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WO2001088130A1 - Molecular cloning and identification of a human leukotriene-like receptor ltxr - Google Patents

Molecular cloning and identification of a human leukotriene-like receptor ltxr Download PDF

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Publication number
WO2001088130A1
WO2001088130A1 PCT/IB2000/001682 IB0001682W WO0188130A1 WO 2001088130 A1 WO2001088130 A1 WO 2001088130A1 IB 0001682 W IB0001682 W IB 0001682W WO 0188130 A1 WO0188130 A1 WO 0188130A1
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Prior art keywords
receptor
sequence
nucleic acid
heptahelix
cell
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PCT/IB2000/001682
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French (fr)
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Christer Owman
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Christer Owman
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Priority to AU11699/01A priority Critical patent/AU1169901A/en
Publication of WO2001088130A1 publication Critical patent/WO2001088130A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates, in general, to genes encoding receptors of the G-protein coupled receptor superfamily.
  • the present invention relates to molecular cloning and expression of a novel cDNA sequence that codes for a heptahelix receptor whose deduced amino acid sequence is related to leukotriene receptors.
  • the present invention also relates to the inflammatory mediators called leukotrienes (which are leukocyte chemoattractants) and other molecules that bind to G-protein coupled receptors, such as the inflammatory receptors.
  • the leukotrienes were discovered in the late nineteen-seventies and shown to comprise a family of widely distributed and biologically highly active inflammatory mediators originating from arachidonic acid through the 5- lipoxygenase pathway (Samuelson, 1983).
  • the name leukotriene derives from the notion that leukotrienes can be produced by leukocytes and that a conjugated triene forms a common structural entity. They are members of the group of eicosanoids, which also contains prostaglandins and thromboxane, for example.
  • the leukotrienes mediate their biological actions via several pharmacolocially-defined high affinity stereoselective membrane-bound receptors.
  • the receptors upon which the leukotrienes are defined are classified into three distinct groups based on the binding and actions of LTB4, LTC4, LTD4, and LTE4, alone or in combination with several antagonists (Alexander et al., 1999). There is pharmacological evidence, however, for one or more additional classes of receptors (Capra et al., 1997).
  • LTB4 is a pro-inflammatory chemoattractant derivative of arachidonic acid that is a product of the 5-lipoxygenase pathway and is formed from LTA4 by a specific hydrolase.
  • LTB4 is a potent activator principally of polymorphonuclear leukocytes (PMN). It has also been shown to be an activator of related myeloid cells and mast cells.
  • LTB4 is one of a number of chemoattractant substances, including the synthetic peptide formyl-methionyl- leucyl-phenylalanine (FMLP), complement component C5a, interleukin 8, and platelet-activating factor, which have similar biological effects and together regulate leukocyte function through interaction with independent receptors.
  • FMLP synthetic peptide formyl-methionyl- leucyl-phenylalanine
  • LTB4 induces chemotaxis, chemokinesis, and aggregation, causing the migration of neutrophils to sites of inflammation. At the site of inflammation, these cells degranulate, resulting in the release of lysosomal enzymes and other antibacterial and microbicidal agents. In addition, they produce superoxide as part of the host's defense response to pathogens. LTB4 also promotes adherence of neutrophils to endothelial cell walls, and, in concert with other mediators, can thereby amplify the inflammatory response.
  • LTB4 LTB4-induced PMN activation in vivo causes neutrophil invasion and accumulation in the lungs, peritoneal cavity, skin, and eyes of experimental animals. Receptor activation, therefore, has been proposed as an important event in pathologies where infiltrating neutrophils are present, for example inflammatory bowel disease (IBD) and psoriasis (Metters, 1995).
  • IBD inflammatory bowel disease
  • psoriasis Methodters, 1995
  • the chemoattractant function of LTB4 distinguishes it from the other (cysteine-containing) leukotrienes, whose main action is exerted in smooth musculature, vascular permeability, and epithelial secretions (Samuelson, 1983).
  • the cysteine-leukotrienes have therefore been implicated as important mediators in the pathophysiololgy of asthma (Drazen et al., 1998).
  • the human BLTR gene is located in chromosome 14 at q11.2-12 (Owman et al., 1996a), close to one of the regions (defined as 14q 11.2-13) reported to be linked to asthma-associated phenotypes (CSGA, 1997).
  • chemoattractant receptors Because of the continuing interest in chemoattractant receptors, there exists a need in the art for the identification of new receptors and the elucidation of their structure so that the function of these receptors in humans can be determined. In particular, there exists a need in the art for sequence information on such receptors, including DNA and amino acid sequences, to enable the isolation and characterization of particular receptors as well as structurally related receptors. The identification of receptors associated with pathogenic conditions would be particularly advantageous in assays for the identification of these conditions in susceptible individuals.
  • this invention aids in fulfilling these needs in the art. More particularly, this invention provides a heptahelix receptor derived from the human genome and which is expressed in human tissues and cells, such as kidney, adrenal gland, stomach, heart, cerebellum, placenta, bladder, lymphoid tissues, and especially liver, lung, and pancreas.
  • the present invention provides isolated heptahelix receptors and nucleic acids encoding these heptahelix receptors.
  • nucleic acids include (a) cDNA clones having a nucleotide sequence derived from the coding region of a native heptahelix gene of the invention; (b) nucleic acids that are capable of hybridizing to the cDNA clones of (a) under moderately stringent conditions and which encode biologically active heptahelix molecules; and (c) nucleic acids that are degenerate as a result of the genetic code to the DNA sequences defined in (a) and (b) and which encode biologically active heptahelix receptor molecules.
  • the present invention also provides recombinant expression vectors comprising the nucleic acids defined above, recombinant heptahelix receptor molecules produced using recombinant expression vectors, and processes for producing recombinant heptahelix receptor molecules using the expression vectors.
  • Recombinant cells containing the recombinant vectors of the invention are also provided, as are methods of using the recombinant cells.
  • the present invention further provides isolated or purified polypeptides and proteins encoded by the nucleic acids of the invention.
  • the invention provides an isolated protein having the amino acid sequence of SEQ ID NO:2, and is encoded by the nucleotide sequence of SEQ ID NO:1 , as follows: ltMACTGGCCCTGGCCCTG CC ATACCTTtMCCCTffiTA CTC ⁇ lOlACTGTCTGTTCTGAGGATA ⁇ CTCTAGCCCACTCATTMGTACAT ⁇
  • 201CTATACTTTTCT (aGGTTCCC GGCCTACTG GGGACTrM ⁇ TACTCTTAATGGCTTTCCT
  • heptahelix receptor compositions of the invention are useful in diagnostic assays for heptahelix receptors, as well as in raising antibodies to heptahelix receptors for use in diagnosis and therapy.
  • the heptahelix compositions can be used directly in therapy to bind or scavenge G-protein coupled receptor agonists, such as leukotrienes, thereby providing means for regulating the functional activity of the agonists.
  • the heptahelix receptor of the invention is, among other things, a leukotriene receptor for leukotriene B4, the invention is useful for detecting LTB4, for example, by immunoassay for heptahelix polypeptides of the invention or by detection of all or part of the polynucleotides encoding the receptor of the invention, such as hybridization assays or amplification reactions.
  • the heptahelix receptor of the invention belongs to a family of receptors that are used by the human immunodeficiency virus (HIV) during its fusion, entry, and infection of human target cells
  • the invention can be useful for preventing infection of such cells by HIV, or by any other virus using the heptahelix receptor of the invention during the infection process.
  • HIV human immunodeficiency virus
  • the heptahelix receptor compositions can be used directly in therapy to bind or scavenge chemoattractants, thereby providing a means for regulating the immune activities of chemoattractants.
  • the heptahelix receptor of the invention can be used to regulate the immune activities of chemoattractants associated with asthma.
  • this invention provides a method for lowering the levels of active leukotriene B4 in a mammal in need thereof, which comprises administering to the mammal a leukotriene B4-lowering amount of a leukotriene B4 receptor comprising the sequence of amino acids of SEQ ID NO:2.
  • this invention provides a method for assaying a ligand or an antagonist or agonist for said ligand.
  • the method comprises: (A) providing a heptahelix receptor of the invention or a fragment thereof comprising a binding domain for the ligand, antagonist, or agonist;
  • the receptor can be in an external cell membrane of a host cell transfected or transduced with DNA encoding the receptor.
  • binding can be detected by intracellular calcium level in the host cell.
  • Figure 1 depicts the nucleotide and amino acid sequences of the cloned LTXR gene (cDNA) and receptor, respectively.
  • the cloned cDNA is 2.8 kb in length, and encodes a 389 amino acid long protein with seven putative transmembrane domains (designated as roman numerals I - VII).
  • Figure 2 depicts the sequencing strategy used to determine the nucleotide sequence of the LTXR cDNA. Arrows indicate approximate location and length of overlapping subclones used in sequencing, as well as the relative direction of sequencing. Sequencing was performed using Sp6 and T7 primers on exo-deleted subclones.
  • Figure 3 shows a representative experiment using HeLa (human uterine) cells transfected with BLTR cDNA to transiently express the corresponding receptor protein in the cell membrane.
  • the cells are co- transfected with a calcium-sensitive reporter construct that emits a luminescence signal from aequorin when cellular calcium is elevated upon activation of the expressed receptor.
  • a calcium-sensitive reporter construct that emits a luminescence signal from aequorin when cellular calcium is elevated upon activation of the expressed receptor.
  • LTB4 extracellular buffer
  • EtOH ethanol solution
  • Figure 4 illustrates the same experimental set-up as in Figure 3, comparing the effects of various leukotrienes together with the buffer (pbs) and ethanol (EtOH) controls.
  • the HeLa cells are transiently expressing the novel LTXR receptor, the previously cloned BLTR receptor, as well as negative control cells not transfected with receptor cDNA. Only LTB4 elicits a response in the LTXR cells. LTB4 also stimulates the BLTR cells, but these also respond to a lower degree to LTC4 and, even less, to LTD4.
  • Figure 5 shows an alignment of the LTXR and the BLTR proteins.
  • the shadowed regions indicate identical amino acids. The greatest similarity is found within the putative transmembrane regions, but can also be found in some intra- and extra-cellular loops and the C-terminus.
  • Figure 6 shows a phylogenetic analysis of the LTXR protein.
  • ClustalX was used to make a protein alignment, that was analyzed with the maximum parsimony method in the PHYLIP package to produce an evolutionary tree. Numbers at the branch points are the results, in percent, of a bootstrap analysis and demonstrates the confidence of the inferred nodes.
  • Figure 7 shows expression profiling of LTXR using an mRNA array.
  • LTXR seems to be expressed at a low level in most tissues. Slightly higher expression can be seen in liver, kidney, adrenal gland, stomach, heart, cerebellum, placenta and bladder.
  • the organization of the array is as follows: A1 , whole brain; A2, cerebellum left; A3, substantia nigra; A4, heart; A5, esophagus; A6, colon transverse; A7, kidney; A8, lung; A9, liver; A10, leukemia HL-60; A11 , fetal brain; A12, yeast total RNA; B1 , cerebral cortex; B2, cerebellum right; B3, accumbens nucleus; B4, aorta; B5, stomach; B6, colon descending; B7, skeletal muscle; B8, placenta; B9, pancreas; B10, HeLa S3; B11 , fetal heart; B12, yeast tRNA; C1 , frontal lobe; C2, corpus callosum; C3, thalamus; C4, atrium left; C5, duodenum; C6, rectum; C7, spleen; C8, bladder; C9, adrenal gland; C10, leukemia K-5
  • D1 parietal lobe
  • D2, amygdala D3, pituitary gland
  • D4, atrium right D5, jejunum; D7, thymus; D8, uterus; D9, thyroid gland
  • D10 leukemia MOLT-4
  • D11 fetal liver
  • D12 E.
  • Figure 8 shows an expression analysis of LTXR by Northern blot. Transcripts were detected in liver, lung, and pancreas. Traces of expression were also detected in heart and placenta.
  • the most common surface receptors on eukaryotic cells belong to the superfamily of G-protein coupled heptahelix receptors. These receptors consist of a protein chain that passes in and out through the cell membrane seven times in a serpentine manner. The clinical significance of this receptor superfamily is illustrated by the fact that the vast majority of pharmaceutical drugs for patient use exert their function by interacting, one way or another, with these heptahelix receptors.
  • the subfamily of leukocyte chemoattractants and their cognate receptors have received particular attention during the last few years because it comprises receptors (chemokine receptors) that are necessary for the AIDS virus, HIV1 , to fuse with and infect the CD4-positive cells of the immune system (Fauci, 1996), and because they are involved in a large number of inflammatory diseases, the prototype being asthma (Drazen et al., 1998).
  • the inflammatory mediators/receptors involved more particularly in this disease are the leukotrienes.
  • Asthma is only one of several severe inflammatory conditions that involve the leukotrienes, which are among the most widely distributed inflammatory mediators in the body. They were discovered and chemically characterized in the nineteen-seventies (Samuelson, 1983) and have since been extensively analyzed for their physiological role and pathophysiological functions. They all derive from arachidonic acid through the lipoxygenase reaction cascade. Naturally, over the years there has been an extensive hunt for the specific receptors that receive the leukotriene signals on the cell surface. The problem had to await its solution until the first receptor was cloned in 1996.
  • CMKRL1 Shortly after CMKRL1 was published, an identical cDNA sequence was obtained by another group working with human erythroleukemia cells and, mainly based on radioligand binding experiments, the corresponding receptor was proposed to be a new member of the P2Y group of purinoceptors (Akbar et al., 1996). The interpretation of the binding data and of the functional assays on transfected cells, as well as the comparatively low relatedness of the deduced amino acid sequence to hitherto cloned P2Y receptors, warranted a reevaluation of the conclusion.
