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GPR17 Is a Negative Regulator of the Cysteinyl Leukotriene 1 Receptor Response to Leukotriene D4 Akiko Maekawaa,B, Barbara Balestrieria,B, K

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GPR17 Is a Negative Regulator of the Cysteinyl Leukotriene 1 Receptor Response to Leukotriene D4 Akiko Maekawaa,B, Barbara Balestrieria,B, K GPR17 is a negative regulator of the cysteinyl leukotriene 1 receptor response to leukotriene D4 Akiko Maekawaa,b, Barbara Balestrieria,b, K. Frank Austena,b,1, and Yoshihide Kanaokaa,b,1 aDepartment of Medicine, Harvard Medical School, Boston, MA 02115; and bDivision of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital, One Jimmy Fund Way, Boston, MA 02115 Contributed by K. Frank Austen, May 20, 2009 (sent for review May 5, 2009) The cysteinyl leukotrienes (cys-LTs) are proinflammatory lipid me- CysLT2RtobeLTD4 Ͼ LTC4 Ͼ LTE4 and LTC4 ϭ LTD4 Ͼ diators acting on the type 1 cys-LT receptor (CysLT1R) to mediate LTE4, respectively. The findings that these receptors are ex- smooth muscle constriction and vascular permeability. GPR17, a G pressed not only on human smooth muscle but also on bone protein-coupled orphan receptor with homology to the P2Y and marrow-derived cells of the innate and adaptive immune systems cys-LT receptors, failed to mediate calcium flux in response to revealed a potential for involvement of the cys-LT/CysLTR leukotriene (LT) D4 with stable transfectants. However, in stable pathway in the infiltrating cells of the inflammatory response cotransfections of 6؋His-tagged GPR17 with Myc-tagged CysLT1R, (18, 19). We and others subsequently reported that the mouse the robust CysLT1R-mediated calcium response to LTD4 was abol- CysLT1R can function as a receptor for LTD4 in transfected cells ished. The membrane expression of the CysLT1R analyzed by FACS with a ligand preference similar to that of the human CysLT1R with anti-Myc Ab was not reduced by the cotransfection, yet both (20, 21) and that the mouse CysLT2R exhibits a ligand profile of LTD4-elicited ERK phosphorylation and the specific binding of LTC4 Ն LTD4 Ͼ LTE4 (21, 22). Targeted disruption of LTC4 3 [ H]LTD4 to microsomal membranes were fully inhibited. CysLT1R synthase, CysLT1R, and CysLT2R in mice confirmed the func- and GPR17 expressed in transfected cells were coimmunoprecipi- tion of the cys-LTs in vascular smooth muscle and in the cellular tated and identified by Western blots, and confocal immunofluo- aspects of allergic or chronic fibrotic pulmonary injury (23–27). rescence microscopy revealed that GPR17 and CysLT1R colocalize An additional receptor in porcine pulmonary arterial rings that on the cell surface of human peripheral blood monocytes. Lenti- is responsive to LTC4 and LTD4, but not LTE4, has been viral knockdown of GPR17 in mouse bone marrow-derived macro- recognized by differential pharmacologic responses or resistance IMMUNOLOGY phages (BMM⌽s) increased both the membrane expression of to available receptor antagonists on various smooth muscle CysLT1R protein by FACS analysis and the LTD4-elicited calcium flux preparations (28, 29). Presumptive evidence for the existence of ⌽ in a dose-dependent manner as compared with control BMM s, a particular LTE4 receptor was suggested by pharmacologic indicating a negative regulatory function of GPR17 for CysLT1Rin studies with guinea pig tissues (30) and has recently been a primary cell. In IgE-dependent passive cutaneous anaphylaxis, supported by the demonstration of LTE4-mediated vascular GPR17-deficient mice showed a marked and significant increase in permeability in mice lacking both the CysLT1R and the CysLT2R vascular permeability as compared with WT littermates, and this through targeted disruption and crossbreeding (31). vascular leak was significantly blocked by pretreatment of the mice A G protein-coupled orphan receptor, GPR17 (originally with the CysLT1R antagonist, MK-571. Taken together, our findings called ‘‘R12’’), was cloned by homologous screening in human suggest that GPR17 is a ligand-independent, constitutive negative genomic DNA with the chemokine IL-8 receptor (32). Subse- regulator for the CysLT1R that suppresses CysLT1R-mediated func- quently, R12 and its variant form of cDNA were cloned by tion at the cell membrane. homologous screening in a human hippocampus cDNA library with the nucleotide chicken P2Y1 and murine P2Y2 receptors inflammation ͉ knockout mice ͉ lipid mediator ͉ macrophage (33). Phylogenic analysis for P2Y-related receptors revealed that human GPR17 is homologous to the CysLTRs (34), with an he cysteinyl leukotrienes (cys-LTs), leukotriene (LT) C4, amino acid sequence that is 31% and 36% identical to that of the human CysLT R and CysLT R, respectively. Human GPR17 TLTD4, and LTE4, are proinflammatory mediators generated 1 2 by the 5-lipoxygenase (5-LO) pathway after activation of par- also is 90.3% identical to both mouse and rat orthologs in amino ticular bone marrow-derived cells to release arachidonic acid acid sequence (34). Human, rat, and mouse GPR17 have recently from the phospholipids of the outer nuclear membrane. In the been