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 Gαs-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 Gαi/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.

Eosinophils Contribute to Intestinal Inflammation via Chemoattractant Receptor-homologous Molecule Expressed on Th2 Cells, CRTH2, in Experimental Crohn’s Disease

“…2D). We also investigated the effect of MK0524, a selective antagonist and inverse agonist at DP 33,34 . As previously also observed in a DSS model 11 , MK0524 (1 mg/kg) worsened inflammation scores in TNBS colitic mice by ~20%.…”

Pharmacological chaperoning: A primer on mechanism and pharmacology

“…In this regard, PC stabilization of A1 adenosine receptor folding intermediates promotes the dissociation of the receptor from HSP 40 protein D1 receptor interacting protein 78 (DRiP78) [91]. Intriguingly, PCs promote the interaction of the prostanoid DP1 receptor and ANKRD13C [92], a cytoplasmically localized molecular chaperone for GPCRs that aids in forward trafficking [93]. …”

“…For nACh receptors, agonists are more potent PCs than antagonists, possibly because heteromeric assembly may occur more efficiently if the receptor is in an activated or desensitized state [72]. In addition to agonist PCs, inverse agonists PC have been identified for the prostanoid DP1 receptor [92]. Moreover, V2R antagonists are “protean agonists” serving as PCs for certain mutants while acting as inverse agonists at wild type receptors [111] further revealing the complexities of pharmacological chaperoning.…”

A G protein–coupled receptor and the intracellular synthase of its agonist functionally cooperate

“…Our previous studies have shown that a large population of DP1 is retained in intracellular compartments after synthesis ( Parent et al, 2010 ; Labrecque et al, 2013 ). Furthermore, we reported that L-PGDS was localized to the ER and other intracellular compartments ( Mathurin et al, 2011 ).…”