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

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Abstract
The cysteinyl leukotrienes (cys-LTs) are proinflammatory lipid mediators acting on the type 1 cys-LT receptor (CysLT 1 R) 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 1 R, the robust CysLT 1 R-mediated calcium response to LTD 4 was abolished. The membrane expression of the CysLT 1 R 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]LTD 4 to microsomal membranes were fully inhibited. CysLT 1 R and GPR17 expressed in transfected cells were coimmunoprecipitated and identified by Western blots, and confocal immunofluorescence microscopy revealed that GPR17 and CysLT 1 R colocalize on the cell surface of human peripheral blood monocytes. Lentiviral knockdown of GPR17 in mouse bone marrow-derived macrophages (BMMΦs) increased both the membrane expression of CysLT 1 R protein by FACS analysis and the LTD 4 -elicited calcium flux in a dose-dependent manner as compared with control BMMΦs, indicating a negative regulatory function of GPR17 for CysLT 1 R 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 1 R antagonist, MK-571. Taken together, our findings suggest that GPR17 is a ligand-independent, constitutive negative regulator for the CysLT 1 R that suppresses CysLT 1 R-mediated function at the cell membrane.

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