Leukotriene B₄ Receptor
Cloning and Intracellular Signaling

TAKEHIKO YOKOMIZO, KAZUYUKI MASUDA, KAZUHIKO KATO, AKIKO TODA, TAKASHI IZUMI, and TAKAO SHIMIZU

Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Tokyo, Tokyo, Japan

	INTRODUCTION

TOP INTRODUCTION BIOSYNTHESIS AND METABOLIC... CLONING AND STRUCTURE OF... PRIMARY STRUCTURES OF BLT... TISSUE DISTRIBUTION OF BLT STRUCTURE OF HUMAN BLT... CHEMOTACTIC ACTIVITY OF LTB4... CONCLUSIONS REFERENCES

Leukotriene B₄ (LTB₄) is a potent chemotactic compound that is synthesized from arachidonic acid, by the action of 5-lipoxygenase and leukotriene A₄ hydrolase (1). While 5-lipoxygenase is present to a limited extent in leukocytes (4), LTA₄ hydrolase is present ubiquitously in many tissues including the lung, kidney, spleen, and gastrointestinal tract (5). Cytosolic phospholipase A₂ (cPLA₂) plays a major role in releasing arachidonic acid from membrane phospholipids (6). We have cloned a human LTB₄ receptor (BLT), a G protein-coupled receptor (GPCR), by subtraction technology using retinoic acid-differentiated HL-60 cell cDNA libraries (7). In this article, we present data on the structure, signal transduction, and chemotactic activity of BLT.

	BIOSYNTHESIS AND METABOLIC INACTIVATION OF LTB4

TOP INTRODUCTION BIOSYNTHESIS AND METABOLIC... CLONING AND STRUCTURE OF... PRIMARY STRUCTURES OF BLT... TISSUE DISTRIBUTION OF BLT STRUCTURE OF HUMAN BLT... CHEMOTACTIC ACTIVITY OF LTB4... CONCLUSIONS REFERENCES

cPLA₂ is a cytosolic enzyme with a CalB domain and a phosphorylation site for mitogen-activated protein kinase (MAP kinase). Although various types of phospholipases are present, the enzyme appears to play a dominant role in releasing arachidonic acid and eicosanoid production on cell stimuli (8, 9). When peritoneal macrophages or neutrophils are stimulated with Ca ionophore or natural ligands (platelet-activating factor [PAF], formyl-methionyl-leucyl-phenylalanine [fMLP], etc.), cPLA₂-null mice failed to produce LTB₄, while wild-type mice produced high amounts of LTB₄ (10-100 ng/10⁶ cells). LTB₄ is also produced in the kidney, lung, and small intestine under inflammatory conditions, where 5-lipoxygenase is practically negligible. The production of LTB₄ is dependent on the infiltration of neutrophils or macrophages rich in 5-lipoxygenase.

Thus produced, LTB₄ is metabolized essentially by two pathways: one is -oxidation and the other is oxidation of the 12-hydroxy moiety. -Oxidation is a predominant route for the metabolic inactivation in human neutrophils (10, 11), while the oxidation of the 12-hydroxy function of LTB₄ occurs in the kidney, lung, and liver (12, 13). We have isolated LTB₄ 12-hydroxydehydrogenase from kidney of various species (14, 15). The product, 12-keto-LTB₄, was at least 100 times less potent than LTB₄ in increasing [Ca²⁺]_i in human leukocytes. However, one finding has indicated that the enzyme is also active on 15-keto-prostaglandins, yielding 13,14-dihydro-15-keto-prostaglandins (16). Although prostaglandin (PG) metabolites are preferable substrates, the bifunctional enzyme plays important roles in inactivation of PGs and LTB₄ in kidney and liver. The synthesis and metabolism of LTB₄ are illustrated in Figure 1.

View larger version (21K): [in this window] [in a new window]   Figure 1.   Biosynthesis and metabolism of LTB4. LOX = lipoxygenase; HD = hydroxydehydrogenase. The compound between 12-keto-LTB4 and 10,11,14,15-tetrahydro-12-keto-LTB4 has not been identified (14).