  • CMKRL1 was the first cloned leukotriene receptor.
  • the receptor clone R2 obtained from a human genomic library (Raport et al., 1996), on the other hand, deviates from the above-mentioned three DNA sequences in its 5' end. This difference led to the identification of a different methionine initiation site in the proposed coding sequence, leading to an amino terminus that was too long in the deduced protein sequence.
  • the leukotriene receptor as it was cloned by Owman et al. has two important implications: (1) the original publication (Owman et al., 1996a) reported that the corresponding receptor gene is located in human chromosome 14, in the q11_2-q12 region, which corresponds precisely to one of the loci for linkage to asthma-associated phenotypes in that chromosome (CSGA, 1997); and (2) there is experimental evidence that the receptor is able to function as a co-receptor for select primary isolates of the AIDS virus, HIV1 (Owman et al., 1998).
  • the present invention provides a novel nucleic acid encoding a putative seven-transmembrane spanning receptor.
  • the nucleic acid is a full-length cDNA which encodes a novel receptor having 37% peptide sequence identity with the original BLTR cloned by Owman et al.
  • the novel nucleic acid and encoded protein have been named LTXR, for leukotriene-like receptor X.
  • the novel LTXR cDNA the cloning of which is disclosed herein, encodes a human leukotriene receptor that responds to leukotriene B4 even more specifically than the previously cloned BLTR.
  • the sequence of the 3' end of the LTXR gene overlaps with the upstream non-coding region of BLTR, implying that the LTXR gene is located within the "asthma locus" of chromosome 14.
  • the nucleic acid can be any nucleic acid capable of encoding a heptahelix receptor of the invention.
  • the nucleic acid is cDNA.
  • the nucleic acid is mRNA.
  • the nucleic acid is DNA, such as genomic DNA and subgenomic DNA.
  • the nucleic acid is isolated or purified. "Isolated” or “purified” refers to a nucleic acid polymer in the form of a separate fragment or as a component of a larger nucleic acid construct, which has been derived from nucleic acid isolated from its natural environment at least once.
  • the heptahelix receptor nucleic acid is in substantially pure form, i.e., free of contaminating endogenous materials. It is also preferable that the nucleic acid be present in a quantity or concentration enabling manipulation by humans or machines, for example in a quantity sufficient to enable identification of the nucleotide sequence using standard biochemical methods. Isolated nucleic acids are preferably provided in the form of an open reading frame uninterrupted by internal, non-translated sequences (introns), which are typically present in eukaryotic genes. The sequence of a preferred isolated nucleic acid of the invention, a cDNA encoding LTXR, is shown in Fig. 1.
  • genomic DNA containing the relevant sequences can be used as a source of coding sequences.
  • subgenomic DNA can be used as a source of coding sequences.
  • mRNA or cDNA can be used as a source of coding sequences. The genomic location of a preferred DNA is described hereinafter.
  • nucleic acids according to the invention comprise a sufficient amount of nucleic acid sequence to encode a heptahelix receptor according to the invention.
  • nucleic acids of the invention comprise only enough heptahelix receptor-specific nucleic acid to encode a biologically active heptahelix receptor.
  • sequences of non-translated heptahelix-specific nucleic acid can be present 5' or 3' from the heptahelix receptor open reading frame. It is preferred that these non-translated sequences do not interfere with manipulation or expression of the coding region.
  • the nucleic acids of the invention comprise the heptahelix-encoding nucleotide sequence as the sole protein- or polypeptide- encoding sequence (i.e., as the sole open reading frame), while in other embodiments the nucleic acid comprises multiple open reading frames.
  • the open reading frames can encode multiple copies of the LTXR receptor, or it can comprise the one or more LTXR open reading frames along with open reading frames encoding other proteins or polypeptides, such as other heptahelix receptors.
  • nucleotide sequence refers to a heteropolymer of deoxyribonucleotides.
  • DNA sequences encoding the heptahelix receptors of this invention can be assembled from DNA fragments, such as short oligonucleotide linkers, or from a series of oligonucleotides, or by amplification reactions to provide a synthetic gene that is capable of being expressed in a recombinant transcriptional unit.
  • nucleic acid encoding the heptahelix receptor of the invention is not limited to the LTXR receptor clones just described.
  • additional cDNA clones can be isolated from cDNA libraries of other mammalian species by cross-species hybridization.
  • Mammalian heptahelix receptors of the present invention include, by way of example, primate, human, murine, canine, feline, bovine, ovine, equine, and porcine heptahelix receptors.
  • Mammalian heptahelix receptors can be obtained, for example, by cross-species hybridization using a single-stranded cDNA derived from the human heptahelix receptor DNA sequence as a hybridization probe to isolate heptahelix receptor cDNAs from mammalian cDNA libraries.
  • DNA encoding a heptahelix receptor can be covalently labeled with a detectable substance, such as a fluorescent group, a radioactive atom, or a chemiluminescent group, by methods well known to those skilled in the art.
  • a detectable substance such as a fluorescent group, a radioactive atom, or a chemiluminescent group
  • the invention provides probes and primers for detection and isolation of heptahelix receptors.
  • the probes and/or primers can comprise the entire coding sequence of a heptahelix receptor, or can comprise a portion of the heptahelix coding region.
  • the sequences of certain preferred probes and primers are disclosed herein, however, it is to be understood that any nucleotide sequence that is sufficiently specific for heptahelix receptor nucleic acids can be used. Selection of such sequences is well within the skill of the ordinary artisan, and can be based on the sequences disclosed herein.
  • the present invention provides vectors comprising a nucleic acid of the invention.
  • the vectors are recombinant expression vectors to amplify or express DNA encoding at least one heptahelix receptor.
  • Recombinant expression vectors are replicable nucleic acid constructs that have synthetic or cDNA-derived nucleic acid sequences encoding mammalian heptahelix receptor or bioequivalent analogs operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes.
  • a transcriptional unit for double-stranded DNA constructs generally comprises an assembly of (1 ) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers; (2) a structural or coding sequence, which is transcribed into mRNA and translated into the heptahelix receptor; and (3) appropriate transcription and translation initiation and termination sequences.
  • Such regulatory elements can include an operator sequence to control transcription and a sequence encoding suitable mRNA ribosomal binding sites.
  • the ability to replicate in a host usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated.
  • Promoters commonly used in recombinant microbial expression vectors include the ⁇ -lactamase (penicillinase) and lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281 :544, 1979), the tryptophan (tp) promoter system (Goeddel et al., Nucl. Acids Res. 8:40576, 1980; and EPA 36,776), and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982).
  • the transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells can be provided by viral sources.
  • promoters and enhancers are derived from polyoma, adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.
  • DNA sequences derived from the SV40 viral genome for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites, can be used to provide the other genetic elements required for expression of a heterologous DNA sequence.
  • DNA regions are operably linked when they are functionally related to each other.
  • DNA for a signal peptide secretory leader
  • a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.
  • operably linked means contiguous and, in the case of secretory leaders, contiguous and in reading frame.
  • Structural elements intended for use in yeast expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • recombinant protein is expressed without a leader or transport sequence, it can include an N- terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.
  • DNA sequences encoding mammalian heptahelix receptors which are to be expressed in a microorganism, preferably contain no introns that could prematurely terminate transcription of DNA into mRNA; however, premature termination of transcription may be desirable, for example, where it would result in mutants having advantageous C-terminal truncations. Due to code degeneracy, there can be considerable variation in nucleotide sequences encoding the same amino acid sequence. Other embodiments include sequences capable of hybridizing to the sequences of the provided cDNA under moderately stringent conditions and other sequences hybridizing or degenerate to those that encode biologically active heptahelix receptor polypeptides.
  • the present invention provides recombinant cells containing a nucleic acid according to the invention.
  • Recombinant heptahelix receptor DNA can be expressed or amplified in a recombinant expression system comprising a substantially homogenous monoculture of suitable host microorganisms, for example, bacteria such as E. coli, or yeast such as S. cerevisiae, which have stably integrated (by transduction or transfection) a recombinant transcriptional unit into chromosomal DNA, or carry the recombinant transcriptional unit as a component of a resident plasmid or other extrachromosomal element.
  • suitable host microorganisms for example, bacteria such as E. coli, or yeast such as S. cerevisiae, which have stably integrated (by transduction or transfection) a recombinant transcriptional unit into chromosomal DNA, or carry the recombinant transcriptional unit as a component of a resident plasmid or other extrachromosomal
  • Transformed host cells are cells that have been transduced or transfected with heptahelix receptor vectors constructed using recombinant DNA techniques.
  • Transformed host cells ordinarily express heptahelix receptor, but host cells transformed for purposes of cloning or amplifying heptahelix receptor DNA do not need to express heptahelix receptor.
  • Expressed heptahelix receptor will be deposited in the cell membrane or secreted into the culture supernatant, depending on the heptahelix receptor DNA selected.
  • Suitable host cells for expression of heptahelix receptor include prokaryotes, yeast, or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram-negative or gram-positive organisms, for example E.
  • coli or bacilli Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems can also be employed to produce heptahelix receptor using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985), the relevant disclosure of which is hereby incorporated by reference.
  • Recombinant heptahelix receptor proteins can also be expressed in yeast hosts, preferably from the Saccharomyces species, such as S. cerevisiae. Yeast of other genera, such as Pichia or Kluyveromyces, can also be employed. Yeast vectors will generally contain an origin of replication from the 2 ⁇ yeast plasmid or an autonomously replicating sequence (ARS), promoter, DNA encoding heptahelix receptor, sequences for polyadenylation, transcription, termination, and a selection gene. Suitable promoter sequences in yeast vectors include the promoters for metallothionein, 3- phosphoglycerate kinase (Hitzeman et al. J. Biol. Chem.
  • glycolytic enzymes Hess et al. J. Adv. Enzyme Reg. 7:149, 1968); and Holland et al., Biochem. 17:4900, 1978
  • enolase glyceraldehyde-3- phosphate dehydrogenase
  • hexokinase hexokinase
  • pyruvate decarboxylase phosphofructokinase
  • glucose-6-phosphate isomerase 3-phosphoglycerate mutase
  • pyruvate kinase phosphoglucose isomerase
  • glucokinase glucokinase
  • mammalian or insect cell culture systems are also advantageously employed to express recombinant protein.
  • Expression of recombinant proteins in mammalian cells is particularly preferred because such proteins are generally correctly folded, appropriately modified, and completely functional.
  • suitable mammalian host cell lines include the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175, 1981 ), and other cell lines capable of expressing an appropriate vector including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines.
  • Mammalian expression vectors can comprise nontranscribed elements, such as an origin of replication, a suitable promoter, and an enhancer linked to the gene to be expressed, and other 5' or 3' nontranslated sequences, such as ribosome binding sites and transcriptional termination sequences.
  • nontranscribed elements such as an origin of replication, a suitable promoter, and an enhancer linked to the gene to be expressed, and other 5' or 3' nontranslated sequences, such as ribosome binding sites and transcriptional termination sequences.
  • Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).
  • heptahelix receptor refers to proteins and polypeptides having amino acid sequences that are substantially similar to the native, mature, mammalian heptahelix receptor amino acid sequence of the invention, and which are biologically active.
  • LTXR chemoattractant receptor
  • temV'mature means a protein expressed in a form lacking a leader sequence as may be present in full-length transcripts of a native gene.
  • heptahelix receptor includes, but is not limited to, analogs or subunits of native proteins having seven membrane-spanning or hydrophobic regions and which exhibit at least some biological activity in common with LTXR.
  • soluble heptahelix receptor constructs which possess the seven hydrophobic regions (and are secreted from the cell), and retain the ability to bind chemoattractants are deemed to be heptahelix receptors of the invention.
  • heptahelix receptor refers generically to mammalian heptahelix receptor.
  • heptahelix receptor means all forms of heptahelix receptor, including mutants and analogs that possess heptahelix receptor biological activity.
  • Soluble heptahelix receptor refers to proteins and polypeptides, or substantially equivalent analogs, having an amino acid sequence corresponding to all or part of the heptahelix receptor, and which are biologically active in that they bind to chemoattractants and other related ligands. Soluble heptahelix receptors include polypeptides that vary from those sequences by one or more substitutions, deletions, or additions, and which retain the ability to bind chemoattractant or inhibit chemoattractant signal transduction activity via cell surface bound heptahelix receptor proteins.
  • Inhibition of chemoattractant signal transduction activity can be determined by transfecting cells with recombinant heptahelix receptor nucleic acids to obtain recombinant receptor expression. The cells can then be contacted with chemoattractant or other ligand and the resulting metabolic or signal transduction effects examined. If an effect results that is attributable to the action of the chemoattractant or other ligand, then the recombinant receptor has signal transduction activity. Procedures for determining whether a polypeptide has signal transduction activity include measurement of adenyl cyclase activity and intracellular calcium measurements.
  • chemoattractant or "chemokine-like" substances as used in the context of the present invention comprises both the “classical” chemoattractants and the chemokines.
  • the "classical” chemoattractants include but are not limited to LTB4, PAF, fMLP, C3a, and C5a.
  • all subtypes of the chemokines i.e., alpha, beta, etc., are within the context of the present invention.
  • Th e term "isolated” or “purified”, as used in the context of this invention to define the purity of heptahelix receptor protein or protein compositions means that the protein or protein composition is separated from its native environment.
  • the heptahelix receptor can be substantially free of other proteins of natural or endogenous origin.
  • the receptor can contain less than about 1 % by mass of protein contaminants residual of its native environment or production processes.
  • Such compositions can contain other proteins added as stabilizers, excipients, or co-therapeutics.
  • substantially similar when used to define either amino acid or nucleic acid sequences means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which is to retain biological activity of the heptahelix receptor as may be determined, for example, in a chemoattractant assay for heptahelix receptor binding.
