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. 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. Platelet-activating factor (PAF) 1The abbreviations used are: PAFplatelet-activating factorβ2ARβ2-adrenergic receptorAEBSF4-(2-aminoethyl) benzenesulfonyl fluoride hydrochlorideEEAIearly endosome autoantigen IEST(2S,3S) trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl esterGPCRG protein-coupled receptorPAFRhuman platelet-activating factor receptorPSIproteasome inhibitor ITfntransferrinDMEMDulbecco's modified Eagle's mediumPBSphosphate-buffered salineBSAbovine serum albuminHAhemagglutinin. is a potent phospholipid mediator released from activated basophils, platelets, macrophages, polymorphonuclear neutrophils, and many other cell types (1Braquet P. Rola-Pleszczynski M. Prostaglandins. 1987; 34: 143-148Crossref PubMed Scopus (35) Google Scholar). 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 (2Ishii S. Shimizu T. Prog. Lipid Res. 2000; 39: 41-82Crossref PubMed Scopus (330) Google Scholar). 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 A2, C, and D activation (3Nakamura M. Honda Z. Izumi T. Sakanaka C. Mutoh H. Minami M. Bito H. Seyama Y. Matsumoto T. Noma M. et al.J. Biol. Chem. 1991; 266: 20400-20405Abstract Full Text PDF PubMed Google Scholar, 4Kunz D. Gerard N.P. Gerard C. J. Biol. Chem. 1992; 267: 9101-9106Abstract Full Text PDF PubMed Google Scholar, 5Ye R.D. Prossnitz E.R. Zou A.H. Cochrane C.G. Biochem. Biophys. Res. Commun. 1991; 180: 105-111Crossref PubMed Scopus (166) Google Scholar, 6Chase P.B. Halonen M. Regan J.W. Am. J. Respir. Cell Mol. Biol. 1993; 8: 240-244Crossref PubMed Scopus (40) Google Scholar, 7Prescott S.M. Zimmerman G.A. McIntyre T.M. J. Biol. Chem. 1990; 265: 17381-17384Abstract Full Text PDF PubMed Google Scholar, 8Liu B. Nakashima S. Kanoh H. Takano T. Shimizu T. Nozawa Y. J. Biochem. (Tokyo). 1994; 116: 882-891Crossref PubMed Scopus (36) Google Scholar, 9Kuruvilla A. Putcha G. Poulos E. Shearer W.T. J. Immunol. 1993; 151: 637-648PubMed Google Scholar). This receptor also activates the mitogen-activated protein kinase cascade (8Liu B. Nakashima S. Kanoh H. Takano T. Shimizu T. Nozawa Y. J. Biochem. (Tokyo). 1994; 116: 882-891Crossref PubMed Scopus (36) Google Scholar, 10Honda Z. Takano T. Gotoh Y. Nishida E. Ito K. Shimizu T. J. Biol. Chem. 1994; 269: 2307-2315Abstract Full Text PDF PubMed Google Scholar, 11Franklin R.A. Tordai A. Mazer B. Terada N. Lucas J. Gelfand E.W. Biochem. Biophys. Res. Commun. 1995; 209: 1111-1118Crossref PubMed Scopus (23) Google Scholar) and the Jak/STAT pathway (12Lukashova V. Asselin C. Krolewski J.J. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 2001; 276: 24113-24121Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). 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 (13Brocheriou I. Stengel D. Mattsson-Hulten L. Stankova J. Rola-Pleszczynski M. Koskas F. Wiklund O. Le Charpentier Y. Ninio E. Circulation. 2000; 102: 2569-2575Crossref PubMed Scopus (31) Google Scholar). 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 (14Ishii S. Nagase T. Tashiro F. Ikuta K. Sato S. Waga I. Kume K. Miyazaki J. Shimizu T. EMBO J. 1997; 16: 133-142Crossref PubMed Scopus (130) Google Scholar). PAFR knockout mice, on the other hand, are resistant to endotoxic shock (15Ishii S. Kuwaki T. Nagase T. Maki K. Tashiro F. Sunaga S. Cao W.H. Kume K. Fukuchi Y. Ikuta K. Miyazaki J. Kumada M. Shimizu T. J. Exp. Med. 1998; 187: 1779-1788Crossref PubMed Scopus (236) Google Scholar). platelet-activating factor β2-adrenergic receptor 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride early endosome autoantigen I (2S,3S) trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester G protein-coupled receptor human platelet-activating factor receptor proteasome inhibitor I transferrin Dulbecco's modified Eagle's medium phosphate-buffered saline bovine serum albumin hemagglutinin. 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 β2AR, colocalize with the transferrin receptor and the Ras-related rab5 GTPase (16Moore R.H. Tuffaha A. Millman E.E. Dai W. Hall H.S. Dickey B.F. Knoll B.J. J. Cell Sci. 1999; 112: 329-338Crossref PubMed Google Scholar). 