	CLONING AND STRUCTURE OF HUMAN BLT

TOP INTRODUCTION BIOSYNTHESIS AND METABOLIC... CLONING AND STRUCTURE OF... PRIMARY STRUCTURES OF BLT... TISSUE DISTRIBUTION OF BLT STRUCTURE OF HUMAN BLT... CHEMOTACTIC ACTIVITY OF LTB4... CONCLUSIONS REFERENCES

We used a homology-based search technique, with sequences of PG receptors, and a Xenopus oocyte expression system to isolate LT receptors; the effort took almost 10 yr. We cloned BLT by another strategy, a cDNA subtraction method (7). The strategy of the subtraction cloning is summarized in Figure 2. About 100 clones were thus isolated and sequenced (Table 1). An orphan GPCR was present among the 100 genes, and it was later found to be BLT by the following criteria: (1) by transient expression of BLT in Cos-7 cells and HEK293 cells, a specific LTB₄ binding was observed; (2) by Scatchard analysis using Cos-7 cell membranes, the K_d value was about 0.15 nM, which is in the same range as the K_d value of differentiated HL-60 cells; (3) displacement of [³H]LTB₄ binding by various eicosanoid analogs showed a pattern similar to previously described pharmacological data (Figure 3); and (4) several CHO cell lines stably expressing BLT responded to LTB₄ and caused signaling by multiple pathways including adenylate cyclase inhibition, [Ca²⁺]_i elevation, and activation of mitogen-activated protein kinases. All these data strongly suggest that the isolated GPCR is a BLT (Figure 4). We should note that this receptor was previously reported to be a novel P2 purinoceptor, P2Y₇, which responds to ATP when expressed in Cos-7 cells (17). We expressed this receptor in C6-15 glioma cells with a negligible amount of intrinsic purinoceptors, and examined the cellular response to ATP or LTB₄. As a result, these transfected cells did not respond to ATP up to 1 µM, but responded to 10 nM LTB₄ (7), indicating that it is not a purinoceptor. The identification of the gene as a BLT was confirmed by other groups (18, 19), and at a meeting on purinoceptors (20).

View larger version (26K): [in this window] [in a new window]   Figure 2.   Subtraction technology. cDNA from retinoic acid-differentiated HL-60 cells was subtracted, using that from undifferentiated cells. Each cDNA was digested by a 4-base cutter (RsaI) and blunt ended prior to subtraction. Single-stranded tester cDNAs [RA(+)] in separate tubes were ligated to specially designed linkers (black and gray boxes), and hybridized to single-stranded driver cDNAs [RA(-)]. The second hybridization was performed in one tube without prior heat denaturation. After blunting by Klenow fragment, RA-specific cDNAs were amplified by suppressive PCR. In this case, only the cDNA fragments with different linkers of 5' and 3' ends can be amplified. The DNA fragments with the same linkers on both sides cannot be amplified by PCR because these linkers are self-complementary. The amplified DNA was sequenced, and a search for homologous genes in the database was performed.

View larger version (24K): [in this window] [in a new window]   Figure 3.   Displacement curve. Binding of 0.25 nM [3H]LTB4 to the membrane fractions of BLT-expressing Cos-7 cells was examined in the presence and absence of various concentrations of eicosanoids (means, n = 4) (7). The IC50 values of the following compounds were more than 10 µM: PGD2, PGE2, PGF2, 5(S)-HETE, 5(R)-HETE, 15(S)-HETE, LTD4, and 5-oxo-ETE.

View larger version (44K): [in this window] [in a new window]   Figure 4.   Primary structure of BLT. Primary structure of human BLT (7, 18). BLT is a putative seven transmembrane-type receptor with two possible N-glycosylation sites and several possible phosphorylation sites by protein kinase C (arrows).