  • nucleic acid subunits and analogs are "substantially similar" to the specific DNA sequences disclosed herein if: (a) the DNA sequence is derived from the coding region of native, mammalian heptahelix receptor gene of the invention; (b) the DNA sequence is capable of hybridization to DNA sequences of (a) and which encode biologically active heptahelix receptor molecules; or (c) DNA sequences that are degenerate as a result of the genetic code to the DNA sequences defined in (a) or (b) and which encode biologically active heptahelix receptor.
  • the heptahelix receptor of the invention can be obtained from natural sources or by recombinant techniques using eukaryotic or procaryotic host systems.
  • the invention provides recombinant cells containing nucleic acids encoding heptahelix receptors according to the invention.
  • "Recombinant” as used herein means that a protein is derived from recombinant (e.g., microbial or mammalian) expression systems.
  • Microbial refers to recombinant proteins made in bacterial or fungal (e.g., yeast) expression systems.
  • recombinant microbial defines a protein produced in a microbial expression system that is essentially free of native endogenous substances. Protein expressed in most bacterial cultures, e.g., E. coli, will be free of glycan. Protein expressed in yeast may have a glycosylation pattern different from that expressed in mammalian cells.
  • Bioly active as used throughout this specification as a characteristic of heptahelix receptors of the invention means that a particular molecule shares sufficient amino acid sequence similarity with embodiments of the present invention to be capable of binding chemoattractant or other ligand, transmitting a stimulus to a cell, for example, as a component of a hybrid receptor construct, or cross-reacting with anti-heptahelix receptor antibodies raised against heptahelix receptor from natural (i.e., non- recombinant) sources.
  • heptahelix receptor within the scope of the invention include various structural forms of the primary protein, which retain biological activity. Due to the presence of ionizable amino and carboxyl groups, for example, a heptahelix receptor protein can be in the form of acidic or basic salts, or can be in neutral form. Individual amino acid residues can also be modified by oxidation or reduction.
  • the primary amino acid structure can be modified by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups, and the like, or by creating amino acid sequence mutants.
  • Covalent derivatives can be prepared by linking particular functional groups to heptahelix receptor amino acid side chains or at the N- or C-termini.
  • Other derivatives of heptahelix receptor within the scope of this invention include covalent or aggregative conjugates of heptahelix receptor or its fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N- terminal or C-terminal fusions.
  • the conjugated peptide can be a signal (or leader) polypeptide sequence at the N-terminal region of the protein, which co-translationally or post-translationally directs transfer of the protein from its site of synthesis to its site of function inside or outside of the cell membrane or wall (e.g., the yeast ⁇ -factor leader).
  • Heptahelix receptor protein fusions can comprise peptides added to facilitate purification or identification of heptahelix receptor (e.g., poly-His).
  • Heptahelix receptors and derivatives can be used as immunogens, reagents in receptor-based immunoassays, or as binding agents for affinity purification procedures of chemoattractants or other binding ligands.
  • Heptahelix receptor derivatives can also be obtained by cross-linking agents, such as M-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, at cysteine and lysine residues.
  • Heptahelix receptor proteins can also be covalently bound through reactive side groups to various insoluble substrates, such as cyanogen bromide-activated, bisoxirane-activated, carbonyldiimidazole-activated, or tosyl-activated agarose structures, or by adsorbing to polyolefin surfaces (with or without glutaraldehyde cross-linking).
  • substrates such as cyanogen bromide-activated, bisoxirane-activated, carbonyldiimidazole-activated, or tosyl-activated agarose structures
  • heptahelix receptor can be used to selectively bind (for purposes of assay or purification) anti-heptahelix receptor antibodies or chemoattractant.
  • the present invention also includes heptahelix receptor, with or without associated native-pattern glycosylation.
  • Heptahelix receptor expressed in yeast or mammalian expression systems can be similar or slightly different in molecular weight and glycosylation pattern than the native molecules, depending upon the expression system.
  • Expression of heptahelix receptor DNAs in bacteria, such as E. coli provides non-glycosylated molecules.
  • Functional mutant analogs of mammalian heptahelix receptor having inactivated N-glycosylation sites can be produced by oligonucleotide synthesis and ligation or by site-specific mutagenesis techniques. These analog proteins can be produced in a homogeneous, reduced-carbohydrate form in good yield using yeast expression systems.
  • Heptahelix receptor derivatives can also be obtained by mutations of heptahelix receptor or its subunits.
  • a heptahelix receptor mutant as referred to herein, is a polypeptide homologous to heptahelix receptor, but which has an amino acid sequence different from native heptahelix receptor because of a deletion, insertion, or substitution.
  • Bioequivalent analogs of heptahelix receptor proteins can be constructed by, for example, making various substitutions of residues or sequences, or deleting terminal or internal residues or sequences, or deleting terminal or internal residues or sequences not needed for biological activity.
  • cysteine residues can be deleted (e.g., Cys178) or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation.
  • substitutions should be made conservatively; i.e., the most preferred substitute amino acids are those having physiochemical characteristics resembling those of the residue to be replaced.
  • the potential effect of the deletion or insertion on biological activity should be considered.
  • Substantially similar polypeptide sequences, as defined above, generally comprise a like number of amino acids.
  • deletions and substitutions will preferably result in homologously or conservatively substituted sequences, meaning that a given residue is replaced by a biologically similar residue.
  • conservative substitutions include substitution of one aliphatic residue for another, such as lie, Val, Leu, or Ala for one another, or substitution of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn.
  • Other such conservative substitutions for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known.
  • Mutations in nucleotide sequences constructed for expression of analog heptahelix receptor must, of course, preserve the reading frame phase of the coding sequences and preferably will not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hairpins, which would adversely affect translation of the receptor mRNA.
  • a mutation site may be predetermined, it is not necessary that the nature of the mutation per se be predetermined. For example, in order to select for optimum characteristics of mutants at a given site, random mutagenesis may be conducted at the target codon and the expressed heptahelix receptor mutants screened for the desired activity.
  • Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.
  • oligonucleotide- directed site-specific mutagenesis procedures can be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required.
  • Purified mammalian heptahelix receptors or analogs can be prepared by culturing suitable host/vector systems to express the recombinant translation products of the DNAs of the present invention, which are then purified from culture media or cell extracts.
  • supematants from systems that secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix.
  • a suitable affinity matrix can comprise a heptahelix or lectin or antibody molecule bound to a suitable support.
  • an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups.
  • the matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification.
  • RP-HPLC reverse-phase high performance liquid chromatography
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • Recombinant heptahelix receptor produced in bacterial culture can be isolated by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange, or size exclusion chromatography steps. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • Microbial cells employed in expression of recombinant the receptor can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
  • Heptahelix receptors synthesized in recombinant culture are characterized by the presence of non-human cell components, including proteins, in amounts and of a character which depend upon the purification steps taken to recover the receptor from the culture. These components ordinarily will be of yeast, prokaryotic, or non-human higher eukaryotic origin and preferably are present in innocuous contaminant quantities, on the order of less than about 1 percent by weight. Further, recombinant cell culture enables the production of heptahelix receptor free of proteins that may be normally associated with receptor as it is found in nature in its species of origin, e.g., in cells, cell exudates, or body fluids.
  • the preferred heptahelix receptor of the invention is a novel chemoattractant, which has turned out to have leukotriene (LT) receptor-like activity.
  • the present invention provides methods of using therapeutic compositions comprising an effective amount of soluble heptahelix receptor and a suitable diluent or carrier in the treatment of a mammal, including primate, human, murine, canine, feline, bovine, ovine, equine, or porcine species.
  • the invention includes methods for modulating heptahelix receptor-dependent responses in humans comprising administering an effective amount of soluble heptahelix receptor to a patient. Appropriate dosages can be determined in trials. The amount and frequency of administration will depend, of course, on such factors as the nature and severity of the indication being treated, the desired response, the condition of the patient, and so forth.
  • the invention provides methods of treating diseases or disorders associated with LTXR.
  • the disorder is asthma.
  • the methods can comprise administering the LTXR to an individual suffering from asthma.
  • the methods can comprise administering to an individual antibodies that specifically react to LTXR.
  • the methods can comprise administering at least one agonist of LTXR to an individual suffering from asthma.
  • the methods can comprise administering an agonist of LTXR to an individual suffering from asthma.
  • the methods include administering the selected compound in an amount sufficient to bring about the desired level of treatment of asthma.
  • the invention provides methods of modulating the activity of LTXR receptors.
  • the immunologically important phagocytes include polymorphonuclear leukocytes (e.g., neutrophils) and mononuclear phagocytes (e.g., monocytes and macrophages).
  • Phagocyte hypofunction is a cause of recurrent pyogenic infection.
  • neutrophils and monocytes respond to chemotactic factors by moving toward the source of infection, where they ingest microorganisms and kill them.
  • a main function of polymorphonuclear leukocytes and monocytes is to kill bacteria and other infectious agents by phagocytosis.
  • the first stage in the ingestion and digestion of a particulate substance by these cells involves the process of bringing the cells and the particles together, usually through chemotaxis. This response is an essential part of host defense against infection. The extensive migration and activity of these cells are manifested by inflammation at the site of injury or invasion of the host.
  • the heptahelix receptor of the invention is capable of modulating the chemoattractant effect of LTB4 chemotaxis. This can be demonstrated by carrying out an assay for neutrophil chemotaxis using LTB4 and the heptahelix receptor of the invention, as disclosed herein.
  • the heptahelix receptor of the invention it is possible to inhibit chemotaxis of granulocytes, monocytes, and macrophages.
  • the receptor of the invention is capable of modulating directional movement of cells. This invention makes it possible to inhibit inflammation associated with cell chemotaxis.
  • the receptor can be administered to a patient to inhibit the effects of chemotactic factors of bacterial or viral origin, or components of plasma activation systems, or factors elaborated by cells of the immune system.
  • Leukocyte response to an acute inflammatory stimulus involves a complex series of events, including adherence to endothelium near the stimulus. Inhibition of leukocyte adherence can be expected to reduce the degree of inflammation seen in conditions, such a septic shock and adult respiratory distress syndrome.
  • LTB4 promotes adherence of polymorphonuclear leukocytes (PMN), such as neutrophils, to endothelial cell walls. Since the heptahelix receptor of this invention can act as a receptor for LTB4, the receptor can effectively block such adherence.
  • PMN polymorphonuclear leukocytes
  • PMN cells can be incubated with a lipopolysaccharide- stimulated mononuclear leukocyte conditioned medium containing LTB4.
  • PMN adherence to nylon can be determined without the receptor of this invention, and then with the receptor of this invention.
  • Polymorphonuclear leukocyte (PMN) adherence to nylon can be readily demonstrated.
  • the compounds employed in the process of this invention are effective in blocking adherence of leukocytes and thereby aiding in reducing the degree of inflammation.
  • Mature phagocytes are in a metabolically dormant state. It is currently believed that recognition of certain objects and substances by phagocytes, such as the attachment of an ingestible particle to the cell surface, changes this situation, and the cell enters a stage of increased metabolic activity, which is referred to as metabolic or respiratory burst. The transition is associated with a series of characteristic changes, including the production of a superoxide anion. LTB4 is capable of producing a similar effect. In addition to its significance for phagocytic function related to inactivation of ingested microbes, activation of oxygen metabolism is a useful indirect marker for the ingestion process per se. It would be desirable to be able to modulate the effect of LTB4 on respiratory burst.
  • the heptahelix receptor employed in this invention is capable of modulating respiratory burst in stimulated polymorphonuclear leukocytes (PMN) as determined using these methods.
  • the heptahelix receptor used in the process of this invention is capable of reducing superoxide production and modulating respiratory burst in phagocytes, such as polymorphonuclear leukocytes and monocytes, and thereby aid in reducing the degree of inflammation.
  • granules in the cytoplasm of the cell fuse with the membrane of a vacuole that was formed around the foreign substance.
  • the granules discharge their contents into the vacuole. Some of this material ends up in the medium surrounding the phagocyte. Since the granules disappear during this process, it is called degranulation.
  • the granule contents include hydrolytic enzymes, lysozyme, bactericidal proteins, and, in the neutrophil, myleoperoxidase.
  • Degranulation can be assessed by measuring the rate of appearance of granule-associated enzymes in the extracellular medium.
  • PMN polymorphonuclear leukocytes
  • degranulation can be assayed by determining release of lysozyme.
  • LTB4 induces the release of lysosomal enzymes.
  • the heptahelix receptor employed in the process of this invention is capable of modulating the release of lysozyme from stimulated PMN and thereby aid in reducing the degree of inflammation.
  • the heptahelix receptor of the invention is capable of modulating the effects of leukotrienes, such as LTB4, on polymorphonuclear leukocytes and mononuclear phagocytes.
  • the receptor is capable of inhibiting cell chemotaxis.
  • the receptor can block adherence of cells.
  • the compounds can decrease oxidative damage to host tissues by phagocytes as evidenced by modulation of respiratory burst in cells stimulated by LTB4.
  • the receptor can modulate the effects of LTB4 on degranulation in stimulated phagocytes.
  • LTB4 Among the conditions that can be treated or alleviated by the inhibition of LTB4 are: sepsis, septic shock, endotoxic shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress, cachexia secondary to AIDS, rheumatoid arthritis, and gouty arthritis.
  • the more generic term “trauma” can be used.
  • the term “trauma” refers broadly to cellular attack by foreign bodies, such as microorganisms, particulate matter, chemical agents, and the like. Included among physical injuries are mechanical injuries, such as those caused by contact with sources of electrical injuries, such as those caused by contact with sources of electrical potential; and radiation damage caused, for example, by prolonged, extensive exposure to infrared, ultraviolet, or ionizing radiations.
