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. 2012 Dec;6(4):205-16.
doi: 10.1007/s12079-012-0175-1. Epub 2012 Aug 10.

Rescue of internalization-defective platelet-activating factor receptor function by EBP50/NHERF1

Denis J Dupré  1 , Marek Rola-Pleszczynski, Jana Stankova
Affiliations

Rescue of internalization-defective platelet-activating factor receptor function by EBP50/NHERF1

Denis J Dupré et al. J Cell Commun Signal. 2012 Dec.

Abstract

Platelet-activating factor (PAF) is a potent phospholipid mediator involved in specific disease states such as allergic asthma, atherosclerosis and psoriasis. The human PAF receptor (PAFR) is a member of the G protein-coupled receptor (GPCR) family. Following PAF stimulation, cells become rapidly desensitized; this refractory state can be maintained for hours and is dependent on PAFR phosphorylation, internalization and trafficking. EBP50/NHERF1 has been found to interact with a variety of proteins and these interactions are involved in a growing range of functions including the assembly of signalling complexes, receptor recycling and transport of proteins to the cell surface. Crucial roles of EBP50 in GPCR physiology include its involvement in internalization, recycling, and downregulation. We were interested in identifying the role of EBP50 in PAFR trafficking. Our results showed that EBP50 binds the PAFR in its basal state, while stimulation decreased the ratio of interaction between the two proteins. We also demonstrated that EBP50 could bind PAFR via its PDZ 2 domain. In addition, we studied the role of EBP50 in various functions of the PAFR such as PAF-induced inositol phosphate accumulation and receptor internalization: EBP50 decreased the WT PAFR response and rescued the function of internalization-deficient mutant receptors, as previously described for the arrestins and the GRKs. These results suggest new roles for EBP50, some of which could help understanding the complex formation after receptor activation.

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Figures

Fig. 1
Fig. 1
Co-immunoprecipitation of EBP50 and PAFR. a 48 h post-transfection, HEK293 cells expressing cmyc-tagged PAFR and HA-tagged EBP50 or pcDNA3 cDNAs were immunoprecipitated in RIPA buffer using an anti-cmyc antibody, separated on SDS-PAGE, transferred to a nitrocellulose membranes. A Western blot analysis was done with antibodies for HA or cmyc epitopes. Results are representative of three independent experiments. b Results are showing the co-immunoprecipitation of cmyc-tagged PAFR and HA-tagged EBP50 PDZ1, PDZ2, ERM domains or pcDNA3 cDNAs in HEK293 cells. Stimulations were performed using PAF at a concentration of 10−6M for 15 min
Fig. 2
Fig. 2
EBP50 effect of PAFR response. HEK293 cells transiently expressing the WT or STOP4 mutant PAFR and EBP50 were used 48 h post-transfection for IP accumulation and binding reactions. a Effect of various concentrations of PAF on the response of PAFR in presence of various EBP50 constructs in HEK293 cells. The results are expressed as the means of three independent experiments, each done in triplicate. b The effect on inositol phosphate production of the co-expression of EBP50 on WT and STOP4 mutant PAFR was measured. Cells were stimulated for 15 min at 37 °C with PAF 10−6M. The results are expressed as the means ± s.e. of three independent experiments, each done in triplicate. T-test statistics were done using Graphpad Prism; where ** = p < 0.01. c Binding reactions were performed in presence of 3H-WEB2086 and PAF as described in the methods. The results are expressed as the means of three independent experiments, each done in duplicate
Fig. 3
Fig. 3
Effect of EBP50 on the internalization of PDZ-binding motif PAFR mutants. a Effect of WT EBP50 on PAFR (WT, N338A, S339A, L340A, K341R) internalization, . Cells were stimulated 48 h post-transfection with PAF 10-6M. The results are expressed as the means ± s.e. of three independent experiments, each done in triplicate. T-test statistics were done using Graphpad Prism; where * = p < 0.05. b Kinetics were done for various time points ranging from 5 min (no internalization) to 60 min (maximal internalization). A ratio of internalization at each time point is represented over basal levels of internalization (5 min). The S339A mutant PAFR was co-expressed with pcDNA3, EBP50 or dominant negative EBP50. The results are expressed as the means of three independent experiments, each done in triplicate. c GST-fusion constructs of the WT and S339A mutant receptor c-tails were used for a histidine pull-down assay conducted between the GST-fusion protein cleaved by thrombin and 25 μg of histidine-tagged EBP50. Results are representative of three independent experiments
Fig. 4
Fig. 4
Effect of EBP50 and arrestin2 on PAFR internalization. To evaluate the effect of EBP50 on receptor internalization, we co-expressed the PAFR, WT or dominant negative EBP50 constructs, as well as arrestin 2. Kinetics were done for time points between 5 min (no internalization) and 60 min (maximal internalization). A ratio of internalization at each time point is represented over basal levels of internalization (5 min). a Internalization of WT PAFR co-expressed with pcDNA3, EBP50, EBP50 dominant negative or arrestin 2. b Internalization of STOP4 PAFR mutant co-expressed with pcDNA3, EBP50, dominant negative EBP50 or arrestin 2. The results are expressed as the means of at least three independent experiments, each done in triplicate. T-test statistics were done using Graphpad Prism; where * = p < 0.05 and ** = p < 0.01
Fig. 5
Fig. 5
Effect of EBP50 and arrestin2 on the internalization of uncoupled PAFR mutants. To evaluate the effect of EBP50 on PAFR mutant receptors internalization, D63N or D289A PAFR were co-expressed with WT or dominant negative EBP50 constructs, or arrestin 2. Kinetics were performed for time points between 5 min (no internalization) and 60 min (maximal internalization). A ratio of internalization at each time point is represented over basal levels of internalization (5 min). a Internalization of D63N mutant PAFR co-expressed with pcDNA3, EBP50, dominant negative EBP50 or arrestin 2. b Internalization of D289A mutant PAFR co-expressed with pcDNA3, EBP50, dominant negative EBP50 or arrestin 2. c Internalization of D289A/S339A mutant PAFR co-expressed with pcDNA3, EBP50, dominant negative EBP50 or arrestin 2. The results are expressed as the means of at least three independent experiments, each done in triplicate. T-test statistics were done using Graphpad Prism; where ** = p < 0.01
Fig. 6
Fig. 6
Effect of EBP50, arrestins and GRKs on the internalization D63N mutant receptor. a Effect of WT EBP50, arrestin 2/3 and GRK2/3/5/6 on the internalization of D63N mutant PAFR. Cells were stimulated 48 h post-transfection with PAF 10−6M. Effect of WT EBP50, WT arrestin 2 or dominant negative isoforms (V53D and Arrestin2Δ318–419) expressed alone or co-expressed on the internalization of WT PAFR (b), D63N (c) or STOP4 PAFR (d). Cells were stimulated 48 h post-transfection with PAF 10−6M. The results are expressed as the means ± s.e. of three independent experiments, each done in triplicate. T-test statistics were done using Graphpad Prism; where * = p < 0.05; ** = p < 0.01; *** = p < 0.001
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