 3C). Fig. 3, D and E show the transferrin and PAF receptor distribution following stimulation; Fig. 3F illustrates their colocalization. Although transferrin receptor trafficking is known to proceed through early sorting endosomes following internalization, it is also transiting through recycling endosomes (36,37). Therefore, localization of the TfnR does not distinguish among segregated endosomal compartments. The Early Endosome Antigen I (EEAI) localizes selectively to early endosomes (38). To determine whether PAFR traffics to early endosomes following endocytosis, we assessed its possible colocalization with EEAI. In Fig. 4, A and B, the basal distribution of EEAI and PAFR, respectively, is illustrated. Fig. 4C shows no colocalization without PAF stimulation. Following PAF stimulation, EEAI expression is unchanged (Fig. 4D) while PAFR distribution shows internalization (Fig. 4E). The merging of the images demonstrates colocalization of EEAI and PAFR after 30 min of PAF stimulation (Fig. 4F).
Effect of Lysosome and Proteasome Inhibitors on PAFR Degradation-Studies of GPCR down-regulation have demonstrated the degradation of most of the receptors via two major mechanisms: lysosomal or proteasomal. We examined the effects of the proteasome inhibitor PSI and lysosomal enzyme inhibitor EST on methylcarbamyl-PAF induced PAFR degradation. When cells were pretreated with the proteasome inhibitor PSI, the level of degradation was diminished by half (p Ͻ 0.04), and similar results were obtained with clastolactacystin, another proteasome inhibitor (data not shown). Pretreatment of PAFR expressing COS-7 cells with EST significantly (p Ͻ 0.04) decreased receptor degradation, compared with cells without inhibitor (Fig. 5). Chloroquine, another lysosome degradation inhibitor had a similar effect (data not shown). When used together, EST and PSI completely blocked PAF-induced receptor degradation. The inhibitors had no significant effect on receptor expression in unstimulated cells.
Rab5 Involvement in PAFR Trafficking-Trafficking of clathrin-coated pits from the plasma membrane to early sorting endosomes, one of the first steps in the endocytic pathway, is mediated by Rab5 (29). We examined the involvement of Rab5 in the targeting of PAFR for degradation (Fig. 6). The coexpression of PAFR and Rab5[WT] induced a massive loss of total receptors, however, PAF still stimulated a further loss of 44 Ϯ 3% of after 8 h. When the PAFR was coexpressed with a dominant negative form of Rab5, Rab5[S34N] (39 -41), the ligand-induced degradation was prevented (Fig. 6A), suggesting a role for Rab5 in the trafficking of PAF receptors toward their degradation. The expression of cell surface receptors (Fig.  6C) and PAFR internalization (Fig. 6D); however, was not changed with Rab5 coexpression indicating that Rab5 did not promote internalization but rather promoted the degradation of intracellular receptors. Rab7 Involvement in PAFR Trafficking-Rab7 is important in membrane transport from early sorting endosome to late endosomes (42,43) and from late endosome to lysosomes (44). We examined the involvement of Rab7 in PAFR trafficking toward its degradation by coexpression with the wild-type Rab7 or the dominant-negative mutant Rab7[N125I] (Fig. 6B).
The total concentration of receptors was decreased with wildtype Rab7 cotransfection but PAF stimulated a further downregulation of PAFR. The co-expression of the dominant-negative form of Rab7[N125I] prevented PAF-stimulated PAFR down-regulation. As with Rab5, the co-expression of Rab7 cDNAs did not affect cell surface expression (Fig. 6C)

FIG. 3. Transferrin receptor colocalization with internalized PAFR.
To visualize internalization of PAFR and TfnR simultaneously, PAFR-transfected COS-7 cells were prelabeled with transferrin conjugated to Alexa-568, rinsed, and incubated with or without PAF (10 Ϫ6 M). PAFR was revealed with anti-c-Myc antibodies followed by secondary rhodamine-conjugated goat anti-mouse antibodies. A, basal expression of transferrin receptor (green). B, basal expression of PAFR (red). C, merged images of transferrin and PAFR show no colocalization of receptors. D, expression of transferrin receptor (green) following a 60-min stimulation. E, expression of PAFR (red) following a 60-min stimulation. F, merged images of transferrin and PAFR shows intracellular colocalization of receptors into early endosomes. internalization (Fig. 6D). These results suggest that PAFR down-regulation involves Rab7-dependent trafficking, and Rab7 does not promote receptor internalization but rather supports degradation of intracellular receptors.
