GPR17 is a negative regulator of the cysteinyl leukotriene 1 receptor response to leukotriene D ₄

Abstract

The cysteinyl leukotrienes (cys-LTs) are proinflammatory lipid mediators acting on the type 1 cys-LT receptor (CysLT 1R) to mediate smooth muscle constriction and vascular permeability. GPR17, a G protein-coupled orphan receptor with homology to the P2Y and cys-LT receptors, failed to mediate calcium flux in response to leukotriene (LT) D 4 with stable transfectants. However, in stable cotransfections of 6؋His-tagged GPR17 with Myc-tagged CysLT 1R, the robust CysLT 1R-mediated calcium response to LTD4 was abolished. The membrane expression of the CysLT 1R analyzed by FACS with anti-Myc Ab was not reduced by the cotransfection, yet both LTD 4-elicited ERK phosphorylation and the specific binding of [ 3 H]LTD4 to microsomal membranes were fully inhibited. CysLT1R and GPR17 expressed in transfected cells were coimmunoprecipitated and identified by Western blots, and confocal immunofluorescence microscopy revealed that GPR17 and CysLT 1R colocalize on the cell surface of human peripheral blood monocytes. Lentiviral knockdown of GPR17 in mouse bone marrow-derived macrophages (BMM⌽s) increased both the membrane expression of CysLT 1R protein by FACS analysis and the LTD4-elicited calcium flux in a dose-dependent manner as compared with control BMM⌽s, indicating a negative regulatory function of GPR17 for CysLT 1R in a primary cell. In IgE-dependent passive cutaneous anaphylaxis, GPR17-deficient mice showed a marked and significant increase in vascular permeability as compared with WT littermates, and this vascular leak was significantly blocked by pretreatment of the mice with the CysLT 1R antagonist, MK-571. Taken together, our findings suggest that GPR17 is a ligand-independent, constitutive negative regulator for the CysLT 1R that suppresses CysLT1R-mediated function at the cell membrane.inflammation ͉ knockout mice ͉ lipid mediator ͉ macrophage T he cysteinyl leukotrienes (cys-LTs), leukotriene (LT) C 4 , LTD 4 , and LTE 4 , are proinflammatory mediators generated by the 5-lipoxygenase (5-LO) pathway after activation of particular bone marrow-derived cells to release arachidonic acid from the phospholipids of the outer nuclear membrane. In the presence of the 5-LO-activating protein (1, 2), 5-LO converts arachidonic acid to LTA 4 (3), which can be conjugated to reduced glutathione to form LTC 4 by an integral trimeric nuclear membrane enzyme, LTC 4 synthase (4 -6). After energydependent export of LTC 4 , glutamic acid and glycine are sequentially cleaved by ␥-glutamyl transpeptidase (7) or ␥-glutamyl leukotrienase (8) and dipeptidases (9, 10) to form LTD 4 and LTE 4 , respectively. The cys-LTs are implicated in human bronchial asthma by their pharmacologic actions to constrict airway and vascular smooth muscle (11-13) and by the clinical efficacy of agents that block 5-LO or the type 1 receptor for the cys-LTs (CysLT 1 R) (14, 15).Two types of human receptors for the cys-LTs, designated CysLT 1 R (16) and CysLT 2 R (17), which belong to the 7-transmembrane, G protein-coupled receptor family, were cloned and shown ...

Is GPR17 a P2Y/Leukotriene Receptor? Examination of Uracil Nucleotides, Nucleotide Sugars, and Cysteinyl Leukotrienes as Agonists of GPR17

“…Our data indicate that GPR17 is neither activated nor internalized by these ligands, whereas receptors that are established targets of these ligands demonstrate robust activation and internalization. Moreover, our data are consistent with GPR17 acting as a negative regulator of CysLTR1 as reported previously (Maekawa et al, 2009). …”

“…The orphan GPCR GPR17 was reported to be activated by uracil nucleotides (UDP), nucleotide sugars (UDP-glucose and UDP-galactose), and cysteinyl leukotrienes (LTC4 and leukotriene D4), and coupled to both inhibition of cAMP accumulation and intracellular Ca 21 mobilization (Ciana et al, 2006;Pugliese et al, 2009), although other studies have reported different agonist selectivity (Benned-Jensen and Rosenkilde, 2010) or no activation of GPR17 by these ligands (Heise et al, 2000;Maekawa et al, 2009). To investigate whether these compounds were agonists at GPR17, HA-tagged GPR17 was stably expressed in C6 and CHO cells by retroviral infection, and its capacity to promote inhibition of cAMP accumulation was assessed.…”

“…35 S]thio)triphosphate binding, and Maekawa et al (2009) concluded that GPR17 was not activated by any of these presumed agonists but instead was a negative regulator of CysLTR1. Given these disparate results, we have reexamined the potential activation of GPR17 in a broad range of cell types that either stably or transiently express this GPCR.…”

Development of [3H]2-Carboxy-4,6-dichloro-1H-indole-3-propionic Acid ([3H]PSB-12150): A Useful Tool for Studying GPR17

“…Instead, GPR17 was identified as a negative regulator of the Cys-LT1R suppressing CysLT1R-mediated function at the cell membrane. 12 Because of these contradictory results, the activation of GPR17 by nucleotides and cysteinyl-leukotrienes is still a subject of intense discussion and controversy.Recently Hennen et al 5 identified 2-carboxy-4,6-dichloro-1H-indole-3-propionic acid (1, MDL29,951) and 3-[(E)-3-(anilino)-3-oxoprop-1-enyl]-4,6-dichloro-1H-indole-2-carboxylic acid (GV150526A, gavestinel, 2) as synthetic, small molecule GPR17 agonists and characterized them in different functional assays including G q -mediated intracellular calcium mobilization, G i -coupled inhibition of adenylate cyclase (AC), G s -coupled stimulation of AC, and dynamic mass redistribution (Figure 1). Activation of GPR17 by 1 was demonstrated in multiple cell types including recombinant cells as well as primary rat oligodendrocytes.…”

Lipid Mediators in Life Science

“…Accordingly, mice deficient in 5-LOX, FLAP, or LTC 4 synthase are all highly resistant to asthmatic challenge [14,31], and an antagonist of CysLT1 is now clinically used as an anti-asthmatic drug worldwide. in addition, it has been recently shown that CysLT1 and CysLT2 can form a heterodimer, that GPr17 counteracts the CysLT1 signaling, and that P2Y12, a purinergic GPCr, is important for the biological action of LTE 4 , the most stable and abundant cys-LT in biological fluid [43,62]. The first LTB 4 receptor, BLT1, is a fundamental T H1 and T H2 immune regulator which acts through recruitment of T lymphocytes [80].…”