GPR17 is a negative regulator of the cysteinyl leukotriene 1 receptor response to leukotriene D4

  1. Akiko Maekawaa,b,
  2. Barbara Balestrieria,b,
  3. K. Frank Austena,b,1 and
  4. Yoshihide Kanaokaa,b,1
  1. aDepartment of Medicine, Harvard Medical School, Boston, MA 02115; and
  2. bDivision of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, One Jimmy Fund Way, Boston, MA 02115

  1. Contributed by K. Frank Austen, May 20, 2009 (received for review May 5, 2009)

 
Next Section

Abstract

The cysteinyl leukotrienes (cys-LTs) are proinflammatory lipid mediators acting on the type 1 cys-LT receptor (CysLT1R) 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) D4 with stable transfectants. However, in stable cotransfections of 6×His-tagged GPR17 with Myc-tagged CysLT1R, the robust CysLT1R-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 [3H]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 CysLT1R 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 CysLT1R protein by FACS analysis and the LTD4-elicited calcium flux in a dose-dependent manner as compared with control BMMΦs, indicating a negative regulatory function of GPR17 for CysLT1R 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 CysLT1R antagonist, MK-571. Taken together, our findings suggest that GPR17 is a ligand-independent, constitutive negative regulator for the CysLT1R that suppresses CysLT1R-mediated function at the cell membrane.

The cysteinyl leukotrienes (cys-LTs), leukotriene (LT) C4, LTD4, and LTE4, 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 LTA4 (3), which can be conjugated to reduced glutathione to form LTC4 by an integral trimeric nuclear membrane enzyme, LTC4 synthase (4–6). After energy-dependent export of LTC4, glutamic acid and glycine are sequentially cleaved by γ-glutamyl transpeptidase (7) or γ-glutamyl leukotrienase (8) and dipeptidases (9, 10) to form LTD4 and LTE4, 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 (CysLT1R) (14, 15).

Two types of human receptors for the cys-LTs, designated CysLT1R (16) and CysLT2R (17), which belong to the 7-transmembrane, G protein-coupled receptor family, were cloned and shown to be 38% homologous in their amino acid sequences. The radio-labeled ligand-binding assay using microsomal membranes from CysLT1R and CysLT2R transfectants revealed the rank order of affinities of the cys-LTs for the CysLT1R and the CysLT2R to be LTD4 > LTC4 > LTE4 and LTC4 = LTD4 > LTE4, respectively. The findings that these receptors are expressed not only on human smooth muscle but also on bone marrow-derived cells of the innate and adaptive immune systems revealed a potential for involvement of the cys-LT/CysLTR pathway in the infiltrating cells of the inflammatory response (18, 19). We and others subsequently reported that the mouse CysLT1R can function as a receptor for LTD4 in transfected cells with a ligand preference similar to that of the human CysLT1R (20, 21) and that the mouse CysLT2R exhibits a ligand profile of LTC4 ≥ LTD4 > LTE4 (21, 22). Targeted disruption of LTC4 synthase, CysLT1R, and CysLT2R in mice confirmed the function of the cys-LTs in vascular smooth muscle and in the cellular aspects of allergic or chronic fibrotic pulmonary injury (23–27). An additional receptor in porcine pulmonary arterial rings that is responsive to LTC4 and LTD4, but not LTE4, has been recognized by differential pharmacologic responses or resistance to available receptor antagonists on various smooth muscle preparations (28, 29). Presumptive evidence for the existence of a particular LTE4 receptor was suggested by pharmacologic studies with guinea pig tissues (30) and has recently been supported by the demonstration of LTE4-mediated vascular permeability in mice lacking both the CysLT1R and the CysLT2R through targeted disruption and crossbreeding (31).

A G protein-coupled orphan receptor, GPR17 (originally called “R12”), was cloned by homologous screening in human genomic DNA with the chemokine IL-8 receptor (32). Subsequently, R12 and its variant form of cDNA were cloned by homologous screening in a human hippocampus cDNA library with the nucleotide chicken P2Y1 and murine P2Y2 receptors (33). Phylogenic analysis for P2Y-related receptors revealed that human GPR17 is homologous to the CysLTRs (34), with an amino acid sequence that is 31% and 36% identical to that of the human CysLT1R and CysLT2R, respectively. Human GPR17 also is 90.3% identical to both mouse and rat orthologs in amino acid sequence (34). Human, rat, and mouse GPR17 have recently been identified as dual receptors for uracil nucleotides and the cys-LTs, LTC4, and LTD4, based on [35S]GTPγS binding assays with transfectants (34, 35). The original report (34) also showed by single-cell calcium imaging that 1321N1 cells and COS-7 cells expressing human GPR17 could respond to 100 nM of LTD4 and to 100 μM of UDP-glucose.

We began a study of mouse GPR17 seeking a novel receptor for LTE4. However, we could not obtain significant calcium flux to 1 μM of LTC4, LTD4, or LTE4 in stable human and mouse GPR17 transfectants using 1321N1, CHO, and HEK-293T cells as hosts. In addition, 1321N1 cell clones stably expressing mouse GPR17 did not respond to 100 μM 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.

Previous SectionNext Section

Results and Discussion

Cotransfection with GPR17 Inhibits the Function but Not the Membrane Expression of the CysLT1R.