  • LTB4 is also characteristically a product of blood phagocytes.
  • the leukotrienes bind to specific receptors on smooth muscle cells and cause prolonged bronchoconstriction.
  • the receptor of this invention is useful for alleviating this condition, including asthmatic bronchoconstriction.
  • EST clone 444- J19 (IMAGE ID 223218) was identified by comparing the previously cloned BLTR receptor (Owman et al., 1996a) to the NCBI EST database using searches with the BLAST algorithm (Altshul et al., 1990).
  • the IMAGE EST clone showing the highest level of identity with the BLTR sequence was obtained from the UK HGMP Resource Centre, Hinxton, Cambridge, England.
  • This clone had an almost 2.7 kb human DNA insert in a pT7T3D-Pac vector.
  • the clone was digested with Xhol and Notl to liberate the human insert, and the insert was purified on an agarose gel.
  • the purified insert was ligated to a Notl-digested mammalian expression vector, pcDNA3 (Invitrogen). After transfer of the insert to the pcDNA3 vector, the insert was analyzed in transient transfection assays in which calcium response after addition of potential ligands was monitored. However, no activation could be seen in these assays.
  • the missing C-terminal part of the gene was amplified from a human leukocyte cDNA library (HL4050AH from Clontech) using PCR and Pwo polymerase (Bohringer Mannheim). The following two primers were used:
  • PCR reactions were performed using one primer that was specific for a sequence in the EST clone and one primer specific for a sequence located within the BLTR leader sequence.
  • the PCR product was litigated to the TA-cloning vector pCR2.1 from Invitrogen.
  • the complete pcDNA3 construct was cleaved with BamHI and Kpnl.
  • the linearized DNA was used to make exodeletions using the Erase-a-Base System from Promega.
  • DNA was then prepared from 3 ml of overnight cultures from individual clones, according to the alkaline lysis method (Current Protocols in Molecular Biology). The DNA obtained was treated with RNase A for 30 min at 37°C, before being purified using the QIAquick PCR Purification K from Qiagen.
  • Fig. 1 The nucleotide sequence (SEQ ID NO:1 ) contains an open reading frame that encodes a novel heptahelix receptor, which has been named LTXR, for leukotriene-Iike receptor X.
  • LTXR novel heptahelix receptor
  • Fig. 2 The deduced amino acid sequence of the encoded LTXR receptor is presented in Fig. 1 as SEQ ID NO:2.
  • the cloning and sequencing strategy is depicted in Fig. 2.
  • the full-length cDNA (Fig. 1) contains an open reading frame with two in-frame methionines preceding the first membrane-spanning helix region and an in-frame stop signal. It is likely that the first methionine shown in Figure 1 is used for initiation of translation, since it conforms better to the optimal "Kozak sequence" (Kozak, 1986) for initiation of translation than does Met-22. However, it is also possible that Met-22 also might function as a translational start, but at a lower frequency (Kozak 1986).
  • the predicted polypeptide contains 389 amino acid residues, giving a calculated relative molecular mass of approximately 42 kDa.
  • the receptor protein shows several features common to the G-protein linked heptahelix receptors, namely (a) consensus sequence for one N-linked glycosylation (Asn-X-Ser/Thr) at Asn-41 in the N-terminal extracellular tail; (b) conserved cysteine residues in each of the first (Cys-125) and second (Cys-199) extracellular loops, providing the possibility to form a stabilizing disulfide bond in the protein structure; (c) proline residues in all (but the third) membrane- spanning regions thought to induce flexibility within the helix structures; and (d) a C-terminus with serine and threonine residues that might serve as substrate for serine/threonine protein kinases (Alexander et al., 1999; Owman et al., 1997).
  • Example 4 Cell Transfections
  • the new clone encoding a full length version of a previously unknown receptor (the above-mentioned EST fragment from the public DAN sequence repository had not previously been identified or characterized as a receptor, but merely existed as a piece of sequence in a data bank), was then tested in transient transfection assays to find ligands that could activate the receptor.
  • HeLa cells were transfected with the LTXR-pcDNA3 construct together with an aequorin-pcDNA3 reporter construct as follows:
  • DMEM Dulbecco's MEM
  • - DNA was prepared using the DNA midiprep kit from Genomed. Then, 0.9 ⁇ g of LTXR-pcDNA3 and 0.4 ⁇ g aequorin-pcDNA3 were mixed with 100 ⁇ l of serum-free medium (OptiMEM from Gibco/BRL and 0.5% penicillin, 0.5% streptomycin, 1% Glutamax) and 5 ⁇ l of Plus-reagent in an eppendorff tube.
  • serum-free medium OptiMEM from Gibco/BRL and 0.5% penicillin, 0.5% streptomycin, 1% Glutamax
  • the cells were incubated with the DNA mixture at 37°C for 5.5 and 6h. Half of the transfection solution was then removed and 3 ml of medium was added to each well (DEM and 10% FSB, 1% glutamax, 0.5% penicillin, 0.5% streptomycin).
  • the activity of the cloned cDNA was assayed using recombinant cells expressing the LTXR protein. More specifically, changes in cellular calcium flow and resulting cellular calcium levels were measured using a LUMIstar luminometer from Lab Vision, using a 96-well format. The assay is based on the fact that, when cellular calcium levels are elevated, for example as a consequence of receptor activation, aequorin together with the substance coelentrazine, emits a transient light signal that can be detected in a luminometer.
  • LTXR was activated by leukotriene B4.
  • the nature of the response in this system resembled that of the previously cloned receptor of leukotriene B4, BLTR.
  • the response differed in that the activation by LTB4 was exclusive with LTXR, whereas the receptor BLTR could be activated also by LTC4 and LTD4 (Fig. 4).
  • LTB4 was exclusive with LTXR
  • BLTR could be activated also by LTC4 and LTD4
  • Fig. 4 As a negative control, HeLa cells that had been mock transfected with an EGFP-pcDNA3 construct together with aequorin-pcDNA3 were also analyzed (Figs. 3 and 4).
  • Protein sequences of related human GPCRs and other known leukotriene receptors of different species were obtained from GenBank using BLAST (Altshul et al., 1990) and aligned using ClustalX version 1.8.
  • the maximum parsimony (PROTPARS) method was applied to the alignment using the PHYLIP package to produce the phylogenetic tree (with bootstrap resampling of 100 subreplicates for statistical significance evaluation).
  • LTXR was clearly the receptor that presented the highest degree of similarity with BLTR, although there is only 37% overall amino acid sequence identity between the two receptors (Fig. 5).
  • the transmembrane regions (l-VII in Fig. 5) are very similar, a few of them almost identical.
  • subtypes within the same receptor family would exhibit 50-65% sequence identity, and higher over the transmembrane regions.
  • the NPY receptor family for example, also includes subtypes that are considerably more divergent, with only 31-34% overall sequence identity (Gêtmar et al., 1997).
  • LTXR expression analysis of LTXR was performed by hybridization in an mRNA array blot (Fig. 7). More specifically, a 5'- TCCAGTTTTGCCCAGATGTGCTA-3' (SEQ ID NO:5) and a 5'- TTCCAGCTCAGCAGTGTCTCGTT-3' (SEQ ID NO:6) primer pair was used to amplify a 422 bp PCR fragment (Probe A).
  • the sequence of the probe is mainly from the LTXR untranslated leader sequence, but has an 142 bp overlap with the open reading frame.
  • a second probe, Probe B was amplified using the primers, 5'-GAGAGGGCTGCTTCTTAGTATGT-3' (SEQ ID NO:7) and 5'-TACTCCTGTCCTGTGCCTATCA-3 (SEQ ID NO:8), which yielded a 673 bp product located within the leader sequence only.
  • the PCR fragments were labelled with [ ⁇ -32 PjdCTP using the Mega-prime labelling kit (Amersham) according to standard procedures (Sambrook et al., 1989) and used in an mRNA multiple tissue expression array blot (Clontech) according to the protocol supplied by the manufacturer. The blot was exposed to Kodak BioMax autoradiograph film for four days.
  • the mRNA array showed low expression of LTXR in most tissues, but slightly higher in liver, kidney, adrenal gland, stomach, heart, cerebellum, placenta and bladder (Fig. 7). The amount of mRNA dotted onto the array membrane was varied slightly depending on the transcriptional activity of the tissue. It was normalized to give equally strong signals of housekeeping genes. Similar array experiments made with BLTR probes (data not shown) indicate similarly a low level of expression in all cells, but with higher expression in adrenal gland, kidney, liver, heart and skeletal muscle.
  • Example 8 Expression Analysis Using Northern Blots
  • LTXR LTXR-specific kinase kinase
  • Fig. 8 Northern blots
  • the probes disclosed in Example 7 were used for the Northem blotting experiment.
  • the Northern blot was performed according to the protocol supplied by the manufacturer (Invitrogen).
  • the blot was exposed to Kodak BioMax autoradiograph film for four days.
  • the results of the mRNA array can be used to give a rough estimate of the expression pattern of the LTXR gene, but the results of the Northern blot should give a more reliable answer because it is more sensitive to subtle differences in expression level.
  • Previous Northern blots with BLTR (Owman et al., 1996a) showed high expression in leukocytes, bone marrow, lymph node, spleen, and thymus, but also in pancreas, skeletal muscle, and heart.
  • LTXR is expressed in lung tissue, along with the location of its gene in the "asthma locus" of chromosome 14, indicates that this heptahelix receptor might play a significant role in asthma, and that it can be used to develop compositions and treatments for this important and costly disorder.
  • Epstein-Barr virus-induced genes First lymphocyte-specific G protein- coupled peptide receptors. J. Virol. 67: 2209-2220.
  • HIV-1 entry cofactor Functional cDNA cloning of a seven-trans-membrane G protein-coupled receptor. Science 272: 872-876.
  • Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44:283-292.
  • Leukotriene B4 is the functional ligand binding to and activating the cloned chemoattractant receptor, CMKRL1. Biochem, Biophys. Res. Commun. 240:162-166.
  • the leukotriene B4 receptor functions as a novel type of coreceptor mediating entry of primary HIV-1 isolates into CD4-positive cells. Proc. Natl. Acad. Sci. USA. 95:9530-9534.

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Abstract

The present invention relates, in general, to genes encoding inflammatory receptors. In particular, the present invention relates to molecular cloning of a novel cDNA sequence that codes for a heptahelix receptor whose deduced amino acid sequence is related to the group of leukotriene receptors. The present invention also relates to the inflammatory mediators called leukotrienes (which are included in the family named leukocyte chemoattractants), in particular leukotriene B4.

Description

MOLECULAR CLONING AND IDENTIFICATION OF A HUMAN LEUKOTRIENE-L1KE RECEPTOR LTXR
BACKGROUND OF THE INVENTION Field of the Invention
The present invention relates, in general, to genes encoding receptors of the G-protein coupled receptor superfamily. In particular, the present invention relates to molecular cloning and expression of a novel cDNA sequence that codes for a heptahelix receptor whose deduced amino acid sequence is related to leukotriene receptors. The present invention also relates to the inflammatory mediators called leukotrienes (which are leukocyte chemoattractants) and other molecules that bind to G-protein coupled receptors, such as the inflammatory receptors. Description of the Related Art
Members of the superfamily of G-protein-coupled membrane receptors receive signals from molecules that show great variety in chemical structure and originate from a large number of sources (Watson and Arkinstall, 1994). Recent rapid development in the understanding of the molecular mechanisms underlying the functions of these receptors is, to a large extent, based on the cloning and structural analysis of cDNA or genes encoding the receptors, not least the receptors receiving signals from neurotransmitter amines and peptides. Such work was pioneered by the groups of Lefkowitz (for example, Dixon et al., 1986), Numa (for example, Kubo et al., 1986), and Nakanishi (for example, Masu et al., 1987). Indeed, this pioneering work has led to a tremendous increase in the interest within the scientific and medical communities in identifying new members of the superfamily of G-protein- coupled membrane receptors that have useful scientific or health-related characteristics.
The leukotrienes (LTs) were discovered in the late nineteen-seventies and shown to comprise a family of widely distributed and biologically highly active inflammatory mediators originating from arachidonic acid through the 5- lipoxygenase pathway (Samuelson, 1983). The name leukotriene derives from the notion that leukotrienes can be produced by leukocytes and that a conjugated triene forms a common structural entity. They are members of the group of eicosanoids, which also contains prostaglandins and thromboxane, for example. The leukotrienes mediate their biological actions via several pharmacolocially-defined high affinity stereoselective membrane-bound receptors. The receptors upon which the leukotrienes are defined are classified into three distinct groups based on the binding and actions of LTB4, LTC4, LTD4, and LTE4, alone or in combination with several antagonists (Alexander et al., 1999). There is pharmacological evidence, however, for one or more additional classes of receptors (Capra et al., 1997).
LTB4 is a pro-inflammatory chemoattractant derivative of arachidonic acid that is a product of the 5-lipoxygenase pathway and is formed from LTA4 by a specific hydrolase. LTB4 is a potent activator principally of polymorphonuclear leukocytes (PMN). It has also been shown to be an activator of related myeloid cells and mast cells. LTB4 is one of a number of chemoattractant substances, including the synthetic peptide formyl-methionyl- leucyl-phenylalanine (FMLP), complement component C5a, interleukin 8, and platelet-activating factor, which have similar biological effects and together regulate leukocyte function through interaction with independent receptors. LTB4 induces chemotaxis, chemokinesis, and aggregation, causing the migration of neutrophils to sites of inflammation. At the site of inflammation, these cells degranulate, resulting in the release of lysosomal enzymes and other antibacterial and microbicidal agents. In addition, they produce superoxide as part of the host's defense response to pathogens. LTB4 also promotes adherence of neutrophils to endothelial cell walls, and, in concert with other mediators, can thereby amplify the inflammatory response.
