Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 106, Issue 28, Pages 11685-11690Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0905364106
Keywords
inflammation; knockout mice; lipid mediator; macrophage
Categories
Funding
- National Institutes of Health [P01HL36110, R01HL82695, R01HL090630, K08AI064226]
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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 x His-tagged GPR17 with Myc-tagged CysLT(1)R, the robust CysLT(1)R-mediated calcium response to LTD4 was abolished. The membrane expression of the CysLT1R analyzed by FACS with anti-Myc Ab was not reduced by the cotransfection, yet both LTD4-elicited ERK phosphorylation and the specific binding of [H-3]LTD4 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 (BMM Phi s) increased both the membrane expression of CysLT(1)R protein by FACS analysis and the LTD4-elicited calcium flux in a dose-dependent manner as compared with control BMM Phi s, 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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