After failing to find that LTD4-initiated calcium flux in a variety of cell lines transfected with 6×His-GPR17 and expressing membrane protein detected by FACS analysis with mouse monoclonal anti-6×His 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 6×His-tagged GPR17. FACS analysis showed that 6×His-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 Myc-tagged CysLT1R in replicate stable transfectants (Fig. 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 μM of LTD4 in a dose-dependent manner (Fig. 1A Left), as previously reported (20). In contrast, as depicted for the highest dose of LTD4 (1 μM), there was no CysLT1R-mediated calcium signal in clones coexpressing GPR17, although they responded to 100 μM of histamine used as a positive control for function of a constitutive receptor (40) (Fig. 1A Right). Inhibition of the CysLT1R response to LTD4 by coexpression of GPR17 was significant (P < 0.005) at all doses of LTD4 as compared to CysLT1R alone (Fig. 1B). Repetition of these transfection protocols 3 to 5 times in CHO or HEK-293T cells again showed a robust dose-dependent response to LTD4 (0.001, 0.01, 0.1, and 1 μM) with CysLT1R alone, and no response to these doses of LTD4 with GPR17 cotransfection or GPR17 alone (data not shown). We also examined the effect of coexpression of GPR17 with CysLT1R in stable 1321N1 transfectants on another ligand, LTC4. We again observed loss of the CysLT1R-mediated calcium flux function (Fig. S1B).

Fig. 1.
View larger version:
Fig. 1.

Stable cotransfection of 1321N1 cells with GPR17 inhibits CysLT1R-mediated calcium flux to LTD4. (A) Intracellular calcium mobilization by LTD4 in these transfectants labeled with fura-2. Arrows indicate point of injection of LTD4 or histamine. Experiments were performed 3 times with 12 individual clones for each combination, and data from 1 representative clone are shown. (B) Peak relative fluorescence of intracellular calcium mobilization by LTD4 in 1321N1 cells stably expressing CysLT1R (closed circles), GPR17 (closed squares), and cells coexpressing CysLT1R and GPR17 (open triangles). Data represent the mean ± SE from 5 independent experiments. *, P < 0.005 vs. cells coexpressing CysLT1R and GPR17.

To exclude the possibility that only LTD4 nonresponsive, stable cotransfectants had selectively survived, we performed the same experiments in 1321N1 cells transiently expressing Myc-tagged mouse CysLT1R, 6×His-tagged mouse GPR17, or both together. We observed that the CysLT1R/GPR17 coexpression did not alter the cell surface expression of the Myc-tagged CysLT1R or of the 6×His-tagged GPR17 as compared to single transient transfectants but that it completely inhibited the LTD4-induced, CysLT1R-mediated calcium signal (Fig. S2 A and B). The control response to histamine-induced calcium flux was intact (Fig. S2B). These findings indicate that GPR17 expression inhibits the CysLT1R response to LTD4 without affecting cell surface expression of CysLT1R or the function of a constitutive receptor.

Cotransfection with GPR17 Inhibits CysLT1R-Mediated ERK Phosphorylation in Response to LTD4.

To further assess the effect of cotransfection with GPR17 on CysLT1R functions, we performed ERK phosphorylation assays (41) in CHO cell stable transfectants expressing CysLT1R with or without GPR17. The bands corresponding to phospho-p44/42 ERK were detected in the cells expressing CysLT1R alone after 10-min stimulation with 1 μM (Fig. 2A) or 0.01 μM (data not shown) of LTD4, but not in the cells coexpressing CysLT1R and GPR17. These findings indicate that GPR17 expression inhibits the CysLT1R response to LTD4 for not only the calcium signaling but also the ERK signaling pathway, which leads to induction of cytokines/chemokines (41). It should be noted that we did not detect any LTD4-mediated ERK phosphorylation in LTD4-stimulated cells expressing GPR17 alone.

Fig. 2.
View larger version:
Fig. 2.

Cotransfection with GPR17 inhibits ERK phosphorylation and [3H]LTD4 binding in microsomal membranes from cells stably expressing CysLT1R. (A) Western blot with anti-phospho p44/42-ERK (Upper panels) or anti-total p44/42-ERK (Lower panels) in CHO cell stable transfectants stimulated for 10 min with or without 1 μM of LTD4. Results are representative from 3 independent experiments. (B) Microsomal membranes from CHO cells transfected with CysLT1R alone (Left) or cotransfected with GPR17 (Right) were incubated with 0–1,000 pM [3H]LTD4 in the presence (open circles) or absence (closed circles) of an excess amount of cold LTD4. Bound [3H]LTD4 was separated from free [3H]LTD4 by filtration through glass fiber filters. The residual membrane-associated [3H]LTD4 on the filter was determined by liquid scintillation counting. The insert shows cell surface staining with isotype control as a shaded histogram and with anti-CysLT1R Ab as a solid line. Data represent the mean ± SE from 3 independent experiments. *, P < 0.005 vs. cells coexpressing CysLT1R and GPR17.

Cotransfection with GPR17 Suppresses [3H]LTD4 Binding by Membranes from Cells Expressing CysLT1R.