Most of the effects of LTB4 have been studied using PMNs, which are also the major site of synthesis for LTB4, although similar effects on monocytes and eosinophils have been reported. LTB4 might also play a role in the immune system through modulation of both T- and B-lymphocyte function (Metters, 1995). LTB4-induced PMN activation in vivo causes neutrophil invasion and accumulation in the lungs, peritoneal cavity, skin, and eyes of experimental animals. Receptor activation, therefore, has been proposed as an important event in pathologies where infiltrating neutrophils are present, for example inflammatory bowel disease (IBD) and psoriasis (Metters, 1995).
Despite the fact that binding assays and functional experiments conclusively identified a receptor for leukotriene B4, and despite the fact that the LTB4 receptor has been extensively investigated as a pharmacological entity (Metters, 1995) and is known under the BLT receptor designation (Alexander et al., 1997), no sequence information relating to LT receptors was available at either the amino acid or nucleotide level until the cDNA encoding an LT receptor was cloned in the nineteen-nineties (Owman et al., 1996a; Yokomizo et al., 1997). More specifically, it was not until 1996 that the first LT receptor cDNA was cloned and characterized (Owman et al., 1996a), and subsequently found to encode the LTB4 receptor, BLTR (Owman et al., 1997; Yokomizo et al., 1997). This receptor is a member of the leukocyte chemoattractant receptors (Murphy, 1994) which belong to the superfamily of seven-transmembrane (7TM or heptahelix) G-protein coupled receptors (Watson and Arkinstall, 1994).
In the inflammatory response the chemoattractant function of LTB4 distinguishes it from the other (cysteine-containing) leukotrienes, whose main action is exerted in smooth musculature, vascular permeability, and epithelial secretions (Samuelson, 1983). The cysteine-leukotrienes have therefore been implicated as important mediators in the pathophysiololgy of asthma (Drazen et al., 1998). Interestingly, the human BLTR gene is located in chromosome 14 at q11.2-12 (Owman et al., 1996a), close to one of the regions (defined as 14q 11.2-13) reported to be linked to asthma-associated phenotypes (CSGA, 1997).
Because of the continuing interest in chemoattractant receptors, there exists a need in the art for the identification of new receptors and the elucidation of their structure so that the function of these receptors in humans can be determined. In particular, there exists a need in the art for sequence information on such receptors, including DNA and amino acid sequences, to enable the isolation and characterization of particular receptors as well as structurally related receptors. The identification of receptors associated with pathogenic conditions would be particularly advantageous in assays for the identification of these conditions in susceptible individuals.
SUMMARY OF THE INVENTION
Accordingly, this invention aids in fulfilling these needs in the art. More particularly, this invention provides a heptahelix receptor derived from the human genome and which is expressed in human tissues and cells, such as kidney, adrenal gland, stomach, heart, cerebellum, placenta, bladder, lymphoid tissues, and especially liver, lung, and pancreas.
More particularly, the present invention provides isolated heptahelix receptors and nucleic acids encoding these heptahelix receptors. Such nucleic acids include (a) cDNA clones having a nucleotide sequence derived from the coding region of a native heptahelix gene of the invention; (b) nucleic acids that are capable of hybridizing to the cDNA clones of (a) under moderately stringent conditions and which encode biologically active heptahelix molecules; and (c) nucleic acids that are degenerate as a result of the genetic code to the DNA sequences defined in (a) and (b) and which encode biologically active heptahelix receptor molecules.
The present invention also provides recombinant expression vectors comprising the nucleic acids defined above, recombinant heptahelix receptor molecules produced using recombinant expression vectors, and processes for producing recombinant heptahelix receptor molecules using the expression vectors. Recombinant cells containing the recombinant vectors of the invention are also provided, as are methods of using the recombinant cells.
The present invention further provides isolated or purified polypeptides and proteins encoded by the nucleic acids of the invention. For example, the invention provides an isolated protein having the amino acid sequence of SEQ ID NO:2, and is encoded by the nucleotide sequence of SEQ ID NO:1 , as follows: ltMACTGGCCCTGGCCCTG CC ATACCTTtMCCCTffiTA CTCα^^ lOlACTGTCTGTTCTGAGGATA∞CTCTAGCCCACTCATTMGTACAT^
201CTATACTTTTCTα(aGGTTCCC GGCCTACTG GGGACTrMαTACTCTTAATGGCTTTCCT
301(HT (rTCCCA TACAGGATCACCTrø^
401 CCCMACT GACACT«HTCTGGTGCCCTCCCCMGCAACCTCMCTT^
501TCCATCTGCCAGGC(X(XαC ATCTOT( rTC(MCTTTTC
601CCTTraTGTCTTGTATCOGGTGCAGCCTGGTAATα£GCCT
701OCCTGGMCCTAG(MATGCCmCATG(3AMAGαGTCATTGACAGCCrø
801GGTGG(aGGAGGTAO5AGGCCAGGGGCTCAGCffi(aC(aGGAGACTffiMACAGGαAGGAT GG∞
901CAGGCCAGAGAGACCAGG(M(MACACACTGCA^^
1001 TOO: AGAGTCCTCCTATTICCTTCacaCCAGGGMTCTTACTGCCCCACTTCAGOTCT llOlGCCTGGGTGOTTGGTGATGGfflAGGAOaGGGTfflGGGAGGGGCCCCAGGAGAGGCCCAGGATGAGCC 1201CICTGCAMTCTGATAGGαCAGfflaGfflCTAGGCACCTCGCCTACTGCTGCπ CCTπαC^ DOl GGTAAGTAGATCTGTGCA∞TCCCTmCACCCCACCATCCACT HOlffiGTGGrffiTAGAGATAGTfflαGCCTGGGGTfflGGACrπATGCClGTTTACαCTfflGCT 1501GGGAGαGGG(»TCTGGGTICCAAGMGGAGTTGTGmGAGGTGGGGTC
1 M A P S H R A S Q V G F C P T P E R P L R P P T C 28
1601«£ CT G»GGAGGCATGGCACCTTCT(M:GG(£ATCACAG(nGGGOT
29 P R R M S V C Y R P P G N E T L L S K T S R A T G T A F L L L A 61 1701C£CCAGAAG3ATCTCCSTCT(rrAOCC^CCCCAGGM
I
62 A L L G L P G N G F V V W S L A G W R P A R G R P L A A T L V L H 94 1801 GGCGCTGCTGGGGCTCCITGGCAACGGOT∞TGGTΘGffiGCTTC ffi 1900 II
95L A L A D G A V L L L T P L F V A F L T R Q A W P L G Q A G C K A V128 1901CTGGCθTGGCCfflCGGCGCGCTGCTG(πGCTCAOGCXBCTCTTTC^GCOT in
129 Y Y V C A L S M Y A S V L L T G L L S L Q R C L A V T R P F L A P 161 2001TGTACTA∞TCTGCGCGCTCAGCATGTA∞(rAGα3TCCTGCTαCCGGCσGCT IV
162 R L R S P A L A R R L L L A V L A A L L L A V P A A V Y R H L 194 2101 TCGGCTG CG QG CC CGGC CCTGG CC ( COG CCTGCTGCTffiCGGTCIGGCTGGCCKCCTCT V
195R D R V C Q L C H P S P V H A A A H L S L E T L T A F V L P F G L M228 2201 GGffiCCGC(MGCCAGCTCTGCCACCCGTCGCOGOT
229 L G C Y S V T L A R L R G A R G S G R H G A R V G R L V S A I V 261 2301TGCTCGGCTGCTAC G(]GTGACGCTGGCKGGCT(£rø^ j
262 L A F G L L W A P Y H A V N L L Q A V A A L A P P E G A L A K L G 294 2401CCTTGCCTTrøCTTGCTCTCfflCCθ:CTACCACGCA(mACCTTCTGCA^ yn
295G A G Q A A R A G T T A L A F F S S S V N P V L Y V F T A G D L L P328 2501GGAC CGGCCAGGCGGOG(mGCGGGAACTACGGCCTIGGCCTTCTICAGTICTAGCGIC
329 R A G P R F L T R L F E G S G E A R G G G R S R E G T M E L R T T 361 2601(ICffiGCAGC^CC∞πTCCTCACGCCCCTCTTCGAAGGCTCTGGGGAGGCCCGAGGGGGC∞
362 P Q L K V V G Q G R G N G D P G G G M E K D G P E W D L * 389
2701CCCTCAGCT(MAGTGCTGGG(IAGGGCCG∞GCMTGGAGAC(iα GGGTGGGATGaGA^ 2799 The heptahelix receptor compositions of the invention are useful in diagnostic assays for heptahelix receptors, as well as in raising antibodies to heptahelix receptors for use in diagnosis and therapy. In addition, the heptahelix compositions can be used directly in therapy to bind or scavenge G-protein coupled receptor agonists, such as leukotrienes, thereby providing means for regulating the functional activity of the agonists.
Also, because the heptahelix receptor of the invention is, among other things, a leukotriene receptor for leukotriene B4, the invention is useful for detecting LTB4, for example, by immunoassay for heptahelix polypeptides of the invention or by detection of all or part of the polynucleotides encoding the receptor of the invention, such as hybridization assays or amplification reactions.
In particular, because the heptahelix receptor of the invention belongs to a family of receptors that are used by the human immunodeficiency virus (HIV) during its fusion, entry, and infection of human target cells, the invention can be useful for preventing infection of such cells by HIV, or by any other virus using the heptahelix receptor of the invention during the infection process.
In addition, the heptahelix receptor compositions can be used directly in therapy to bind or scavenge chemoattractants, thereby providing a means for regulating the immune activities of chemoattractants. For example, the heptahelix receptor of the invention can be used to regulate the immune activities of chemoattractants associated with asthma.
More particularly, this invention provides a method for lowering the levels of active leukotriene B4 in a mammal in need thereof, which comprises administering to the mammal a leukotriene B4-lowering amount of a leukotriene B4 receptor comprising the sequence of amino acids of SEQ ID NO:2.
In another embodiment, this invention provides a method for assaying a ligand or an antagonist or agonist for said ligand. The method comprises: (A) providing a heptahelix receptor of the invention or a fragment thereof comprising a binding domain for the ligand, antagonist, or agonist;
(B) incubating the receptor with a test sample suspected to contain the ligand, antagonist, or agonist; and
(C) detecting binding between the receptor and the ligand, antagonist, or agonist.
The receptor can be in an external cell membrane of a host cell transfected or transduced with DNA encoding the receptor. In addition, binding can be detected by intracellular calcium level in the host cell.
BRIEF DESCRIPTION OF THE DRAWINGS
This invention will be more fully described with reference to the drawings in which:
Figure 1 depicts the nucleotide and amino acid sequences of the cloned LTXR gene (cDNA) and receptor, respectively. The cloned cDNA is 2.8 kb in length, and encodes a 389 amino acid long protein with seven putative transmembrane domains (designated as roman numerals I - VII).
Figure 2 depicts the sequencing strategy used to determine the nucleotide sequence of the LTXR cDNA. Arrows indicate approximate location and length of overlapping subclones used in sequencing, as well as the relative direction of sequencing. Sequencing was performed using Sp6 and T7 primers on exo-deleted subclones.
Figure 3 shows a representative experiment using HeLa (human uterine) cells transfected with BLTR cDNA to transiently express the corresponding receptor protein in the cell membrane. The cells are co- transfected with a calcium-sensitive reporter construct that emits a luminescence signal from aequorin when cellular calcium is elevated upon activation of the expressed receptor. When the cells are exposed to LTB4 there is a pronounced increase in cellular calcium levels. The response is significantly different from the control levels recorded when cells receive the extracellular buffer (ECB) in which they are suspended, or ethanol solution (EtOH) corresponding to the solvent for LTB4. The curves are the means (± standard errors) of triplicate readings, the x axis shows time in seconds after administration of test solution, the y axis shows relative light intensity.
Figure 4 illustrates the same experimental set-up as in Figure 3, comparing the effects of various leukotrienes together with the buffer (pbs) and ethanol (EtOH) controls. The HeLa cells are transiently expressing the novel LTXR receptor, the previously cloned BLTR receptor, as well as negative control cells not transfected with receptor cDNA. Only LTB4 elicits a response in the LTXR cells. LTB4 also stimulates the BLTR cells, but these also respond to a lower degree to LTC4 and, even less, to LTD4.
Figure 5 shows an alignment of the LTXR and the BLTR proteins. The shadowed regions indicate identical amino acids. The greatest similarity is found within the putative transmembrane regions, but can also be found in some intra- and extra-cellular loops and the C-terminus.