Ubiquitination of the PAFR-It has recently been shown that some GPCRs can be ubiquitinated. Ubiquitination has been shown to be important in lysosome-mediated degradation of several plasma proteins as well as in the proteasome degradation pathway. Since our results suggested a role for both lysosomes and proteasomes in the degradation of PAFR, we assessed the possible ubiquitination of the receptor. Fig. 7A illustrates that PAFR was ubiquitinated when coexpressed with a mono-ubiquitin fusion protein containing a triple HA epitope at the N terminus. In this experiment, dynamin K44A was expressed to abolish receptor internalization and limit degradation. First, PAFR was immunoprecipitated with an anti-c-Myc antibody and analyzed by Western blotting with an anti-HA antibody, revealing the tagged mono-ubiquitin construct. Ubiquitination of the PAFR was observed in both stimulated and unstimulated cells, but was absent when the monoubiquitin construct or the receptor was not expressed. To further characterize PAFR ubiquitination, the wild-type receptor was coexpressed with the wild-type or dominant-negative ubiquitin ligase Cbl. Fig. 7B illustrates that PAFR ubiquitina-tion is not modulated when coexpressed with the wild-type Cbl which indicates that the concentration of the endogenous ubiquitin ligase is not limiting. However, in the presence of the dominant negative Cbl (NCbl) ubiquitination of both the stimulated and unstimulated receptor was decreased by ϳ50%. Next, the role of ubiquitination in basal or stimulated receptor down-regulation was examined. The expression of wild-type or NCbl did not change basal down-regulation of the receptor, however the PAF-stimulated receptor degradation was abolished in NCbl-expressing cells (Fig. 7C). DISCUSSION In the present study, we demonstrated that PAF-induced receptor endocytosis is a prerequisite for PAFR down-regulation. Using labeled transferrin, anti-EEA1 antibodies, Rab5/ Rab7 wild-type or dominant-negative constructs, we identified the trafficking pathways involved in receptor down-regulation. A role for both proteasomes and lysosomes in this phenomenon was suggested by the use of inhibitors. Agonist-independent mono-ubiquitination and its role in agonist-dependent downregulation of the PAFR was also demonstrated.
Upon stimulation, PAFR is rapidly desensitized and internalized via clathrin-coated vesicles. Previous results, from our laboratory, showed that ϳ80% of stimulated receptors are recycled back to the cell surface within 1 h while 20% disappear from the cell (32). Although PAFR internalization has been studied, nothing is currently known about its trafficking and degradation following agonist stimulation.
One of the first steps in the endocytic pathway, trafficking of clathrin-coated pits from the plasma membrane to early sorting endosomes, is mediated by Rab5 (29). Mutations that alter Rab5 activity can affect the targeting of the transferrin receptor from the cell surface to early sorting endosomes (39). We demonstrated that PAFR could colocalize with TfnR, following stimulation with PAF, whereas unstimulated cells showed no colocalization. Although TfnR trafficking is known to proceed through early sorting endosomes following internalization, it is also known to transit through distinct recycling endosomes (36,37). Therefore, localization of TfnR does not distinguish among segregated endosomal compartments. We investigated whether PAFR would show any distribution to the early endosomes where EEAI is selectively located. Following stimulation with PAF, the receptor colocalized with EEAI whereas untreated cells showed no colocalization, demonstrating a role for early endosomes in PAFR trafficking. Our results are in agreement with previous studies, which demonstrated the role of the early endosome compartments for the trafficking of ␤ 2 AR, the neurotensin receptor (NT1) and the M4 subtype of muscarinic acetylcholine receptor (41, 45, 46). FIG. 4. Early endosome autoantigen I colocalization with internalized PAFR. 40 h after transfection with PAFR cDNA, COS-7 cells were incubated with or without PAF (10 Ϫ6 M) and anti-c-Myc antibodies, then fixed with 3% paraformaldehyde before labeling with anti-EEA1 antibodies. Cells were then incubated with biotin-conjugated donkey anti-goat antibodies, followed by a mixture of rhodamine-conjugated goat anti-mouse IgG antibodies and streptavidin-FITC. A, basal expression of EEAI (green). B, basal expression of PAFR (red). C, merged image of EEAI and PAFR shows no colocalization. D, expression of EEAI (green) following a 30-min stimulation with PAF. E, expression of PAFR (red) following a 30min stimulation with PAF. F, merged image of EEAI and PAFR shows intracellular colocalization. Receptors were immunoprecipitated, separated on SDS-PAGE, dried gels were exposed to film, and the bands were quantified by densitometry. Results are expressed as means Ϯ S.E. of three independent experiments. *, p Ͻ 0.04; **, p Ͻ 0.0004.
Endocytosed receptor trafficking can also proceed through late endosomes toward lysosomal degradation. First, Rab7 is essential for the transport from early to late endosomes (30). In addition, Rab7 plays a role in the transport of internalized receptors from late endosomes to lysosomes (44). Thus, Rab7 is thought to control the aggregation and fusion of late endocytic structures/lysosomes, which is essential for maintenance of the perinuclear lysosomal compartment (29). Our results indicate that Rab7 has an important role in PAFR trafficking since a Rab7 dominant-negative mutant completely blocked receptor degradation.