We performed [3H]LTD4 binding assays using microsomal membranes prepared from CHO cell clones stably expressing mouse CysLT1R with or without cotransfection with mouse GPR17. Microsomal membranes from CHO cell clones expressing CysLT1R alone showed increased binding with increasing inputs of [3H]LTD4 alone (Fig. 2B, Left). This binding was prevented by inclusion of excess cold ligand, thereby indicating its specificity as we had previously demonstrated (20). In contrast, there was no total or specific [3H]LTD4 binding in microsomal membranes from clones cotransfected with GPR17 and CysLT1R (Fig. 2B, Right). The cell surface expression of CysLT1R with and without GPR17 cotransfection was similar by FACS analysis with rabbit polyclonal anti-CysLT1R peptide Ab (RB34) (Fig. 2B, Insets). There was no [3H]LTD4 specific binding in microsomal membranes from stable clones expressing GPR17 alone (data not shown). Specific [3H]LTD4 binding on microsomal membranes expressing CysLT1R was not inhibited when they were mixed with microsomal membranes from clones expressing only GPR17, indicating that GPR17 needed to be coexpressed with CysLT1R to suppress its ligand binding (data not shown). [3H]LTD4 binding was also robust in 1321N1 cells transiently expressing Myc-tagged mouse CysLT1R and absent with co-transfection with 6×His-tagged GPR17 (Fig. S2C).

CysLT1R and GPR17 Coimmunoprecipitate in CHO Cells Cotransfected with both Receptors.

To determine if GPR17 can physically interact with CysLT1R, we used a fusion protein of mouse GPR17 and the YFP (GPR17-YFP) and a Myc-tagged-mouse CysLT1R (CysLT1R-Myc). The GPR17-YFP and CysLT1R-Myc constructs were transiently transfected alone or in combination into CHO cells, and the cell lysates were immunoprecipitated with mouse anti-Myc mAb or with rabbit antiserum against GFP, which recognizes denatured recombinant GFP and YFP. The immunoprecipitates were separated by SDS/PAGE and analyzed by Western blot. Mouse anti-GFP mAb (JL-8) was used for Western blots of the immunoprecipitates obtained with anti-Myc Ab, and rabbit polyclonal anti-Myc Ab was used for immunoprecipitates obtained with anti-GFP Ab. We detected bands corresponding to dimers at ≈70 kDa for CysLT1R-Myc and ≈120 kDa for GPR17-YFP only in immunoprecipitates prepared from the cells cotransfected with CysLT1R-Myc and GPR17-YFP (Fig. 3 A and B, lane 4). We only detected the bands corresponding to each monomer when we oversaturated the signals, suggesting that the major form of both receptors for interaction may be dimers that can progress to oligomers as observed in bands with higher molecular weights. The direct Western blots of these lysates show comparable bands. These findings indicate that GPR17 and CysLT1R can form heterodimers or heteromultimers.

Fig. 3.
View larger version:
Fig. 3.

GPR17 and CysLT1R coimmunoprecipitate in CHO cells cotransfected with both receptors and GPR17 and CysLT1R colocalize at the plasma membrane in human monocytes. (A and B) HEK-293T cells were transfected with pEYFP-N1 vector (YFP) + pCXN vector (lanes 1 and 5), GPR17-YFP + pCXN (lane 2), YFP + CysLT1R-Myc (lane 3), or GPR17-YFP + CysLT1R-Myc (lanes 4 and 6). (A) Lysates from each transfectant were immunoprecipitated with rabbit antiserum against GFP, and the precipitates (Left) and total cell lysates (Right) were separated by SDS/PAGE and analyzed by Western blot with rabbit polyclonal anti-Myc Ab. (B) Lysates from each transfectant were immunoprecipitated with agarose conjugated with mouse anti-Myc mAb and the precipitates (Left) and total cell lysates (Right) were separated by SDS/PAGE and analyzed by Western blot with mouse anti-GFP mAb. Molecular weight markers are shown at Left. An arrowhead and an asterisk indicate CysLT1R-Myc dimers and GPR17-YFP dimers, respectively. Results are representative from 3 independent experiments. IP, immunoprecipitation; WB, Western blot analysis; IgH, Ig heavy chains. (C) Human peripheral blood monocytes were stained with anti-human GPR17 N-terminal peptide Ab and anti-human CysLT1R C-terminal peptide Ab. Immunoreactivities to GPR17 and CysLT1R were visualized with secondary Abs conjugated with Cy3 (red) for GPR17 and with FITC (green) for CysLT1R. Colocalization is visualized in the merged image as yellow-white, because of overlap of the colors for Cy3 and FITC. Results are representative from 4 independent experiments.

CysLT1R and GPR17 Colocalize on Human Monocytes.

To examine if GPR17 and CysLT1R colocalize on the plasma cell membrane in primary cells, we stained, fixed, and permeabilized human monocytes with rabbit polyclonal anti-human GPR17 N-terminal peptide Ab and goat polyclonal anti-human CysLT1R C-terminal peptide Ab. Visualization with secondary Abs, Cy3-conjugated anti-rabbit IgG (red) and FITC-conjugated anti-goat IgG (green), showed discrete colors, while analysis by confocal immunofluorescence microscopy showed a pseudocolored yellow-white signal resulting from the merged immunostained images with red and green signals. Both GPR17 and CysLT1R were dominantly expressed on the plasma membrane and they were partially colocalized (Fig. 3C, merged image).

GPR17 Silencing in BMMΦs Increases CysLT1R Expression and Function.

