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.
D000818:Animals; D019556:COS Cells; D003546:Cysteine Endopeptidases; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D034281:Dynamins / Q000378:metabolism; D005799:Genes, Dominant; D006801:Humans; D008024:Ligands; D008247:Lysosomes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry; D018613:Microscopy, Confocal; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry*; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D021381:Protein Transport; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000737:chemistry*; D011990:Receptors, Transferrin / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014168:Transferrin / Q000737:chemistry / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
D018076:DNA, Complementary; D008024:Ligands; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D010980:Platelet Membrane Glycoproteins; D043562:Receptors, G-Protein-Coupled; D011990:Receptors, Transferrin; D014168:Transferrin; D025801:Ubiquitin; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; C039351:platelet activating factor receptor; D000090802:rab7 GTP-Binding Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D034281:Dynamins