The expression of both Rab5 and Rab7 resulted in a loss of total receptors from unstimulated cells, however, neither internalization nor cell-surface receptor expression was affected, indicating these proteins affect intracellular targeting of receptors for degradation rather than endocytosis (47). Ligand-stimulated down-regulation was blocked by dominant negative Rab5 and Rab7 but not basal down-regulation suggesting dual mechanisms for the trafficking of PAF-stimulated and unstimulated receptors.
The covalent modification of lysine residues of proteins by ubiquitin is a well known signal for targeting of many cytosolic proteins to proteasomes. Recently, the ubiquitination of cellsurface proteins emerged as a mechanism that could regulate the endocytic trafficking of membrane proteins (48). The yeast Ste2p GPCR was shown to undergo endocytosis and sorting of endocytosed receptors to the vacuole via a ubiquitin-dependent mechanism (49 -51). The ubiquitin ligase Cbl has a role in the lysosomal sorting of the epidermal growth factor (EGF) recep-tor but not in its internalization (52). In addition, recent studies of CXCR4 and ␤ 2 AR provided evidence that ubiquitination of cytoplasmic lysine residues is essential for lysosomal trafficking of those GPCRs (23,31). However, this phenomenon does not appear to be essential for all GPCRs since murine delta opioid receptor trafficking was shown to be independent of ubiquitination (53). Our results demonstrated that ubiquitination of PAFR was not ligand dependent. When we coexpressed NCbl, ubiquitination of PAFR was reduced in both stimulated and unstimulated cells but degradation of PAFstimulated PAFR was blocked while PAFR down-regulation in unstimulated cells was not affected. This would suggest that ubiquitination of the receptor, in itself, is not sufficient for PAFR down-regulation. Possibly a ligand-induced conformational change may expose the ubiquitin molecule and make it available for binding with a protein(s) involved in intracellular sorting of the receptor. Some GPCRs, as shown for the murine delta-opioid receptor, can proceed through ubiquitin-independent trafficking. The underlying mechanism is still unknown but many studies suggest that there might be modulation of the endocytic trafficking by non-covalent protein interactions with the receptor (54 -58). Although ubiquitination may be important for targeting of some receptors, it may also be essential for other cytosolic proteins implicated in receptor endocytosis and trafficking. Indeed, studies of ligand-induced endocytic trafficking of the EGF receptor indicate that ubiquitinated proteins associate with the endocytic membrane (59). Our results indicate that ϳ50% of receptors disappear from the cell within 5 h of incubation, in the absence of stimulation, suggesting a con- stitutive internalization and degradation process. Ligand-independent down-regulation, unlike PAF-stimulated down-regulation, is not affected by dominant-negative dynamin and NCbl, indicating a definite dichotomy in the trafficking of stimulated and unstimulated PAFR.
Studies have demonstrated that degradation of some GPCRs during agonist-induced down-regulation occurs in lysosomes (19,25,26,28), whereas degradation of the ␤ 2 AR, thrombin, opiate and ␦-opioid receptor can proceed through the proteasome (19,23,(25)(26)(27)(28). Other non-GPCR membrane receptors such as the platelet-derived growth factor receptor and Met tyrosine kinase receptor are also known to undergo rapid agonist-promoted ubiquitination, which targets them for recognition and degradation by proteasomes or lysosomes (21,22,60) These observations indicate that both lysosomes and proteasomes are involved in the degradation of GPCRs as illustrated by the ␤ 2 AR and the -opioid receptor which use both pathways (16,20,24). Our results suggest that both degradation mechanisms could be used for PAFR. Different mechanisms are proposed to explain the dual degradation system. First, some receptors might be targeted toward lysosomal or proteasomal degradation via two distinct signals, either directly on the Receptors were immunoprecipitated, separated on SDS-PAGE, dried gels were exposed to film, and the bands were quantified by densitometry. Results are representative of three independent experiments. receptor or on an associated protein. Alternatively, proteasome degradation of a protein, or proteins, other than the receptor could be required for the targeting and transport of the receptor to lysosomes (61). Also, in membrane-bound proteins such as the IL-2R or the CXCR4, distinct motifs can target the receptor toward degradation (31,62). Such motifs could be regulated, for example by phosphorylation, following receptor activation, and target the receptor to the appropriate compartments.
In conclusion, our results indicate that PAF-induced proteolysis of the PAFR is mediated by lysosomes and proteasomes and suggest that the trafficking pathway toward degradation of the receptor is mediated via the early/late endosomal compartments and dependent on Rab5 and Rab7. PAFR ubiquitination levels are not modulated by ligand stimulation but ligandinduced PAFR down-regulation is dependent on ubiquitination while basal down-regulation is not. These results suggest that other mechanisms of regulation of receptor trafficking might mediate endocytosis or postendocytic sorting of basally ubiquitinated GPCRs in mammalian cells.