Figure 6 shows a phylogenetic analysis of the LTXR protein. ClustalX was used to make a protein alignment, that was analyzed with the maximum parsimony method in the PHYLIP package to produce an evolutionary tree. Numbers at the branch points are the results, in percent, of a bootstrap analysis and demonstrates the confidence of the inferred nodes. Abbreviations used and the GenBank accession numbers (in brackets) of the receptors are as follows: GALR3, Galanin receptor type 3 (NP003605); GALR2, Galanin receptor type 2 (O43603); SSR1 , Somatostatin receptor 1 (NP001040); SSR2, Somatostatin receptor 2 (NP001041 ); SSR3, Somatostatin receptor 3 (NP001042); PAF-R, Platelet activating factor receptor 1 (P25105); CYSLT1 , Cysteinyl leukotriene receptor 1 (NP006630); FPRL1 , Formyl peptide receptor-like 1 (NP001453); FPRL2, Formyl peptide receptor-like 2 (NP002021); BLTR Mus musculus (NP032545), Leukotriene B4 receptor from Mus musculus; BLTR Rattus norvegicus (JC7096), Leukotriene B4 receptor from Rattus norvegicus; BLTR Cavia porcellus (AAD42063), Leukotriene B4 receptor from Cavia porcellus; LTXR, Homo sapiens LTXR (CAA67001 ); CRTH2, Chemoattractant receptor-homologous molecule expressed on TH2 cells (NP004769). Figure 7 shows expression profiling of LTXR using an mRNA array. LTXR seems to be expressed at a low level in most tissues. Slightly higher expression can be seen in liver, kidney, adrenal gland, stomach, heart, cerebellum, placenta and bladder. The organization of the array is as follows: A1 , whole brain; A2, cerebellum left; A3, substantia nigra; A4, heart; A5, esophagus; A6, colon transverse; A7, kidney; A8, lung; A9, liver; A10, leukemia HL-60; A11 , fetal brain; A12, yeast total RNA; B1 , cerebral cortex; B2, cerebellum right; B3, accumbens nucleus; B4, aorta; B5, stomach; B6, colon descending; B7, skeletal muscle; B8, placenta; B9, pancreas; B10, HeLa S3; B11 , fetal heart; B12, yeast tRNA; C1 , frontal lobe; C2, corpus callosum; C3, thalamus; C4, atrium left; C5, duodenum; C6, rectum; C7, spleen; C8, bladder; C9, adrenal gland; C10, leukemia K-562; C11 , fetal kidney; C12, E. coli rRNA; D1 , parietal lobe; D2, amygdala; D3, pituitary gland; D4, atrium right; D5, jejunum; D7, thymus; D8, uterus; D9, thyroid gland; D10, leukemia MOLT-4; D11 , fetal liver; D12, E. coli DNA; E1 , occipital lobe; E2, caudate nucleus; E3, spinal cord; E4, ventricle left; E5, ileum; E7, peripheral blood leukocyte; E8, prostate; E9, salivary gland; E10, Burkitt's lymphoma Raji; E11 , fetal spleen; E12, poly r(A); F1 , temporal lobe; F2, hippocampus; F4, ventricle right; F5, ilocecum; F7, lymph node; F8, testis; F9, mammary gland; F10, Burkitt's lymphoma Daudi; F11 , fetal thymus; F12, human C0t_1 DNA; G1 , paracentral gyms of cerebral cortex; G2, medulla oblongata; G4, interventricular septum; G5, appendix; G7, bone marrow; G8, ovary; G10, colorectal adenocarcinoma SW480; G11 , fetal lung; G12, human DNA 100ng; H1 , pons; H2, putamen; H4, apex of the heart; H5, colon ascending; H7, trachea; H10, lung carcinoma A549; H12, human DNA 500ng.
Figure 8 shows an expression analysis of LTXR by Northern blot. Transcripts were detected in liver, lung, and pancreas. Traces of expression were also detected in heart and placenta.
DETAILED DESCRIPTION OF THE INVENTION
All functions of a multicellular organism, such as a human, require communication between individual cells. This communication is mediated primarily by chemical mediators, which transmit signals to cells via surface receptors on the cells. The surface receptors are often complex proteins. Chemical signalling is thus the basis for the extensive trafficking of inflammatory cells during the host-defense reaction involving the entire immune system.
The most common surface receptors on eukaryotic cells belong to the superfamily of G-protein coupled heptahelix receptors. These receptors consist of a protein chain that passes in and out through the cell membrane seven times in a serpentine manner. The clinical significance of this receptor superfamily is illustrated by the fact that the vast majority of pharmaceutical drugs for patient use exert their function by interacting, one way or another, with these heptahelix receptors.
The subfamily of leukocyte chemoattractants and their cognate receptors have received particular attention during the last few years because it comprises receptors (chemokine receptors) that are necessary for the AIDS virus, HIV1 , to fuse with and infect the CD4-positive cells of the immune system (Fauci, 1996), and because they are involved in a large number of inflammatory diseases, the prototype being asthma (Drazen et al., 1998). The inflammatory mediators/receptors involved more particularly in this disease are the leukotrienes.
Asthma is only one of several severe inflammatory conditions that involve the leukotrienes, which are among the most widely distributed inflammatory mediators in the body. They were discovered and chemically characterized in the nineteen-seventies (Samuelson, 1983) and have since been extensively analyzed for their physiological role and pathophysiological functions. They all derive from arachidonic acid through the lipoxygenase reaction cascade. Naturally, over the years there has been an extensive hunt for the specific receptors that receive the leukotriene signals on the cell surface. The problem had to await its solution until the first receptor was cloned in 1996.
At that time, the cloning of a cDNA encoding a novel (human) G-protein coupled receptor was published. Although it was possible from the deduced protein sequence to suggest that the molecule belonged to the chemoattractant receptor family, the natural ligand could not be established at the time. Hence, this "orphan" receptor was given the provisional name, CMKRL1 (Owman et al., 1996a).
Shortly after CMKRL1 was published, an identical cDNA sequence was obtained by another group working with human erythroleukemia cells and, mainly based on radioligand binding experiments, the corresponding receptor was proposed to be a new member of the P2Y group of purinoceptors (Akbar et al., 1996). The interpretation of the binding data and of the functional assays on transfected cells, as well as the comparatively low relatedness of the deduced amino acid sequence to hitherto cloned P2Y receptors, warranted a reevaluation of the conclusion. Thus, in more extensive studies of the binding and function of the receptor, stably expressed in a human astrocytoma cell line, others were able to assess that the purported P2Y7 receptor is not a member of the P2Y family of signalling molecules (Herald et al., 1997). With the use of a subtraction strategy in retinoic acid-differentiated HL-60 cells, a cDNA sequence identical to the coding sequence of CMKRL1 was again reported in 1997, and the corresponding natural ligand for the receptor identified as leukotriene B4 (Yokomizo et al., 1997).
Meanwhile, it was shown in binding assays and functional studies that cells expressing CMKRL1 respond to leukotriene B4 (Owman et al., 1997). Thus, CMKRL1 was the first cloned leukotriene receptor. The receptor clone R2 obtained from a human genomic library (Raport et al., 1996), on the other hand, deviates from the above-mentioned three DNA sequences in its 5' end. This difference led to the identification of a different methionine initiation site in the proposed coding sequence, leading to an amino terminus that was too long in the deduced protein sequence.
In this context, the leukotriene receptor as it was cloned by Owman et al. has two important implications: (1) the original publication (Owman et al., 1996a) reported that the corresponding receptor gene is located in human chromosome 14, in the q11_2-q12 region, which corresponds precisely to one of the loci for linkage to asthma-associated phenotypes in that chromosome (CSGA, 1997); and (2) there is experimental evidence that the receptor is able to function as a co-receptor for select primary isolates of the AIDS virus, HIV1 (Owman et al., 1998).
The present invention provides a novel nucleic acid encoding a putative seven-transmembrane spanning receptor. In a preferred embodiment, the nucleic acid is a full-length cDNA which encodes a novel receptor having 37% peptide sequence identity with the original BLTR cloned by Owman et al. The novel nucleic acid and encoded protein have been named LTXR, for leukotriene-like receptor X. The novel LTXR cDNA, the cloning of which is disclosed herein, encodes a human leukotriene receptor that responds to leukotriene B4 even more specifically than the previously cloned BLTR. In addition, the sequence of the 3' end of the LTXR gene overlaps with the upstream non-coding region of BLTR, implying that the LTXR gene is located within the "asthma locus" of chromosome 14.
The nucleic acid can be any nucleic acid capable of encoding a heptahelix receptor of the invention. In embodiments, the nucleic acid is cDNA. In other embodiments, the nucleic acid is mRNA. In embodiments, the nucleic acid is DNA, such as genomic DNA and subgenomic DNA. In preferred embodiments, the nucleic acid is isolated or purified. "Isolated" or "purified" refers to a nucleic acid polymer in the form of a separate fragment or as a component of a larger nucleic acid construct, which has been derived from nucleic acid isolated from its natural environment at least once. Preferably, the heptahelix receptor nucleic acid is in substantially pure form, i.e., free of contaminating endogenous materials. It is also preferable that the nucleic acid be present in a quantity or concentration enabling manipulation by humans or machines, for example in a quantity sufficient to enable identification of the nucleotide sequence using standard biochemical methods. Isolated nucleic acids are preferably provided in the form of an open reading frame uninterrupted by internal, non-translated sequences (introns), which are typically present in eukaryotic genes. The sequence of a preferred isolated nucleic acid of the invention, a cDNA encoding LTXR, is shown in Fig. 1.
In embodiments, genomic DNA containing the relevant sequences can be used as a source of coding sequences. In embodiments, subgenomic DNA can be used as a source of coding sequences. In embodiments, mRNA or cDNA can be used as a source of coding sequences. The genomic location of a preferred DNA is described hereinafter.
The nucleic acids according to the invention comprise a sufficient amount of nucleic acid sequence to encode a heptahelix receptor according to the invention. In embodiments, nucleic acids of the invention comprise only enough heptahelix receptor-specific nucleic acid to encode a biologically active heptahelix receptor. In embodiment, sequences of non-translated heptahelix-specific nucleic acid can be present 5' or 3' from the heptahelix receptor open reading frame. It is preferred that these non-translated sequences do not interfere with manipulation or expression of the coding region. In embodiments, the nucleic acids of the invention comprise the heptahelix-encoding nucleotide sequence as the sole protein- or polypeptide- encoding sequence (i.e., as the sole open reading frame), while in other embodiments the nucleic acid comprises multiple open reading frames. Where the nucleic acid comprises multiple open reading frames, the open reading frames can encode multiple copies of the LTXR receptor, or it can comprise the one or more LTXR open reading frames along with open reading frames encoding other proteins or polypeptides, such as other heptahelix receptors.
As used herein, "nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides. DNA sequences encoding the heptahelix receptors of this invention can be assembled from DNA fragments, such as short oligonucleotide linkers, or from a series of oligonucleotides, or by amplification reactions to provide a synthetic gene that is capable of being expressed in a recombinant transcriptional unit.
It will be understood that the nucleic acid encoding the heptahelix receptor of the invention is not limited to the LTXR receptor clones just described. For example, additional cDNA clones can be isolated from cDNA libraries of other mammalian species by cross-species hybridization. Mammalian heptahelix receptors of the present invention include, by way of example, primate, human, murine, canine, feline, bovine, ovine, equine, and porcine heptahelix receptors. Mammalian heptahelix receptors can be obtained, for example, by cross-species hybridization using a single-stranded cDNA derived from the human heptahelix receptor DNA sequence as a hybridization probe to isolate heptahelix receptor cDNAs from mammalian cDNA libraries. For use in hybridization, DNA encoding a heptahelix receptor can be covalently labeled with a detectable substance, such as a fluorescent group, a radioactive atom, or a chemiluminescent group, by methods well known to those skilled in the art. Such probes can also be used for in vitro diagnosis of particular conditions.
In this regard, the invention provides probes and primers for detection and isolation of heptahelix receptors. The probes and/or primers can comprise the entire coding sequence of a heptahelix receptor, or can comprise a portion of the heptahelix coding region. The sequences of certain preferred probes and primers are disclosed herein, however, it is to be understood that any nucleotide sequence that is sufficiently specific for heptahelix receptor nucleic acids can be used. Selection of such sequences is well within the skill of the ordinary artisan, and can be based on the sequences disclosed herein.
Alternative mRNA constructs, which can be attributed to different mRNA splicing events following transcription and which share large regions of identity or similarity with the heptahelix receptors disclosed herein, are considered to be within the scope of the present invention.
Thus, the present invention provides vectors comprising a nucleic acid of the invention. In embodiments, the vectors are recombinant expression vectors to amplify or express DNA encoding at least one heptahelix receptor. Recombinant expression vectors are replicable nucleic acid constructs that have synthetic or cDNA-derived nucleic acid sequences encoding mammalian heptahelix receptor or bioequivalent analogs operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. A transcriptional unit for double-stranded DNA constructs generally comprises an assembly of (1 ) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers; (2) a structural or coding sequence, which is transcribed into mRNA and translated into the heptahelix receptor; and (3) appropriate transcription and translation initiation and termination sequences. Such regulatory elements can include an operator sequence to control transcription and a sequence encoding suitable mRNA ribosomal binding sites. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated.
Promoters commonly used in recombinant microbial expression vectors include the β-lactamase (penicillinase) and lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281 :544, 1979), the tryptophan (tp) promoter system (Goeddel et al., Nucl. Acids Res. 8:40576, 1980; and EPA 36,776), and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982). The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells can be provided by viral sources. For example, commonly used promoters and enhancers are derived from polyoma, adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites, can be used to provide the other genetic elements required for expression of a heterologous DNA sequence.
DNA regions are operably linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor, which participates in the secretion of the polypeptide. A promoter is operably linked to a coding sequence if it controls the transcription of the sequence. A ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous and, in the case of secretory leaders, contiguous and in reading frame. Structural elements intended for use in yeast expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it can include an N- terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.
DNA sequences encoding mammalian heptahelix receptors, which are to be expressed in a microorganism, preferably contain no introns that could prematurely terminate transcription of DNA into mRNA; however, premature termination of transcription may be desirable, for example, where it would result in mutants having advantageous C-terminal truncations. Due to code degeneracy, there can be considerable variation in nucleotide sequences encoding the same amino acid sequence. Other embodiments include sequences capable of hybridizing to the sequences of the provided cDNA under moderately stringent conditions and other sequences hybridizing or degenerate to those that encode biologically active heptahelix receptor polypeptides.