identified as dual receptors for uracil nucleotides and the 35 ␥ presence of the 5-LO-activating protein (1, 2), 5-LO converts cys-LTs, LTC4, and LTD4, based on [ S]GTP S binding assays arachidonic acid to LTA4 (3), which can be conjugated to with transfectants (34, 35). The original report (34) also showed reduced glutathione to form LTC4 by an integral trimeric nuclear by single-cell calcium imaging that 1321N1 cells and COS-7 cells membrane enzyme, LTC synthase (4–6). After energy- expressing human GPR17 could respond to 100 nM of LTD4 and 4 ␮ dependent export of LTC4, glutamic acid and glycine are se- to 100 M of UDP-glucose. quentially cleaved by ␥-glutamyl transpeptidase (7) or ␥-glu- We began a study of mouse GPR17 seeking a novel receptor tamyl leukotrienase (8) and dipeptidases (9, 10) to form LTD for LTE4. However, we could not obtain significant calcium flux 4 ␮ and LTE4, respectively. The cys-LTs are implicated in human to 1 M of LTC4, LTD4,orLTE4 in stable human and mouse bronchial asthma by their pharmacologic actions to constrict GPR17 transfectants using 1321N1, CHO, and HEK-293T cells airway and vascular smooth muscle (11–13) and by the clinical as hosts. In addition, 1321N1 cell clones stably expressing mouse efficacy of agents that block 5-LO or the type 1 receptor for the cys-LTs (CysLT1R) (14, 15). Author contributions: A.M., K.F.A., and Y.K. designed research; A.M., B.B., and Y.K. Two types of human receptors for the cys-LTs, designated performed research; A.M., B.B., K.F.A., and Y.K. analyzed data; and A.M., K.F.A., and Y.K. CysLT1R (16) and CysLT2R (17), which belong to the 7-trans- wrote the paper. membrane, G protein-coupled receptor family, were cloned and The authors declare no conflict of interest. shown to be 38% homologous in their amino acid sequences. The 1To whom correspondence may be addressed. E-mail: [email protected] or radio-labeled ligand-binding assay using microsomal membranes [email protected]. from CysLT1R and CysLT2R transfectants revealed the rank This article contains supporting information online at www.pnas.org/cgi/content/full/ order of affinities of the cys-LTs for the CysLT1R and the 0905364106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0905364106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 25, 2021 GPR17 did not respond to 100 ␮M of UDP-glucose. Since heterodimerization of G protein-coupled receptors modulates expression and/or function either negatively (36, 37) or positively (38, 39) in various transfectants, we considered the possibility that GPR17 may associate with CysLT1R to control its calcium signaling function. Here we show that GPR17 may function as a negative regulator for the CysLT1R response to LTD4 not only in cotransfection of transformed cells but also constitutively in primary cells in which its knockdown resulted in increased membrane expression and LTD4-mediated function of CysLT1R. We provide physiologic evidence for this regulatory role of GPR17 by demonstrating that the vascular leak following IgE- dependent, mast cell-mediated passive cutaneous anaphylaxis (PCA) is significantly increased in GPR17-deficient mice and that this response is blocked by administration of a CysLT1R antagonist. Results and Discussion Cotransfection with GPR17 Inhibits the Function but Not the Mem- brane Expression of the CysLT1R. After failing to find that LTD4- initiated calcium flux in a variety of cell lines transfected with 6ϫHis-GPR17 and expressing membrane protein detected by FACS analysis with mouse monoclonal anti-6ϫHis Ab (data not shown), we considered that GPR17 might influence CysLT1R expression and function. We established 12 independent 1321N1 clones stably expressing Myc-tagged mouse CysLT1R with or without mouse 6ϫHis-tagged GPR17. FACS analysis showed that 6ϫHis-GPR17 protein was expressed in all clones as assessed by staining with anti-His Ab in nonpermeabilized cells [supporting information (SI) Fig. S1A] and that the GPR17 expression did not alter the cell surface expression of the Fig. 1. Stable cotransfection of 1321N1 cells with GPR17 inhibits CysLT1R- Myc-tagged CysLT1R in replicate stable transfectants (Fig. mediated calcium flux to LTD4.(A) Intracellular calcium mobilization by LTD4 in these transfectants labeled with fura-2. Arrows indicate point of injection S1A). The clones that expressed Myc-tagged CysLT1R alone and were labeled with fura-2 responded to 0.001, 0.01, 0.1, and 1 ␮M of LTD4 or histamine. Experiments were performed 3 times with 12 individual clones for each combination, and data from 1 representative clone are shown. of LTD in a dose-dependent manner (Fig. 1A Left), as previ- 4 (B) Peak relative fluorescence of intracellular calcium mobilization by LTD4 in ously reported (20). In contrast, as depicted for the highest dose 1321N1 cells stably expressing CysLT1R (closed circles), GPR17 (closed squares), of LTD4 (1 ␮M), there was no CysLT1R-mediated calcium signal and cells coexpressing CysLT1R and GPR17 (open triangles). Data represent the in clones coexpressing GPR17, although they responded to 100 mean Ϯ SE from 5 independent experiments.
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