To seek a regulatory function of GPR17 in primary cells, we used shRNA interference by the lentivirus system to knock down GPR17 expression in mouse bone marrow-derived macrophages (BMMΦs). This cell type expressed both GPR17 and CysLT1R as assessed by RT-PCR (Fig. 4A). Quantitative RT-PCR revealed that lentivirus infection with 2 of 5 shRNA constructs (TRCN124057 and TRCN124056) effectively suppressed the GPR17 mRNA expression in BMMΦs by about 70% and 40%, respectively (Fig. 4B). Mouse BMMΦs were then infected with lentivirus particles carrying a control vector or the GPR17-knockdown constructs and cultured for 72 h. FACS analysis with anti-CysLT1R peptide Ab (RB34) revealed that the cell surface expression of CysLT1R was enhanced 1.5- to 2-fold by the GPR17 knockdown with both constructs as compared with BMMΦs infected by control vector construct (Fig. 4C and Fig. S3A). In a parallel experiment, the LTD4-elicited calcium flux in the GPR17 knockdown BMMΦs was increased in a dose-dependent manner over a 1,000-fold range of the ligand concentrations as compared with BMMΦs infected by control vector construct (Fig. 4D and Fig. S3A). The sensitivity for a comparable signal was increased by about 1–2 logs for each of the knockdown constructs in BMMΦs (Fig. 4E). Importantly, ATP-elicited calcium flux (24) was similar in both GPR17 knockdown and control BMMΦs, suggesting that GPR17 does not affect calcium flux mediated by another intrinsic receptor (Fig. 4D, Lower panels). We also observed the enhanced CysLT1R response to a weaker CysLT1R ligand, LTC4, with GPR17 knockdown (Fig. S3B). These results reveal that GPR17 can negatively regulate the surface expression and function of the CysLT1R in macrophages.

Fig. 4.
View larger version:
Fig. 4.

Knockdown of GPR17 in BMMΦs increases CysLT1R expression and function. (A) RT-PCR analysis for GPR17 and CysLT1R in mouse BMMΦs. (B) Quantitative RT-PCR for mouse GPR17 in total RNA from BMMΦs 2 days after infection with lentiviral particles carrying pLKO1 vector (gray) or individual constructs (TRCN124057 and TRCN124056) of pLKO1-mouse GPR17 shRNA (black). (C) Cell surface expression of CysLT1R on mouse BMMΦs 3 days after infection with lentiviral particles carrying pLKO1 vector (dotted line) or pLKO1-mouse GPR17 shRNA (TRCN124057, thick black line). Isotype control staining is shown in shaded histogram. (D) Dose-dependent intracellular calcium flux response to LTD4 (Upper) or 50 μM of ATP (Lower) in mouse BMMΦs 3 days after infection with lentiviral particles carrying pLKO1 vector or pLKO1-mouse GPR17 shRNA (TRCN124057). Arrows indicate point of injection of LTD4 or ATP. Results are representative from 5 independent experiments. (E) Peak relative fluorescence of intracellular calcium mobilization by LTD4 in mouse BMMΦs infected with lentiviral particles carrying pLKO1 vector (open triangles) or individual constructs [TRCN124057 (closed circles) and TRCN124056 (closed squares)] of pLKO1-mouse GPR17 shRNA. Data represent the mean ± SE (n = 5–7) from 5 independent experiments. *, P < 0.01; #, P < 0.05 vs. control BMMΦs.

In contrast to the enhanced CysLT1R expression in our knockdown studies (Fig. 4C), cooverexpression of GPR17 did not affect the cell surface expression of CysLT1R (Fig. S1A and S2A). Not all potential G protein-coupled receptors expressed on the cell surface are associated with G proteins according to the allosteric ternary complex model (42). As we overexpressed CysLT1R under the control of a strong promoter, the chicken β-actin promoter, it is likely that most of the CysLT1R is not associated with G proteins, and thus not functional, but still immunoreactive. Perhaps GPR17 only interacts with the functional CysLT1R. It is also conceivable that cooverexpression of GPR17 may stabilize the CysLT1R localization on the cell surface by forming heterodimers. Indeed, cooverexpression of CysLT1R also did not affect the cell surface expression of GPR17 (Fig. S2A). The knockdown study avoids the problem of induced CysLT1R overexpression by decreasing GPR17 transcription in the presence of a native expression system for CysLT1R. In this setting, there was both increased membrane expression of CysLT1R and its G protein-coupled calcium flux.

Viability of a GPR17-Deficient Mouse Strain.

To demonstrate that dampening of the CysLT1R function was a physiological role of GPR17 in vivo, we obtained a GPR17-deficient mouse strain. Although this strain is commercially available (Deltagen), its phenotypic characterization has not been reported. The targeting vector was designed so that a lacZ-neo gene casette interrupts 76 bp of the coding region on exon 2 of the mouse GPR17 (Fig. S4A). The genotypes of the pups from intercrossing of heterozygotes (N7, C57BL/6) determined by PCR with genomic tail DNA using GPR17 gene-specific primers and a neo gene primer demonstrated the 440-bp and 299-bp bands of the mutant and WT alleles, respectively, as illustrated for 1 litter in Fig. S4B. The ratio, homozygotes:heterozygotes:WT, was consistent with the expected Mendelian frequency, and similar numbers of male and female homozygotes were produced. The GPR17-deficient mice developed normally and exhibited no apparent clinical abnormalities up to 9 months of age. Because mouse GPR17 is abundantly expressed in the brain (35), we examined whether the lacZ-neo gene casette insertion disrupted expression of the GPR17 mRNA in this tissue. By Northern blot analysis with total RNAs, a 6-kb band was detected in the brain of WT mice, whereas the band was not detected in the sample from GPR17-deficient mice (Fig. S4C).

