Abstract
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.
Highlights
Platelet-activating factor (PAF)1 is a potent phospholipid mediator released from activated basophils, platelets, macrophages, polymorphonuclear neutrophils, and many other cell
Using markers such as labeled transferrin, EEA1, rab5, and rab7 and specific inhibitors, we examined the trafficking pathways involved in receptor down-regulation and evaluated the role of proteasomes and lysosomes in this phenomenon
PAF receptor (PAFR) was immunoprecipitated, proteins were separated by electrophoresis, and bands were quantified by densitometric analysis after gels were exposed to film
Summary
Materials—Reagents were obtained from the following sources: methylcarbamyl-PAF from Cayman Chemical Company (Ann Arbor, MI), DMEM high glucose and DMEM high glucose without L-methionine and L-cysteine from Invitrogen Canada Inc. (Burlington, ON, Canada), bovine serum albumin, fetal bovine serum, and protein A-Sepharose from Sigma-Aldrich, FuGENE-6 Transfection reagent from Roche Applied Science, rhodamine-conjugated goat anti-mouse IgG, biotinconjugated donkey anti-goat antibodies and streptavidin-FITC from Bio Can Scientific (Mississauga, ON, Canada), Redivue Pro-Mix [35S]methionine and Hyperfilm MP from Amersham Biosciences (Baie d’Urfe, QC, Canada), transferrin-Alexa568 from Molecular Probes (Eugene, OR), and goat anti-EEA1 antibody from Santa Cruz Biotechnology. Metabolic Labeling and Immunoprecipitation of the PAF Receptor—24 h after transfection, cells were washed with PBS and incubated in DMEM high glucose without L-methionine and L-cysteine for 30 min, at 37 °C. The cells were grown on coverslips (22 mm), 40 h post-transfection, incubated with serum-free DMEM containing PAF (10Ϫ7 M), anti-cMyc antibodies and 0.1% BSA at 4 °C for 1 h, at 37 °C for indicated times. Confocal Microscopy Analysis of Transferrin—The cells were grown on coverslips (22 mm), 40 h post-transfection, incubated with serumfree DMEM containing PAF 10Ϫ7 M, anti-cMyc antibodies, 50 g/ml transferrin-Alexa568 and 0.1% BSA at 4 °C for 1 h, at 37 °C for indicated times, and fixed with 3% paraformaldehyde for 20 min at room temperature. Statistics—Student’s t test was performed to determine statistical significance
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