title: 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.
authors.affiliation: Département de Médecine, Université de Sherbrooke, Sherbrooke, Quebec, Canada.
authors.forename: Pascale
authors.initials: P
authors.lastname: Labrecque
authors.forename: Sébastien J
authors.initials: SJ
authors.lastname: Roy
authors.forename: Louis
authors.initials: L
authors.lastname: Fréchette
authors.forename: Christian
authors.initials: C
authors.lastname: Iorio-Morin
authors.forename: Maxime A
authors.initials: MA
authors.lastname: Gallant
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Parent
mesh_terms: D015153:Blotting, Western; D020126:Brefeldin A / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D007211:Indoles / Q000494:pharmacology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D018832:Molecular Chaperones / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D015230:Prostaglandin D2 / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011982:Receptors, Prostaglandin / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D007211:Indoles; C518174:MK-0524; D018832:Molecular Chaperones; D011500:Protein Synthesis Inhibitors; D011982:Receptors, Prostaglandin; C501562:prostanoid D receptor 1, human; D020126:Brefeldin A; D000242:Cyclic AMP; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D015230:Prostaglandin D2