GPR17 Deficiency in Mice Increases CysLT1R-Mediated Vascular Permeability in PCA.

We previously reported that the vascular leak accompanying IgE-dependent, mast cell-mediated PCA is highly dependent on the integrity of LTC4 synthase (23) and the CysLT1R (24). To demonstrate a physiologic regulatory role of GPR17 in this CysLT1R-mediated inflammatory response in vivo, we repeated such PCA studies in GPR17-deficient mice and their WT littermates. After local sensitization of ear mast cells with 10 ng of monoclonal IgE and i.v. challenge with hapten-specific antigen, the extravasation of plasma proteins associated with Evans blue dye was markedly and significantly increased in GPR17-deficient as compared to their sufficient littermates (Fig. 5). Furthermore, this protein leak was significantly (≈80%) blocked by pretreatment of the mice with the CysLT1R antagonist, MK-571. This enhanced vascular leak in GPR17-deficient mice was also evident when the dose of monoclonal IgE per ear was reduced by a log so that there was virtually no response in the WT littermates. Again, the vascular leak in GPR17-deficient mice was markedly and significantly suppressed by MK-571 (Fig. S4D).

Fig. 5.
View larger version:
Fig. 5.

GPR17-deficient mice show markedly increased CysLT1R-mediated vascular permeability in PCA. GPR17-deficient mice (KO) and their WT littermates (WT) were sensitized with 10 ng anti-DNP IgE or saline and challenged with 100 μg DNP-human serum albumin with 1% Evans blue dye. These mice had (black columns) or had not (white columns) been pretreated with MK-571. The net Evans blue dye extravasation is expressed as the mean ± SE (n = 8 mice per group) from 3 independent experiments. *, P < 0.01; #, P < 0.05.

Our findings of a lack of LTC4, LTD4, and LTE4 recognition by GPR17 transfectants are consistent with the previous observation by Heise et al. that there was no Cl− conductance in response to LTD4 in Xenopus oocytes when the cRNA for GPR17 was microinjected (17). Instead, we found that cotransfection of GPR17 eliminated CysLT1R binding and signaling in response to a full range of LTD4 concentrations without preventing expression at the membrane, as assessed by FACS analysis. Importantly, knockdown of GPR17 in mouse BMMΦs increased membrane expression of CysLT1R and increased the magnitude and sensitivity to LTD4-induced calcium flux. Finally, GPR17-deficient mice showed an enhanced vascular permeability in IgE-dependent PCA as compared to their WT littermates, and this enhanced response was MK-571 sensitive. These results indicate that GPR17 is a negative regulator of CysLT1R-mediated responses in vitro and in vivo.

In a recent study of another class of orphan G protein-coupled receptor, Levoye et al. reported that the orphan receptor GPR50 and the melatonin 1 (MT1) receptor formed a heterodimer and showed that coexpression of GPR50 and MT1 receptor specifically abolished MT1 receptor function in the transfectants (43). This inhibition was suppressed by deletion of a large portion of the C-terminal tail of GPR50 although the heterodimerization of GPR50 and MT1 receptor was retained, thereby suggesting that the C-terminal domain of GPR50 modulates a regulatory portion of MT1. These authors consider this finding to be an example of constitutive regulation of a receptor of the neuroendocrine system by a nonligand-binding G protein-coupled orphan receptor. Our observation that GPR17, an orphan receptor with homology to the CysLTRs, negatively regulates the expression and function of CysLT1R provides the initial example of such nonligand-dependent, constitutive regulation in the 5-LO/LTC4 synthase pathway. Importantly, we have validated this possibility by showing that in the absence of GPR17 the CysLT1R-mediated component of the PCA response is profoundly aggravated.

Previous SectionNext Section

Materials and Methods

cDNA Cloning of the Mouse and Human GPR17.

Methods to clone the full-length GPR17 cDNAs from mouse brain and human placenta cDNA libraries by PCR are described in SI Materials and Methods.

Cell Culture and Stable Expression of the Mouse CysLT1R With or Without Mouse GPR17.

Stable transfectants of the mouse CysLT1R with or without mouse GPR17 were established as described in SI Materials and Methods.

Intracellular Calcium Mobilization.

The 1321N1 or CHO cell transfectants and mouse BMMΦs were labeled with fura-2, stimulated with LTC4, LTD4, or LTE4, and assayed as described (20).

ERK Phosphorylation.

After 6 h of serum starvation, the CHO cell stable transfectants were stimulated with vehicle (ethanol) or LTD4 at 1 μM and 0.01 μM for 10 min at 37 °C and assayed as described in SI Materials and Methods.

[3H]LTD4 Specific Binding.

[3H]LTD4 binding to the microsomal membranes from the CHO cell transfectants was assayed as described (20).

Flow Cytometry.