	PRIMARY STRUCTURES OF BLT FROM VARIOUS ANIMALS

TOP INTRODUCTION BIOSYNTHESIS AND METABOLIC... CLONING AND STRUCTURE OF... PRIMARY STRUCTURES OF BLT... TISSUE DISTRIBUTION OF BLT STRUCTURE OF HUMAN BLT... CHEMOTACTIC ACTIVITY OF LTB4... CONCLUSIONS REFERENCES

We and others have cloned BLTs from human (7), guinea pig (21, 22), mouse (23, 24), and rat (25). Table 2 shows the similarities of the sequences of BLTs from these four species. Two possible N-glycosylation sites and several possible sites of phosphorylation by protein kinase C were well conserved among these four species. The homology was higher in the putative transmembrane (TM) domains, especially in TM2, -3, and -7. The amino acids in the intracellular loops and tail were also conserved, especially in the third intracellular loop and the proximal portion of the cytoplasmic tail. The third intracellular loop contains a unique cluster of basic amino acid residues (Figure 4), suggesting that this region might be involved in LTB₄-specific signal transduction. It has only low homology (less than 20%) with prostaglandin receptors.

	TISSUE DISTRIBUTION OF BLT

TOP INTRODUCTION BIOSYNTHESIS AND METABOLIC... CLONING AND STRUCTURE OF... PRIMARY STRUCTURES OF BLT... TISSUE DISTRIBUTION OF BLT STRUCTURE OF HUMAN BLT... CHEMOTACTIC ACTIVITY OF LTB4... CONCLUSIONS REFERENCES

BLT is almost exclusively expressed in peripheral leukocytes, with some expression in the thymus and spleen in humans. There are several transcripts different in size, depending on the tissue (7). The nature of the other bands seen by Northern blotting has remained unclear. In guinea pig and rat, the highest expression was in leukocytes, and the weaker expression was in lung and spleen. In mice, peripheral eosinophils and casein-elicited peritoneal macrophages also express BLT message as much as neutrophils, suggesting novel roles of LTB₄ in eosinophil functions (23). Human BLT has been reported to act as a coreceptor for macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains (26) in the same manner as several chemokine receptors, CCR5 (27), and CXCR4 (33). BLT expressed on human macrophages can be a target for the inhibition of HIV entry, and various BLT antagonists should be examined for their ability to prevent HIV infection.

	STRUCTURE OF HUMAN BLT GENE

TOP INTRODUCTION BIOSYNTHESIS AND METABOLIC... CLONING AND STRUCTURE OF... PRIMARY STRUCTURES OF BLT... TISSUE DISTRIBUTION OF BLT STRUCTURE OF HUMAN BLT... CHEMOTACTIC ACTIVITY OF LTB4... CONCLUSIONS REFERENCES

We have isolated genomic clones containing human or mouse BLT gene and are analyzing the structure and mechanism of the transcriptional regulation of BLT. The human BLT gene is reported to be located on chromosome 14q11.2-q12 (34). The human BLT gene is relatively small (about 5 kb in length), and consists of three exons (K. Kato and T. Yokomizo, unpublished observation, 1999). The open reading frame of BLT is in exon 3, and two distinct 5' untranslated regions of HL-5 and -1 (human BLT cDNA [7]) are in exon 1 and 2, respectively. Analyses of the promoter functions of BLT are now ongoing to reveal the molecular mechanism of the unique expression pattern of BLT.

	CHEMOTACTIC ACTIVITY OF LTB4 AND BLT

TOP INTRODUCTION BIOSYNTHESIS AND METABOLIC... CLONING AND STRUCTURE OF... PRIMARY STRUCTURES OF BLT... TISSUE DISTRIBUTION OF BLT STRUCTURE OF HUMAN BLT... CHEMOTACTIC ACTIVITY OF LTB4... CONCLUSIONS REFERENCES