Accordingly, the present invention provides recombinant cells containing a nucleic acid according to the invention. Recombinant heptahelix receptor DNA can be expressed or amplified in a recombinant expression system comprising a substantially homogenous monoculture of suitable host microorganisms, for example, bacteria such as E. coli, or yeast such as S. cerevisiae, which have stably integrated (by transduction or transfection) a recombinant transcriptional unit into chromosomal DNA, or carry the recombinant transcriptional unit as a component of a resident plasmid or other extrachromosomal element. Generally, cells constituting the system are the progeny of a single ancestral transformant. Recombinant expression systems as defined herein will express heterologous protein upon induction of the regulatory elements linked to the DNA sequence or synthetic gene to be expressed.
Transformed host cells are cells that have been transduced or transfected with heptahelix receptor vectors constructed using recombinant DNA techniques. Transformed host cells ordinarily express heptahelix receptor, but host cells transformed for purposes of cloning or amplifying heptahelix receptor DNA do not need to express heptahelix receptor. Expressed heptahelix receptor will be deposited in the cell membrane or secreted into the culture supernatant, depending on the heptahelix receptor DNA selected. Suitable host cells for expression of heptahelix receptor include prokaryotes, yeast, or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram-negative or gram-positive organisms, for example E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems can also be employed to produce heptahelix receptor using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985), the relevant disclosure of which is hereby incorporated by reference.
Recombinant heptahelix receptor proteins can also be expressed in yeast hosts, preferably from the Saccharomyces species, such as S. cerevisiae. Yeast of other genera, such as Pichia or Kluyveromyces, can also be employed. Yeast vectors will generally contain an origin of replication from the 2μ yeast plasmid or an autonomously replicating sequence (ARS), promoter, DNA encoding heptahelix receptor, sequences for polyadenylation, transcription, termination, and a selection gene. Suitable promoter sequences in yeast vectors include the promoters for metallothionein, 3- phosphoglycerate kinase (Hitzeman et al. J. Biol. Chem. 255:2073, 1980), or other glycolytic enzymes (Hess et al. J. Adv. Enzyme Reg. 7:149, 1968); and Holland et al., Biochem. 17:4900, 1978), such as enolase, glyceraldehyde-3- phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, phosphoglucose isomerase, and glucokinase.
Various mammalian or insect cell culture systems are also advantageously employed to express recombinant protein. Expression of recombinant proteins in mammalian cells is particularly preferred because such proteins are generally correctly folded, appropriately modified, and completely functional. Examples of suitable mammalian host cell lines include the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175, 1981 ), and other cell lines capable of expressing an appropriate vector including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines. Mammalian expression vectors can comprise nontranscribed elements, such as an origin of replication, a suitable promoter, and an enhancer linked to the gene to be expressed, and other 5' or 3' nontranslated sequences, such as ribosome binding sites and transcriptional termination sequences. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).
It should be clear from the above disclosure that this invention also relates to the discovery of a new heptahelix receptor. A preferred sequence for the heptahelix receptor is disclosed in Fig. 1 (SEQ ID NO:2). As used herein, the term "heptahelix receptor" refers to proteins and polypeptides having amino acid sequences that are substantially similar to the native, mature, mammalian heptahelix receptor amino acid sequence of the invention, and which are biologically active. For example, they are capable of binding chemoattractant molecules or transducing a biological signal initiated by a chemoattractant or other ligand binding to a cell as LTXR is able to do, or cross-reacting with anti-heptahelix receptor antibodies raised against heptahelix receptor from natural (i.e., non-recombinant) sources. As used throughout the specification, the temV'mature" means a protein expressed in a form lacking a leader sequence as may be present in full-length transcripts of a native gene.
The term "heptahelix receptor" includes, but is not limited to, analogs or subunits of native proteins having seven membrane-spanning or hydrophobic regions and which exhibit at least some biological activity in common with LTXR. For example, soluble heptahelix receptor constructs, which possess the seven hydrophobic regions (and are secreted from the cell), and retain the ability to bind chemoattractants are deemed to be heptahelix receptors of the invention.
In the absence of any species designation, heptahelix receptor refers generically to mammalian heptahelix receptor. Similarly, in the absence of any specific designation for deletion mutants, the term heptahelix receptor means all forms of heptahelix receptor, including mutants and analogs that possess heptahelix receptor biological activity.
"Soluble heptahelix receptor" as used in the context of the present invention refers to proteins and polypeptides, or substantially equivalent analogs, having an amino acid sequence corresponding to all or part of the heptahelix receptor, and which are biologically active in that they bind to chemoattractants and other related ligands. Soluble heptahelix receptors include polypeptides that vary from those sequences by one or more substitutions, deletions, or additions, and which retain the ability to bind chemoattractant or inhibit chemoattractant signal transduction activity via cell surface bound heptahelix receptor proteins. Inhibition of chemoattractant signal transduction activity can be determined by transfecting cells with recombinant heptahelix receptor nucleic acids to obtain recombinant receptor expression. The cells can then be contacted with chemoattractant or other ligand and the resulting metabolic or signal transduction effects examined. If an effect results that is attributable to the action of the chemoattractant or other ligand, then the recombinant receptor has signal transduction activity. Procedures for determining whether a polypeptide has signal transduction activity include measurement of adenyl cyclase activity and intracellular calcium measurements.
The group of chemoattractant or "chemokine-like" substances as used in the context of the present invention comprises both the "classical" chemoattractants and the chemokines. Examples of the "classical" chemoattractants, as one having ordinary skill in the art would appreciate, include but are not limited to LTB4, PAF, fMLP, C3a, and C5a. In addition, all subtypes of the chemokines, i.e., alpha, beta, etc., are within the context of the present invention. Th e term "isolated" or "purified", as used in the context of this invention to define the purity of heptahelix receptor protein or protein compositions, means that the protein or protein composition is separated from its native environment. In one embodiment, the heptahelix receptor can be substantially free of other proteins of natural or endogenous origin. In a preferred embodiment, the receptor can contain less than about 1 % by mass of protein contaminants residual of its native environment or production processes. Such compositions, however, can contain other proteins added as stabilizers, excipients, or co-therapeutics.
The term "substantially similar" when used to define either amino acid or nucleic acid sequences means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which is to retain biological activity of the heptahelix receptor as may be determined, for example, in a chemoattractant assay for heptahelix receptor binding. Alternatively, nucleic acid subunits and analogs are "substantially similar" to the specific DNA sequences disclosed herein if: (a) the DNA sequence is derived from the coding region of native, mammalian heptahelix receptor gene of the invention; (b) the DNA sequence is capable of hybridization to DNA sequences of (a) and which encode biologically active heptahelix receptor molecules; or (c) DNA sequences that are degenerate as a result of the genetic code to the DNA sequences defined in (a) or (b) and which encode biologically active heptahelix receptor.
The heptahelix receptor of the invention can be obtained from natural sources or by recombinant techniques using eukaryotic or procaryotic host systems. Thus, as disclosed above, the invention provides recombinant cells containing nucleic acids encoding heptahelix receptors according to the invention. "Recombinant" as used herein means that a protein is derived from recombinant (e.g., microbial or mammalian) expression systems. "Microbial" refers to recombinant proteins made in bacterial or fungal (e.g., yeast) expression systems. As a product, "recombinant microbial" defines a protein produced in a microbial expression system that is essentially free of native endogenous substances. Protein expressed in most bacterial cultures, e.g., E. coli, will be free of glycan. Protein expressed in yeast may have a glycosylation pattern different from that expressed in mammalian cells.
"Biologically active" as used throughout this specification as a characteristic of heptahelix receptors of the invention means that a particular molecule shares sufficient amino acid sequence similarity with embodiments of the present invention to be capable of binding chemoattractant or other ligand, transmitting a stimulus to a cell, for example, as a component of a hybrid receptor construct, or cross-reacting with anti-heptahelix receptor antibodies raised against heptahelix receptor from natural (i.e., non- recombinant) sources.
Derivatives of heptahelix receptor within the scope of the invention include various structural forms of the primary protein, which retain biological activity. Due to the presence of ionizable amino and carboxyl groups, for example, a heptahelix receptor protein can be in the form of acidic or basic salts, or can be in neutral form. Individual amino acid residues can also be modified by oxidation or reduction.
The primary amino acid structure can be modified by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups, and the like, or by creating amino acid sequence mutants. Covalent derivatives can be prepared by linking particular functional groups to heptahelix receptor amino acid side chains or at the N- or C-termini. Other derivatives of heptahelix receptor within the scope of this invention include covalent or aggregative conjugates of heptahelix receptor or its fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N- terminal or C-terminal fusions. For example, the conjugated peptide can be a signal (or leader) polypeptide sequence at the N-terminal region of the protein, which co-translationally or post-translationally directs transfer of the protein from its site of synthesis to its site of function inside or outside of the cell membrane or wall (e.g., the yeast α-factor leader). Heptahelix receptor protein fusions can comprise peptides added to facilitate purification or identification of heptahelix receptor (e.g., poly-His).
Heptahelix receptors and derivatives can be used as immunogens, reagents in receptor-based immunoassays, or as binding agents for affinity purification procedures of chemoattractants or other binding ligands. Heptahelix receptor derivatives can also be obtained by cross-linking agents, such as M-maleimidobenzoyl succinimide ester and N-hydroxysuccinimide, at cysteine and lysine residues. Heptahelix receptor proteins can also be covalently bound through reactive side groups to various insoluble substrates, such as cyanogen bromide-activated, bisoxirane-activated, carbonyldiimidazole-activated, or tosyl-activated agarose structures, or by adsorbing to polyolefin surfaces (with or without glutaraldehyde cross-linking). Once bound to a substrate, heptahelix receptor can be used to selectively bind (for purposes of assay or purification) anti-heptahelix receptor antibodies or chemoattractant.
The present invention also includes heptahelix receptor, with or without associated native-pattern glycosylation. Heptahelix receptor expressed in yeast or mammalian expression systems can be similar or slightly different in molecular weight and glycosylation pattern than the native molecules, depending upon the expression system. Expression of heptahelix receptor DNAs in bacteria, such as E. coli, provides non-glycosylated molecules. Functional mutant analogs of mammalian heptahelix receptor having inactivated N-glycosylation sites can be produced by oligonucleotide synthesis and ligation or by site-specific mutagenesis techniques. These analog proteins can be produced in a homogeneous, reduced-carbohydrate form in good yield using yeast expression systems.
Heptahelix receptor derivatives can also be obtained by mutations of heptahelix receptor or its subunits. A heptahelix receptor mutant, as referred to herein, is a polypeptide homologous to heptahelix receptor, but which has an amino acid sequence different from native heptahelix receptor because of a deletion, insertion, or substitution. Bioequivalent analogs of heptahelix receptor proteins can be constructed by, for example, making various substitutions of residues or sequences, or deleting terminal or internal residues or sequences, or deleting terminal or internal residues or sequences not needed for biological activity. For example, cysteine residues can be deleted (e.g., Cys178) or replaced with other amino acids to prevent formation of unnecessary or incorrect intramolecular disulfide bridges upon renaturation. Generally, substitutions should be made conservatively; i.e., the most preferred substitute amino acids are those having physiochemical characteristics resembling those of the residue to be replaced. Similarly, when a deletion or insertion strategy is adopted, the potential effect of the deletion or insertion on biological activity should be considered. Substantially similar polypeptide sequences, as defined above, generally comprise a like number of amino acids.
In order to preserve the biological activity of heptahelix receptors, deletions and substitutions will preferably result in homologously or conservatively substituted sequences, meaning that a given residue is replaced by a biologically similar residue. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as lie, Val, Leu, or Ala for one another, or substitution of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known.
Mutations in nucleotide sequences constructed for expression of analog heptahelix receptor must, of course, preserve the reading frame phase of the coding sequences and preferably will not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hairpins, which would adversely affect translation of the receptor mRNA. Although a mutation site may be predetermined, it is not necessary that the nature of the mutation per se be predetermined. For example, in order to select for optimum characteristics of mutants at a given site, random mutagenesis may be conducted at the target codon and the expressed heptahelix receptor mutants screened for the desired activity. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide- directed site-specific mutagenesis procedures can be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required.
Purified mammalian heptahelix receptors or analogs can be prepared by culturing suitable host/vector systems to express the recombinant translation products of the DNAs of the present invention, which are then purified from culture media or cell extracts. For example, supematants from systems that secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix. For example, a suitable affinity matrix can comprise a heptahelix or lectin or antibody molecule bound to a suitable support. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification.
Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify a heptahelix receptor-containing composition. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous heptahelix receptor.
Recombinant heptahelix receptor produced in bacterial culture can be isolated by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange, or size exclusion chromatography steps. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of recombinant the receptor can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
Heptahelix receptors synthesized in recombinant culture are characterized by the presence of non-human cell components, including proteins, in amounts and of a character which depend upon the purification steps taken to recover the receptor from the culture. These components ordinarily will be of yeast, prokaryotic, or non-human higher eukaryotic origin and preferably are present in innocuous contaminant quantities, on the order of less than about 1 percent by weight. Further, recombinant cell culture enables the production of heptahelix receptor free of proteins that may be normally associated with receptor as it is found in nature in its species of origin, e.g., in cells, cell exudates, or body fluids.
The preferred heptahelix receptor of the invention is a novel chemoattractant, which has turned out to have leukotriene (LT) receptor-like activity. Accordingly, the present invention provides methods of using therapeutic compositions comprising an effective amount of soluble heptahelix receptor and a suitable diluent or carrier in the treatment of a mammal, including primate, human, murine, canine, feline, bovine, ovine, equine, or porcine species. The invention includes methods for modulating heptahelix receptor-dependent responses in humans comprising administering an effective amount of soluble heptahelix receptor to a patient. Appropriate dosages can be determined in trials. The amount and frequency of administration will depend, of course, on such factors as the nature and severity of the indication being treated, the desired response, the condition of the patient, and so forth.