To confirm the expression of epitope-tagged GPR17 and CysLT1R in 1321N1 cells, Alexa Fluor 647-conjugated mouse Penta-His mAb (Qiagen, 5 μg/mL) and Alexa Fluor 488-conjugated mouse anti-Myc tag mAb (4A6, Upstate, 5 μg/mL), respectively, was used. Rabbit polyclonal anti-CysLT1R peptide Ab (RB34, against the conserved sequence DEKNNTKCFEPPQNN of extracellular loop 3 of both mouse and human CysLT1R, 5 μg/mL) (37) and allophycocyanin-conjugated donkey anti-rabbit IgG were used on CHO cells stably expressing CysLT1R. Nonspecific rabbit and mouse IgG (Jackson Immunochemical) were used as controls. Analyses were performed on a FACSCanto flow cytometer (BD Biosciences), and data were analyzed with the FlowJo software.

Immunoprecipitation and Confocal Immunofluorescence Microscopy.

Methods for immunoprecipitation with transfectants and confocal immunofluorescence microscopy with human monocytes are described in SI Materials and Methods.

Preparation of Lentiviral Particles and Infection.

Five shRNA constructs for the mouse GPR17 in a pLKO1 vector (TRCN124054–8) were purchased from Open Biosystems. Infectious viral particles were prepared by cotransfection of HEK-293T cells with each shRNA construct, a packing vector (psPAX2), and an envelope vector (pMD2.G) according to the manufacturer's protocol. The viral stocks were titrated in HT1080 cells. Mouse BMMΦs were cultured from BALB/c mouse bone marrow cells as described (44). BMMΦs were incubated with viral particles in culture medium to achieve a multiplicity of infection of 10. After 24 h, the medium was replaced and the cells were cultured for an additional 48 h before the functional studies were performed.

Conventional and Quantitative RT-PCR.

Methods for conventional and quantitative RT-PCR are described in SI Materials and Methods.

GPR17-Deficient Mice and Northern Blot Analysis.

Detailed information of GPR17-deficient mice and methods for Northern blot analysis are described in SI Materials and Methods.

PCA.

PCA was performed in the ear of GPR17−/− and their WT littermates as described (24) except that the intradermal dose of mouse monoclonal anti-dinitrophenyl (DNP) IgE (Sigma) was reduced from 25 ng to 10 or 2.5 ng. The details are described in SI Materials and Methods.

Statistical Analysis.

Results were expressed as mean ± SE. Student's t test was used for the statistical analysis. A value of P < 0.05 was considered significant.

Previous SectionNext Section

Acknowledgments

We thank Tyler Hickman for technical assistance and Dr. Joshua Boyce for critical discussions of this work. These studies were supported by National Institutes of Health grants P01HL36110, R01HL82695, R01HL090630 (to Y.K.), and K08AI064226 (to B.B.).

Previous SectionNext Section

Footnotes

  • 1To whom correspondence may be addressed. E-mail: ykanaoka{at}rics.bwh.harvard.edu or fausten{at}rics.bwh.harvard.edu

  • Author contributions: A.M., K.F.A., and Y.K. designed research; A.M., B.B., and Y.K. performed research; A.M., B.B., K.F.A., and Y.K. analyzed data; and A.M., K.F.A., and Y.K. wrote the paper.

  • The authors declare no conflict of interest.

  • This article contains supporting information online at www.pnas.org/cgi/content/full/0905364106/DCSupplemental.

