Inverse agonist and pharmacochaperone properties of MK-0524 on the prostanoid DP1 receptor

Abstract

Prostaglandin D₂ (PGD₂) acts through two G protein-coupled receptors (GPCRs), the prostanoid DP receptor and CRTH2 also known as DP1 and DP2, respectively. Several previously characterized GPCR antagonists are now classified as inverse agonists and a number of GPCR ligands are known to display pharmacochaperone activity towards a given receptor. Here, we demonstrate that a DP1 specific antagonist, MK-0524 (also known as laropiprant), decreased basal levels of intracellular cAMP produced by DP1, a Gα(s)-coupled receptor, in HEK293 cells. This reduction in cAMP levels was not altered by pertussis toxin treatment, indicating that MK-0524 did not induce coupling of DP1 to Gα(i/o) proteins and that this ligand is a DP1 inverse agonist. Basal ERK1/2 activation by DP1 was not modulated by MK-0524. Interestingly, treatment of HEK293 cells expressing Flag-tagged DP1 with MK-0524 promoted DP1 cell surface expression time-dependently to reach a maximum increase of 50% compared to control after 24 h. In contrast, PGD₂ induced the internalization of 75% of cell surface DP1 after the same time of stimulation. The increase in DP1 cell surface targeting by MK-0524 was inhibited by Brefeldin A, an inhibitor of transport from the endoplasmic reticulum-Golgi to the plasma membrane. Confocal microscopy confirmed that a large population of DP1 remained trapped intracellularly and co-localized with calnexin, an endoplasmic reticulum marker. Redistribution of DP1 from intracellular compartments to the plasma membrane was observed following treatment with MK-0524 for 24 h. Furthermore, MK-0524 promoted the interaction between DP1 and the ANKRD13C protein, which we showed previously to display chaperone-like effects towards the receptor. We thus report that MK-0524 is an inverse agonist and a pharmacochaperone of DP1. Our findings may have important implications during therapeutic treatments with MK-0524 and for the development of new molecules targeting DP1.

Figure 1. MK-0524 reduces DP1 cAMP signaling below basal levels.

HEK293 cells transiently expressing Flag-DP1 were stimulated with 10 nM (A) or increasing concentrations (B) of the indicated ligands for 10 min and cAMP levels were measured as described in “Materials and Methods”. Results are presented in fmol/well above or below basal cAMP production by DP1 in absence of ligand (set at 0) (A) or as the % of the maximal response obtained with PGD₂ stimulation (B). Data are the mean ± S.E. of at least three independent experiments. *** is P<0.001.

Figure 2. The MK-0524-mediated reduction in DP1 cAMP signaling below basal levels is unaffected by pertussis toxin.

HEK293 cells transiently expressing Flag-DP1 pretreated or not with 1 µg/ml of pertussis toxin (PTX) for 10 min were incubated with increasing concentrations of MK-0524 and cAMP levels were measured as described in “Materials and Methods”. Results are presented as fmol of cAMP generated per well. Data are the mean ± S.E. of at least three independent experiments.

Figure 3. MK-0524 does not modulate ERK1/2 activation by DP1.

HEK293 cells transiently expressing Flag-DP1 were stimulated with 1 µM PGD₂ or MK-0524 for the indicated times. ERK1/2 activation was analyzed by Western blot using a phospho-ERK1/2 (pERK1/2) antibody as described under “Materials and Methods”. Total amounts of ERK1/2 in the loaded samples were revealed by an anti-ERK1/2 antibody. The blots shown are representative of three separate experiments. Densitometry analyses (pERK1/2/ERK) of at least three different experiments were performed. IB, immunoblotting. * is P<0.05 and *** is P<0.001.

Figure 4. MK-0524 promotes cell surface expression of DP1.

Cell surface expression of the receptor was measured by ELISA as described under “Materials and Methods” in HEK293 cells transiently expressing Flag-DP1 incubated for the indicated times with 1 µM of PGD₂, BW245C, BWA868C or MK-0524. The results are shown as the percentage of cell surface expression of Flag-DP1 in cells stimulated with the ligands compared with cells treated with control vehicles. Results are the mean ± S.E. of at least four independent experiments.

Figure 5. The total expression of the DP1 protein is not modulated by MK-0524.

Lysates of HEK293 cells transiently expressing Flag-DP1 incubated for 24 h with vehicle or 1 µM of MK-0524 were analyzed by Western blot using a monoclonal Flag antibody. The blot shown is representative of three separate experiments. IB, immunoblotting.

Figure 6. DP1 does not undergo constitutive internalization.

Cell surface receptor expression was measured by ELISA as described under “Materials and Methods” in HEK293 transiently co-transfected with pcDNA3-Flag-DP1 or pcDNA3-Flag-TPβ in combination with pcDNA3 or pcDNA3-dynamin-K44A, a dominant negative mutant of dynamin. Data are shown as the percentage of cell surface receptor expression in cells co-transfected with pcDNA3 for each receptor. Results are the mean ± S.E. of at least three independent experiments. ** is P<0.01.

Figure 7. MK-0524 induces translocation of DP1 from intracellular compartments to the plasma membrane.

The distribution of DP1 in HEK293 cells was determined by immunofluorescence confocal microscopy. HEK293 cells transfected with Flag-DP1 were treated with vehicle or 1 µM of MK-0524 alone or in presence of 20 µM Brefeldin A for 90 min. Cells were labeled with mouse anti-FLAG and either a rabbit anti-calnexin antibody (top and midlle panels) or a rabbit anti-protein disulfide isomerase (PDI) antibody (lower panel) as described under “Materials and Methods”. Secondary antibodies were Alexa Fluor 488 donkey anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG. Merge images of the green-labelled DP1 and red-labeled calnexin or PDI are shown. Bars, 10 µM.

Figure 8. The promotion of DP1 cell surface targeting by MK-0524 is inhibited by Brefeldin A.

Cell surface receptor expression was measured by ELISA as described under “Materials and Methods” in HEK293 cells transiently expressing Flag-DP1 that were pre-incubated with vehicle or 20 µM of Brefeldin A (BFA) for 30 min, and then treated with 1 µM of MK-0524 or its control vehicle for 90 min. The results are shown as the percentage increase of DP1 cell surface expression when compared to control cells treated with vehicle. Results are the mean ± S.E. of three independent experiments. * is P<0.05.

Figure 9. MK-0524 promotes the interaction between DP1 and the ANKRD13C protein.

HEK293 cells transiently co-expressing Flag-DP1 and ANKRD13C-myc were treated with vehicle (ethanol) or 1 µM MK-0524 for 24 h. Flag-DP1 was immunoprecipitated as described under “Materials and Methods” and immunoprecipitated samples as well as cell lysates were analyzed by Western blot with anti-Flag and anti-Myc antibodies. The blots shown are representative of three separate experiments. The ratio of the amount of ANKRD13C that was co-immunoprecipitated on the quantity of receptor immunoprecipitated was calculated by densitometry analyses from the three separate experiments and the results are shown in the bottom panel as the mean ± S.E. * is P<0.05.