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 (17Kojro E. Fahrenholz F. J. Biol. Chem. 1995; 270: 6476-6481Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), β2AR (18Jockers R. Angers S. Da Silva A. Benaroch P. Strosberg A.D. Bouvier M. Marullo S. J. Biol. Chem. 1999; 274: 28900-28908Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar)) or via a clathrin-mediated endocytosis pathway as described for the β2AR or the kappa opioid (16Moore R.H. Tuffaha A. Millman E.E. Dai W. Hall H.S. Dickey B.F. Knoll B.J. J. Cell Sci. 1999; 112: 329-338Crossref PubMed Google Scholar, 19Gagnon A.W. Kallal L. Benovic J.L. J. Biol. Chem. 1998; 273: 6976-6981Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 20Li J.G. Benovic J.L. Liu-Chen L.Y. Mol. Pharmacol. 2000; 58: 795-801Crossref PubMed Scopus (92) Google Scholar). Studies with various receptors demonstrate that for the platelet-derived growth factor receptor, Met tyrosine kinase receptor, β2AR, and the receptors S. K. S. Y. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, M. G.A. S. Mol. Biol. 1997; PubMed Scopus (202) Google Scholar, 2001; PubMed Scopus Google Scholar, M. A. S. P. Bouvier M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar) degradation can occur via the proteasome the of receptors are degraded by lysosomal proteases A.W. Kallal L. Benovic J.L. J. Biol. Chem. 1998; 273: 6976-6981Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, L. A.W. Benovic J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, L. K. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, J.L. R. Res. Mol. Res. 1999; PubMed Scopus Google Scholar). pathways of degradation for some GPCRs are the of the of receptors toward degradation is not well Following in GPCRs are targeted to the early endosome and Rab5 are implicated in or of the early C. H. K. B. M. 1992; Full Text PDF PubMed Scopus Google Scholar). is known to from early to endosomes and be linked to toward lysosomes A. K. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The β2AR can in dependent (16Moore R.H. Tuffaha A. Millman E.E. Dai W. Hall H.S. Dickey B.F. Knoll B.J. J. Cell Sci. 1999; 112: 329-338Crossref PubMed Google Scholar) while and is implicated in receptor toward degradation J.G. Benovic J.L. Liu-Chen L.Y. Mol. Pharmacol. 2000; 58: 795-801Crossref PubMed Scopus (92) Google Scholar). on of the β2AR 2001; PubMed Scopus Google Scholar) and A. Benovic J.L. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar) their targeting to lysosomes. In the we receptor endocytosis is a for PAFR down-regulation. Using such as transferrin, and and specific we also the pathways involved in receptor down-regulation and the of proteasomes and lysosomes in this The or of PAFR was also ubiquitination is known to be linked to degradation by both lysosomes and were from the from high and high and from bovine serum bovine and protein from from and from and from from and from inhibitor I and trans-epoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester were from was from Cell and cells were in high with bovine serum and were at a of in and to of and of were after was by from a human cDNA and and and in while was and and in was from from the of The and and the and were a from M. and I. of and of the PAF after cells were with and in high and for at of was for at was and cells were then for in high with bovine and with were for the were in of and The was by at for with of protein at for and by at for The was with an for then of protein were and the was for After with the proteins were from with of by and were to of was as with some Z. Takano T. Gotoh Y. Nishida E. Ito K. Shimizu T. J. Biol. Chem. 1994; 269: 2307-2315Abstract Full Text PDF PubMed Google Scholar). The cells were on 40 with PAF and at for then at for The were with for at then in in for and with and at for The cells were then with at for by for then with a of and for After the were on The cells were on a system with a were to a with of cells were on 40 with PAF and at for then at for and with for at The were then in in for and then with and at for The cells were then with for After the were on for and binding were on cells the PAFR and as described C. J.L. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). cells were with and in The binding were on cells in a total of of the at for were by The was by of receptor was on cells receptor and as described C. J.L. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). were to medium or PAF in the of at for in in of H. R.A. B. R. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). were then with the and in was then by was to of PAFR hours cells were with for and then in medium for PAFR was then proteins were by and were by after were to with a PAF was to the of receptor degradation. In a a of receptors after the of several and that receptors in to the down-regulation of a degradation was degradation be and to of ligand-induced degradation. receptors were degraded after of and the degradation a by of of studies that PAFR through and is dependent on C. J.L. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, C. J.L. R. L. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, Z. Le C. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In to was for we the of in the degradation of the the of receptor with cells not the with receptor down-regulation to cells However, receptor down-regulation was completely inhibited by the dominant-negative a in basal degradation studies that the of receptors on the cell surface not in cells or dominant-negative but PAF was blocked in cells results indicate that PAFR degradation is dependent on and of we PAFR the pathway of the transferrin receptor of PAFR and cells were with transferrin to and with PAF. and the basal of both and respectively, with colocalization of transferrin and PAFR D and the transferrin and PAF receptor their transferrin receptor is known to through early endosomes internalization, is also through endosomes J. Cell Sci. 1994; PubMed Google Scholar, B. S. E. J. M. J. Cell Biol. 2000; PubMed Scopus Google Scholar). of the not The I (EEAI) to early endosomes M. M. R. M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). PAFR to early endosomes endocytosis, we its colocalization with In and the basal of and respectively, is colocalization PAF Following PAF stimulation, is while PAFR The of the colocalization of and PAFR after of PAF endosome autoantigen I colocalization with PAFR. 40 after with PAFR cells were with or PAF and then with with were then with by a of and basal of basal of PAFR C, of and PAFR of a with PAF. of PAFR a with PAF. of and PAFR of and on PAFR of GPCR down-regulation have demonstrated the degradation of of the receptors via lysosomal or the effects of the proteasome inhibitor and lysosomal inhibitor on PAFR degradation. cells were with the proteasome inhibitor the of degradation was by and results were with proteasome inhibitor not of PAFR cells with receptor with cells inhibitor degradation inhibitor a not used together, and completely blocked receptor degradation. The inhibitors on receptor in Rab5 in PAFR of from the to early of the in the is mediated by Rab5 C. H. K. B. M. 1992; Full Text PDF PubMed Scopus Google Scholar). the of Rab5 in the targeting of PAFR for degradation The of PAFR and a of total PAF a of of after the PAFR was with a dominant negative of H. O. A. J. M. EMBO J. 1994; PubMed Scopus Google Scholar, K. Ito K. A. K. T. Biochem. 1999; PubMed Scopus Google Scholar, J.L. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Google Scholar), the ligand-induced degradation was a for Rab5 in the of PAF receptors toward their degradation. The of cell surface receptors and PAFR was not with Rab5 indicating that Rab5 not but the degradation of in PAFR is in from early endosome to endosomes Y. B. A. J. Cell Biol. 1995; PubMed Scopus Google Scholar, R. M. D. M. M. C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) and from endosome to lysosomes S. P. J. Cell Sci. 1995; Google Scholar). the of in PAFR toward its degradation by with the or the dominant-negative The total of receptors was with but PAF a down-regulation of PAFR. The of the dominant-negative of PAFR down-regulation. with the of not cell surface or PAFR results that PAFR down-regulation involves and not receptor but degradation of of the been that some GPCRs can be been to be in degradation of several proteins as well as in the proteasome degradation results a for both lysosomes and proteasomes in the degradation of we the ubiquitination of the that PAFR was ubiquitinated with a protein a at the In this was to receptor and degradation. PAFR was with an and by with an the of the PAFR was in both and but was the or the receptor was not PAFR the receptor was with the or dominant-negative ubiquitin ligase that PAFR ubiquitination is not with the which that the of the ubiquitin ligase is not However, in the of the dominant negative (NCbl) ubiquitination of both the and receptor was by the of ubiquitination in basal or receptor down-regulation was The of or not basal down-regulation of the receptor, the receptor degradation was in cells In the we demonstrated that receptor endocytosis is a for PAFR