Previous Section
 

References

  1. ↵
    1. Dixon RA,
    2. et al.
    (1990) Requirement of a 5-lipoxygenase-activating protein for leukotriene synthesis. Nature 343:282–284.
    CrossRefMedline
  2. ↵
    1. Ferguson AD,
    2. et al.
    (2007) Crystal structure of inhibitor-bound human 5-lipoxygenase-activating protein. Science 317:510–512.
    Abstract/FREE Full Text
  3. ↵
    1. Rouzer CA,
    2. Matsumoto T,
    3. Samuelsson B
    (1986) Single protein from human leukocytes possesses 5-lipoxygenase and leukotriene A4 synthase activities. Proc Natl Acad Sci USA 83:857–861.
    Abstract/FREE Full Text
  4. ↵
    1. Yoshimoto T,
    2. Soberman RJ,
    3. Spur B,
    4. Austen KF
    (1988) Properties of highly purified leukotriene C4 synthase of guinea pig lung. J Clin Invest 81:866–871.
    MedlineWeb of Science
  5. ↵
    1. Ago H,
    2. et al.
    (2007) Crystal structure of a human membrane protein involved in cysteinyl leukotriene biosynthesis. Nature 448:609–612.
    CrossRefMedline
  6. ↵
    1. Martinez Molina D,
    2. et al.
    (2007) Structural basis for synthesis of inflammatory mediators by human leukotriene C4 synthase. Nature 448:613–616.
    CrossRefMedline
  7. ↵
    1. Anderson ME,
    2. Allison RD,
    3. Meister A
    (1982) Interconversion of leukotrienes catalyzed by purified gamma-glutamyl transpeptidase: Concomitant formation of leukotriene D4 and gamma-glutamyl amino acids. Proc Natl Acad Sci USA 79:1088–1091.
    Abstract/FREE Full Text
  8. ↵
    1. Shi ZZ,
    2. Han B,
    3. Habib GM,
    4. Matzuk MM,
    5. Lieberman MW
    (2001) Disruption of gamma-glutamyl leukotrienase results in disruption of leukotriene D4 synthesis in vivo and attenuation of the acute inflammatory response. Mol Cell Biol 21:5389–5395.
    Abstract/FREE Full Text
  9. ↵
    1. Lee CW,
    2. Lewis RA,
    3. Corey EJ,
    4. Austen KF
    (1983) Conversion of leukotriene D4 to leukotriene E4 by a dipeptidase released from the specific granule of human polymorphonuclear leucocytes. Immunology 48:27–35.
    MedlineWeb of Science
  10. ↵
    1. Habib GM,
    2. Shi ZZ,
    3. Cuevas AA,
    4. Lieberman MW
    (2003) Identification of two additional members of the membrane-bound dipeptidase family. FASEB J 17:1313–1315.
    Abstract/FREE Full Text
  11. ↵
    1. Dahlen SE,
    2. Hedqvist P,
    3. Hammarstrom S,
    4. Samuelsson B
    (1980) Leukotrienes are potent constrictors of human bronchi. Nature 288:484–486.
    CrossRefMedline
  12. ↵
    1. Weiss JW,
    2. et al.
    (1982) Bronchoconstrictor effects of leukotriene C in humans. Science 216:196–198.
    Abstract/FREE Full Text
  13. ↵
    1. Soter NA,
    2. Lewis RA,
    3. Corey EJ,
    4. Austen KF
    (1983) Local effects of synthetic leukotrienes (LTC4, LTD4, LTE4, and LTB4) in human skin. J Invest Dermatol 80:115–119.
    CrossRefMedlineWeb of Science
  14. ↵
    1. Israel E,
    2. et al.
    (1990) The effects of a 5-lipoxygenase inhibitor on asthma induced by cold, dry air. N Engl J Med 323:1740–1744.
    Abstract
  15. ↵
    1. Manning PJ,
    2. et al.
    (1990) Inhibition of exercise-induced bronchoconstriction by MK-571, a potent leukotriene D4-receptor antagonist. N Engl J Med 323:1736–1739.
    Abstract
  16. ↵
    1. Lynch KR,
    2. et al.
    (1999) Characterization of the human cysteinyl leukotriene CysLT1 receptor. Nature 399:789–793.
    CrossRefMedline
  17. ↵
    1. Heise CE,
    2. et al.
    (2000) Characterization of the human cysteinyl leukotriene 2 receptor. J Biol Chem 275:30531–30536.
    Abstract/FREE Full Text
  18. ↵
    1. Figueroa DJ,
    2. et al.
    (2001) Expression of the cysteinyl leukotriene 1 receptor in normal human lung and peripheral blood leukocytes. Am J Respir Crit Care Med 163:226–233.
    Abstract/FREE Full Text
  19. ↵
    1. Figueroa DJ,
    2. et al.
    (2003) Expression of cysteinyl leukotriene synthetic and signalling proteins in inflammatory cells in active seasonal allergic rhinitis. Clin Exp Allergy 33:1380–1388.
    CrossRefMedlineWeb of Science
  20. ↵
    1. Maekawa A,
    2. Kanaoka Y,
    3. Lam BK,
    4. Austen KF
    (2001) Identification in mice of two isoforms of the cysteinyl leukotriene 1 receptor that result from alternative splicing. Proc Natl Acad Sci USA 98:2256–2261.
    Abstract/FREE Full Text
  21. ↵
    1. Ogasawara H,
    2. et al.
    (2002) Characterization of mouse cysteinyl leukotriene receptors mCysLT1 and mCysLT2: Differential pharmacological properties and tissue distribution. J Biol Chem 277:18763–18768.
    Abstract/FREE Full Text
  22. ↵
    1. Hui Y,
    2. et al.
    (2001) The murine cysteinyl leukotriene 2 (CysLT2) receptor. cDNA and genomic cloning, alternative splicing, and in vitro characterization. J Biol Chem 276:47489–47495.
    Abstract/FREE Full Text
  23. ↵
    1. Kanaoka Y,
    2. Maekawa A,
    3. Penrose JF,
    4. Austen KF,
    5. Lam BK
    (2001) Attenuated zymosan-induced peritoneal vascular permeability and IgE-dependent passive cutaneous anaphylaxis in mice lacking leukotriene C4 synthase. J Biol Chem 276:22608–22613.
    Abstract/FREE Full Text
  24. ↵
    1. Maekawa A,
    2. Austen KF,
    3. Kanaoka Y
    (2002) Targeted gene disruption reveals the role of cysteinyl leukotriene 1 receptor in the enhanced vascular permeability of mice undergoing acute inflammatory responses. J Biol Chem 277:20820–20824.