LTB₄ is one of the most potent chemotactic compounds produced in macrophages and neutrophils. Once macrophages and neutrophils are activated, they produce several cytokines and inflammatory mediators, including LTB₄, and recruit additional neutrophils, eosinophils and macrophages to the inflammatory lesion. Previously, P. Hedqvist and collaborators clearly showed the chemotactic activity of this compound, using a hamster cheek pouch preparation (35, 36). We tried to repeat this experiment, using CHO-BLT cells in a Boyden chamber assay (Figure 5A). A filter with 8-µm-diameter pores was coated with fibronectin, and CHO cells were placed in the upper chambers. As shown in Figure 5B, only BLT-carrying cells moved toward the ligand through the 8-µm pores (right), whereas vector-transfected cells did not show such activity. Next, we quantitatively measured chemotactic and chemokinetic activities with a multiwell plate reader. Instead of counting the cells, cells were stained with Diff-Quik (International Reagents, Japan) and the absorbance at 595 nm was measured (Figure 6). Both chemotactic activity and chemokinetic activity showed bell-shaped dose responses, and they were completely blocked by pretreatment of the cells with pertussis toxin (PTX, 100 ng/ml). These data suggest that G-inhibitory (G_i) proteins are required for LTB₄-dependent chemotactic and chemokinetic activities. To our surprise, these data show that CHO cells have an intrinsic ability to move. Once the chemoattractant receptors were introduced, the cells would migrate toward the gradients of the ligands. It will be important to determine the manner in which BLT signals chemotaxis in CHO cells (dominant negative transfection, antibody injection, etc.) and to explore the in vivo use of CHO cells expressing a chemotactic receptor of interest.

View larger version (21K): [in this window] [in a new window]   View larger version (133K): [in this window] [in a new window]   Figure 5.   Boyden chamber assay. (A) Principle of Boyden chamber assay. Polyvinyl pyrolidone (PVP)-free filters with 8-µm pores were precoated with fibronectin 13.3 (µg/ml). The lower chambers were filled with various concentrations of LTB4, and the cells were placed to the upper chambers. For the chemokinetic assay, the upper chambers were filled with the same concentrations of LTB4 as in the lower chambers. After 4 h of incubation, the migrated cells were fixed and stained with Diff-Quik. (B) Typical results of Boyden chamber assay. (Right panel ) CHO cells migrated to 1 nM LTB4. (Left panel ) In the case of CHO cells transfected with the empty vector, only the pores in the filter are observed.

View larger version (33K): [in this window] [in a new window]   View larger version (19K): [in this window] [in a new window]   Figure 6.   Chemotactic activities of CHO cells expressing BLT. (A) Photograph of the lower side of the filter in a Boyden chamber assay. (B) Dose-response curve of CHO cell chemotaxis. The number of migrated cells was quantified by measuring the OD595nm after Diff-Quik staining. Data points represent means ± SE (n = 4).

	CONCLUSIONS

TOP INTRODUCTION BIOSYNTHESIS AND METABOLIC... CLONING AND STRUCTURE OF... PRIMARY STRUCTURES OF BLT... TISSUE DISTRIBUTION OF BLT STRUCTURE OF HUMAN BLT... CHEMOTACTIC ACTIVITY OF LTB4... CONCLUSIONS REFERENCES

LTB₄ has been known for more than 20 years to be one of the most potent chemoattractants for leukocytes. LTB₄ plays important roles in host defense against bacterial infections by recruiting leukocytes to the infectious lesions. LTB₄ is also known as a candidate for initiation and progression of several inflammatory disorders including arthritis (37, 38), asthma (39), psoriasis (40), and ischemic nephritis (41). The synthesis and metabolism of LTB₄ have been summarized, and now we know the primary structure of the receptor, BLT, which will enable us to analyze the biological functions of LTB₄ in vitro and in vivo. In addition, identification of BLT also makes possible the rational design of BLT antagonists as antiinflammatory and anti-HIV drugs.

	Footnotes

Correspondence and requests for reprints should be addressed to Takehiko Yokomizo, M.D., Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: yokomizo{at}m.u-tokyo.ac.jp. URL: http://www.biochem2.m.u-tokyo.ac.jp/web/index.html

	References

TOP INTRODUCTION BIOSYNTHESIS AND METABOLIC... CLONING AND STRUCTURE OF... PRIMARY STRUCTURES OF BLT... TISSUE DISTRIBUTION OF BLT STRUCTURE OF HUMAN BLT... CHEMOTACTIC ACTIVITY OF LTB4... CONCLUSIONS REFERENCES

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