down-regulation. Using transferrin, or dominant-negative we the pathways involved in receptor down-regulation. for both proteasomes and lysosomes in this was by the of and its in down-regulation of the PAFR was also stimulation, PAFR is rapidly and via from that of receptors are recycled to the cell surface while from the cell C. J.L. Rola-Pleszczynski M. Stankova J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). PAFR been is known its and degradation agonist of the in the of from the to early is mediated by Rab5 C. H. K. B. M. 1992; Full Text PDF PubMed Scopus Google Scholar). that Rab5 can the targeting of the transferrin receptor from the cell surface to early endosomes H. O. A. J. M. EMBO J. 1994; PubMed Scopus Google Scholar). demonstrated that PAFR colocalize with with PAF, cells is known to through early endosomes internalization, is also known to through distinct endosomes J. Cell Sci. 1994; PubMed Google Scholar, B. S. E. J. M. J. Cell Biol. 2000; PubMed Scopus Google Scholar). of not PAFR to the early endosomes is Following with PAF, the receptor with cells a for early endosomes in PAFR Our results are in with which demonstrated the of the early endosome for the of β2AR, the receptor and the of receptor J.L. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Google Scholar, F. D. J. A. J. 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Full Text Full Text PDF PubMed Scopus Google Scholar). ubiquitination be for targeting of some also be for other proteins implicated in receptor endocytosis and studies of ligand-induced of the receptor indicate that ubiquitinated proteins with the N. A. P. J. Cell Biol. PubMed Scopus Google Scholar). Our results indicate that of receptors from the cell of in the of stimulation, a and degradation is not by dominant-negative and indicating a in the of and PAFR. Studies have demonstrated that degradation of some GPCRs down-regulation in lysosomes A.W. Kallal L. Benovic J.L. J. Biol. Chem. 1998; 273: 6976-6981Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, L. A.W. Benovic J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, J.L. R. Res. Mol. Res. 1999; PubMed Scopus Google Scholar), degradation of the β2AR, and receptor can through the proteasome A.W. Kallal L. Benovic J.L. J. Biol. Chem. 1998; 273: 6976-6981Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 2001; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, L. A.W. Benovic J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, L. K. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, J.L. R. Res. Mol. Res. 1999; PubMed Scopus Google Scholar). receptors such as the platelet-derived growth factor receptor and Met tyrosine kinase receptor are also known to undergo which for and degradation by proteasomes or lysosomes S. K. S. Y. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, M. G.A. S. Mol. Biol. 1997; PubMed Scopus (202) Google Scholar, N. I. H. J. Biol. Google Scholar) indicate that both lysosomes and proteasomes are involved in the degradation of GPCRs as by the β2AR and the receptor which both pathways (16Moore R.H. Tuffaha A. Millman E.E. Dai W. Hall H.S. Dickey B.F. Knoll B.J. J. Cell Sci. 1999; 112: 329-338Crossref PubMed Google Scholar, 20Li J.G. Benovic J.L. Liu-Chen L.Y. Mol. Pharmacol. 2000; 58: 795-801Crossref PubMed Scopus (92) Google Scholar, M. A. S. P. Bouvier M. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). Our results that both degradation mechanisms be used for PAFR. mechanisms are to the degradation some receptors be targeted toward lysosomal or degradation via distinct on the receptor or on an associated proteasome degradation of a or other the receptor be for the targeting and of the receptor to lysosomes L. Cell Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). in proteins such as the or the distinct can target the receptor toward degradation A. Benovic J.L. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). be for by phosphorylation, receptor and target the receptor to the In results indicate that of the PAFR is mediated by lysosomes and proteasomes and that the pathway toward degradation of the receptor is mediated via the early/late and dependent on Rab5 and PAFR ubiquitination are not by but ligand-induced PAFR down-regulation is dependent on ubiquitination while basal down-regulation is results that other mechanisms of of receptor mediate endocytosis or of ubiquitinated GPCRs in

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