    Abstract/FREE Full Text
  25. ↵
    1. Beller TC,
    2. et al.
    (2004) Cysteinyl leukotriene 1 receptor controls the severity of chronic pulmonary inflammation and fibrosis. Proc Natl Acad Sci USA 101:3047–3052.
    Abstract/FREE Full Text
  26. ↵
    1. Beller TC,
    2. Maekawa A,
    3. Friend DS,
    4. Austen KF,
    5. Kanaoka Y
    (2004) Targeted gene disruption reveals the role of the cysteinyl leukotriene 2 receptor in increased vascular permeability and in bleomycin-induced pulmonary fibrosis in mice. J Biol Chem 279:46129–46134.
    Abstract/FREE Full Text
  27. ↵
    1. Kim DC,
    2. et al.
    (2006) Cysteinyl leukotrienes regulate Th2 cell-dependent pulmonary inflammation. J Immunol 176:4440–4448.
    Abstract/FREE Full Text
  28. ↵
    1. Back M,
    2. et al.
    (2000) Antagonist resistant contractions of the porcine pulmonary artery by cysteinyl-leukotrienes. Eur J Pharmacol 401:381–388.
    CrossRefMedlineWeb of Science
  29. ↵
    1. Back M,
    2. et al.
    (2000) Prostacyclin modulation of contractions of the human pulmonary artery by cysteinyl-leukotrienes. Eur J Pharmacol 401:389–395.
    CrossRefMedlineWeb of Science
  30. ↵
    1. Lee TH,
    2. Austen KF,
    3. Corey EJ,
    4. Drazen JM
    (1984) Leukotriene E4-induced airway hyperresponsiveness of guinea pig tracheal smooth muscle to histamine and evidence for three separate sulfidopeptide leukotriene receptors. Proc Natl Acad Sci USA 81:4922–4925.
    Abstract/FREE Full Text
  31. ↵
    1. Maekawa A,
    2. Kanaoka Y,
    3. Xing W,
    4. Austen KF
    (2008) Functional recognition of a distinct receptor preferential for leukotriene E4 in mice lacking the cysteinyl leukotriene 1 and 2 receptors. Proc Natl Acad Sci USA 105:16695–16700.
    Abstract/FREE Full Text
  32. ↵
    1. Raport CJ,
    2. et al.
    (1996) New members of the chemokine receptor gene family. J Leukoc Biol 59:18–23.
    Abstract
  33. ↵
    1. Blasius R,
    2. Weber RG,
    3. Lichter P,
    4. Ogilvie A
    (1998) A novel orphan G protein-coupled receptor primarily expressed in the brain is localized on human chromosomal band 2q21. J Neurochem 70:1357–1365.
    MedlineWeb of Science
  34. ↵
    1. Ciana P,
    2. et al.
    (2006) The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinyl-leukotrienes receptor. EMBO J 25:4615–4627.
    CrossRefMedlineWeb of Science
  35. ↵
    1. Lecca D,
    2. et al.
    (2008) The recently identified P2Y-like receptor GPR17 is a sensor of brain damage and a new target for brain repair. PLoS ONE 3:e3579.
    CrossRefMedline
  36. ↵
    1. McGraw DW,
    2. et al.
    (2006) Airway smooth muscle prostaglandin-EP1 receptors directly modulate beta2-adrenergic receptors within a unique heterodimeric complex. J Clin Invest 116:1400–1409.
    CrossRefMedlineWeb of Science
  37. ↵
    1. Jiang Y,
    2. Borrelli LA,
    3. Kanaoka Y,
    4. Bacskai BJ,
    5. Boyce JA
    (2007) CysLT2 receptors interact with CysLT1 receptors and down-modulate cysteinyl leukotriene dependent mitogenic responses of mast cells. Blood 110:3263–3270.
    Abstract/FREE Full Text
  38. ↵
    1. White JH,
    2. et al.
    (1998) Heterodimerization is required for the formation of a functional GABA(B) receptor. Nature 396:679–682.
    CrossRefMedline
  39. ↵
    1. Xu H,
    2. et al.
    (2004) Different functional roles of T1R subunits in the heteromeric taste receptors. Proc Natl Acad Sci USA 101:14258–14263.
    Abstract/FREE Full Text
  40. ↵
    1. Yanai K,
    2. Maeyama K,
    3. Nakahata N,
    4. Nakanishi H,
    5. Watanabe T
    (1992) Calcium mobilization and its desensitization induced by endothelins and sarafotoxin in human astrocytoma cells (1321N1): Comparison of histamine-induced calcium mobilization. Naunyn Schmiedebergs Arch Pharmacol 346:51–56.
    CrossRefMedlineWeb of Science
  41. ↵
    1. Mellor EA,
    2. Austen KF,
    3. Boyce JA
    (2002) Cysteinyl leukotrienes and uridine diphosphate induce cytokine generation by human mast cells through an interleukin 4-regulated pathway that is inhibited by leukotriene receptor antagonists. J Exp Med 195:583–592.
    Abstract/FREE Full Text
  42. ↵
    1. Lefkowitz RJ,
    2. Cotecchia S,
    3. Samama P,
    4. Costa T
    (1993) Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins. Trends Pharmacol Sci 14:303–307.
    CrossRefMedline
  43. ↵
    1. Levoye A,
    2. et al.
    (2006) The orphan GPR50 receptor specifically inhibits MT1 melatonin receptor function through heterodimerization. EMBO J 25:3012–3023.
    CrossRefMedlineWeb of Science
  44. ↵
    1. Hume DA,
    2. Gordon S
    (1983) Optimal conditions for proliferation of bone marrow-derived mouse macrophages in culture: The roles of CSF-1, serum, Ca2+, and adherence. J Cell Physiol 117:189–194.
    CrossRefMedlineWeb of Science
« Previous | Next Article »Table of Contents

This Article

  1. Published online before print June 26, 2009, doi: 10.1073/pnas.0905364106 PNAS July 14, 2009 vol. 106 no. 28 11685-11690
  1. AbstractFree
  2. Figures Only
  3. » Full Text
  4. Full Text (PDF)
  5. Supporting Information

Readers Also Viewed

Classifications

Navigate This Article

Link: Advanced Search

This Week's Issue

  1. February 9, 2010, 107 (6)
  1. Current Issue
  1. Alert me to new issues of PNAS

Most