In a preferred embodiment, the invention provides methods of treating diseases or disorders associated with LTXR. In embodiments, the disorder is asthma. The methods can comprise administering the LTXR to an individual suffering from asthma. Alternatively, the methods can comprise administering to an individual antibodies that specifically react to LTXR. In other embodiments, the methods can comprise administering at least one agonist of LTXR to an individual suffering from asthma. Alternatively, the methods can comprise administering an agonist of LTXR to an individual suffering from asthma. Preferably, the methods include administering the selected compound in an amount sufficient to bring about the desired level of treatment of asthma.
In another preferred embodiment, the invention provides methods of modulating the activity of LTXR receptors. More particularly, the immunologically important phagocytes include polymorphonuclear leukocytes (e.g., neutrophils) and mononuclear phagocytes (e.g., monocytes and macrophages). Phagocyte hypofunction is a cause of recurrent pyogenic infection. To combat pyogenic infection, neutrophils and monocytes respond to chemotactic factors by moving toward the source of infection, where they ingest microorganisms and kill them. A main function of polymorphonuclear leukocytes and monocytes is to kill bacteria and other infectious agents by phagocytosis. The first stage in the ingestion and digestion of a particulate substance by these cells involves the process of bringing the cells and the particles together, usually through chemotaxis. This response is an essential part of host defense against infection. The extensive migration and activity of these cells are manifested by inflammation at the site of injury or invasion of the host.
It has been shown that LTB4 induces chemotaxis by neutrophils. The heptahelix receptor of the invention is capable of modulating the chemoattractant effect of LTB4 chemotaxis. This can be demonstrated by carrying out an assay for neutrophil chemotaxis using LTB4 and the heptahelix receptor of the invention, as disclosed herein. By the use of the heptahelix receptor of the invention, it is possible to inhibit chemotaxis of granulocytes, monocytes, and macrophages. Thus, the receptor of the invention is capable of modulating directional movement of cells. This invention makes it possible to inhibit inflammation associated with cell chemotaxis. The receptor can be administered to a patient to inhibit the effects of chemotactic factors of bacterial or viral origin, or components of plasma activation systems, or factors elaborated by cells of the immune system.
Leukocyte response to an acute inflammatory stimulus involves a complex series of events, including adherence to endothelium near the stimulus. Inhibition of leukocyte adherence can be expected to reduce the degree of inflammation seen in conditions, such a septic shock and adult respiratory distress syndrome. LTB4 promotes adherence of polymorphonuclear leukocytes (PMN), such as neutrophils, to endothelial cell walls. Since the heptahelix receptor of this invention can act as a receptor for LTB4, the receptor can effectively block such adherence.
Purified PMN cells can be incubated with a lipopolysaccharide- stimulated mononuclear leukocyte conditioned medium containing LTB4. PMN adherence to nylon can be determined without the receptor of this invention, and then with the receptor of this invention. Polymorphonuclear leukocyte (PMN) adherence to nylon can be readily demonstrated. The compounds employed in the process of this invention are effective in blocking adherence of leukocytes and thereby aiding in reducing the degree of inflammation.
Mature phagocytes are in a metabolically dormant state. It is currently believed that recognition of certain objects and substances by phagocytes, such as the attachment of an ingestible particle to the cell surface, changes this situation, and the cell enters a stage of increased metabolic activity, which is referred to as metabolic or respiratory burst. The transition is associated with a series of characteristic changes, including the production of a superoxide anion. LTB4 is capable of producing a similar effect. In addition to its significance for phagocytic function related to inactivation of ingested microbes, activation of oxygen metabolism is a useful indirect marker for the ingestion process per se. It would be desirable to be able to modulate the effect of LTB4 on respiratory burst.
Quantitative methods for direct measurement of hydrogen peroxide and superoxide anions released into the medium are currently available. The heptahelix receptor employed in this invention is capable of modulating respiratory burst in stimulated polymorphonuclear leukocytes (PMN) as determined using these methods. The heptahelix receptor used in the process of this invention is capable of reducing superoxide production and modulating respiratory burst in phagocytes, such as polymorphonuclear leukocytes and monocytes, and thereby aid in reducing the degree of inflammation.
During ingestion, granules in the cytoplasm of the cell fuse with the membrane of a vacuole that was formed around the foreign substance. The granules discharge their contents into the vacuole. Some of this material ends up in the medium surrounding the phagocyte. Since the granules disappear during this process, it is called degranulation. The granule contents include hydrolytic enzymes, lysozyme, bactericidal proteins, and, in the neutrophil, myleoperoxidase.
Degranulation can be assessed by measuring the rate of appearance of granule-associated enzymes in the extracellular medium. In the case of polymorphonuclear leukocytes (PMN), degranulation can be assayed by determining release of lysozyme. LTB4 induces the release of lysosomal enzymes. The heptahelix receptor employed in the process of this invention is capable of modulating the release of lysozyme from stimulated PMN and thereby aid in reducing the degree of inflammation.
In summary, the heptahelix receptor of the invention is capable of modulating the effects of leukotrienes, such as LTB4, on polymorphonuclear leukocytes and mononuclear phagocytes. The receptor is capable of inhibiting cell chemotaxis. In addition, the receptor can block adherence of cells. The compounds can decrease oxidative damage to host tissues by phagocytes as evidenced by modulation of respiratory burst in cells stimulated by LTB4. Finally, the receptor can modulate the effects of LTB4 on degranulation in stimulated phagocytes. These effects are suggestive of clinical effectiveness in at least the following areas and conditions.
Among the conditions that can be treated or alleviated by the inhibition of LTB4 are: sepsis, septic shock, endotoxic shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress, cachexia secondary to AIDS, rheumatoid arthritis, and gouty arthritis.
By reference to the specific cause of the disease condition, the more generic term "trauma" can be used. The term "trauma" refers broadly to cellular attack by foreign bodies, such as microorganisms, particulate matter, chemical agents, and the like. Included among physical injuries are mechanical injuries, such as those caused by contact with sources of electrical injuries, such as those caused by contact with sources of electrical potential; and radiation damage caused, for example, by prolonged, extensive exposure to infrared, ultraviolet, or ionizing radiations.
LTB4 is also characteristically a product of blood phagocytes. The leukotrienes bind to specific receptors on smooth muscle cells and cause prolonged bronchoconstriction. The receptor of this invention is useful for alleviating this condition, including asthmatic bronchoconstriction.
EXAMPLES
A preferred embodiment of the invention will now be described in still greater detail in the following Examples. These examples are offered by way of illustration, and not by way of limitation. Example 1 : Cloning of LTXR
In search of additional members of the leukotriene receptor family, the sequence encoding the first cloned leukotriene receptor (Owman et al., 1996a) was run against various databases. More specifically, EST clone 444- J19 (IMAGE ID 223218) was identified by comparing the previously cloned BLTR receptor (Owman et al., 1996a) to the NCBI EST database using searches with the BLAST algorithm (Altshul et al., 1990). The IMAGE EST clone showing the highest level of identity with the BLTR sequence was obtained from the UK HGMP Resource Centre, Hinxton, Cambridge, England. This clone had an almost 2.7 kb human DNA insert in a pT7T3D-Pac vector. The clone was digested with Xhol and Notl to liberate the human insert, and the insert was purified on an agarose gel. The purified insert was ligated to a Notl-digested mammalian expression vector, pcDNA3 (Invitrogen). After transfer of the insert to the pcDNA3 vector, the insert was analyzed in transient transfection assays in which calcium response after addition of potential ligands was monitored. However, no activation could be seen in these assays. Subsequent sequence analysis revealed that the human DNA insert from the EST clone contained an open reading frame having identity to the original BLTR heptahelix receptor sequence. However, the newly isolated sequence did not contain a stop codon in the correct reading frame, and thus encoded a truncated version of a putative new heptahelix receptor, lacking sequences encoding the C-terminal part of the receptor.
Because the EST clone that was identified as containing heptahelix receptor coding sequences did not encode a functional heptahelix receptor, the missing C-terminal part of the gene was amplified from a human leukocyte cDNA library (HL4050AH from Clontech) using PCR and Pwo polymerase (Bohringer Mannheim). The following two primers were used:
5'-TTCGTGCTTCCTTTCGGGCTGAT-3' (SEQ ID NO:3) and
5'-AGTCTAGAGGGTCTGCTGTCAAAGG-3' (SEQ ID NO:4). One primer was designed from a sequence motif from the cloned EST sequence, and the other primer was designed from a clone encoding a potential C-terminal sequence (AF075012).
More specifically, in sequencing the original EST human DNA, it was observed that 100 bases in the 3' end of the EST clone was identical to the 5' end of a BLTR leader sequence (Akbar et al., 1996). During the synthesis of the cDNA library, from which the EST had been made (Hillier et al., 1995), the cDNA had been cut with the enzyme Notl, before being ligated to the cloning vector. A Notl site is also present 100 bp into the BLTR leader sequence. Thus, it was hypothesized that the novel putative heptahelix receptor and the BLTR gene might be linked in the genome. Therefore, the above-described PCR reactions were performed using one primer that was specific for a sequence in the EST clone and one primer specific for a sequence located within the BLTR leader sequence. The PCR product was litigated to the TA-cloning vector pCR2.1 from Invitrogen. A Notl/Xbal fragment from the PCR product, containing the desired C-terminal part of the receptor, was cleaved out, purified on an agarose gel, and subsequently ligated to the Notl/Xbal sites in the original (truncated) pcDNA3 construct containing the human EST sequence, yielding a 2.8 kb cDNA clone containing a complete coding sequence for the putative heptahelix receptor (see Fig. 1). Example 2: Exodeletions and Subcloning
The complete pcDNA3 construct was cleaved with BamHI and Kpnl. The linearized DNA was used to make exodeletions using the Erase-a-Base System from Promega. DNA was then prepared from 3 ml of overnight cultures from individual clones, according to the alkaline lysis method (Current Protocols in Molecular Biology). The DNA obtained was treated with RNase A for 30 min at 37°C, before being purified using the QIAquick PCR Purification K from Qiagen. The exodeletion clones were used in sequencing reactions along with a 1.3 kb Apal fragment from the pcDNA3 construct, which was cleaved out and cloned into an Apal site in pBluescrip SK+. Example 3: Sequencing and Sequence Comparisons
Sequencing was performed using the Big Dye TM Terminator Cycle Sequencing kit from PE Applied Biosystems. Sp6 and T7 primers were used to sequence the exodeleted and the full-length pcDNA3 construct. T3 and T7 primers were used to sequence the Apal fragment in pBluescript SK+. The nucleotide sequence of the cDNA is depicted in Fig. 1. The nucleotide sequence (SEQ ID NO:1 ) contains an open reading frame that encodes a novel heptahelix receptor, which has been named LTXR, for leukotriene-Iike receptor X. The deduced amino acid sequence of the encoded LTXR receptor is presented in Fig. 1 as SEQ ID NO:2. The cloning and sequencing strategy is depicted in Fig. 2.
The full-length cDNA (Fig. 1) contains an open reading frame with two in-frame methionines preceding the first membrane-spanning helix region and an in-frame stop signal. It is likely that the first methionine shown in Figure 1 is used for initiation of translation, since it conforms better to the optimal "Kozak sequence" (Kozak, 1986) for initiation of translation than does Met-22. However, it is also possible that Met-22 also might function as a translational start, but at a lower frequency (Kozak 1986).
The predicted polypeptide contains 389 amino acid residues, giving a calculated relative molecular mass of approximately 42 kDa. The receptor protein shows several features common to the G-protein linked heptahelix receptors, namely (a) consensus sequence for one N-linked glycosylation (Asn-X-Ser/Thr) at Asn-41 in the N-terminal extracellular tail; (b) conserved cysteine residues in each of the first (Cys-125) and second (Cys-199) extracellular loops, providing the possibility to form a stabilizing disulfide bond in the protein structure; (c) proline residues in all (but the third) membrane- spanning regions thought to induce flexibility within the helix structures; and (d) a C-terminus with serine and threonine residues that might serve as substrate for serine/threonine protein kinases (Alexander et al., 1999; Owman et al., 1997). Example 4: Cell Transfections
The new clone, encoding a full length version of a previously unknown receptor (the above-mentioned EST fragment from the public DAN sequence repository had not previously been identified or characterized as a receptor, but merely existed as a piece of sequence in a data bank), was then tested in transient transfection assays to find ligands that could activate the receptor.
More specifically, HeLa cells were transfected with the LTXR-pcDNA3 construct together with an aequorin-pcDNA3 reporter construct as follows:
- HeLa cells were seeded in 6-well plates and incubated in Dulbecco's MEM (DMEM) from Gibco/BRL with 1.5% penicillin, 0.5% streptomycin, 10% FSB, and 1T glutamax at 37°C. After 2-3 days, when the cells were 60-80% confluent, they were transfected using Lipofectamine Plus (Gibcoc/BRL).
- DNA was prepared using the DNA midiprep kit from Genomed. Then, 0.9 μg of LTXR-pcDNA3 and 0.4 μg aequorin-pcDNA3 were mixed with 100 μl of serum-free medium (OptiMEM from Gibco/BRL and 0.5% penicillin, 0.5% streptomycin, 1% Glutamax) and 5 μl of Plus-reagent in an eppendorff tube.
In a second eppendorff tube, 100 μl of the same serum-free medium and 9 μl of Lipofectamine were mixed. The two tubes were incubated at room temperature for 15 min and subsequently mixed. After 15 min incubation at room temperature, 800 μl serum-free medium was added to the tube, and the mixture was added to a well with HeLa cells that previously had been washed with serum-free medium.
- The cells were incubated with the DNA mixture at 37°C for 5.5 and 6h. Half of the transfection solution was then removed and 3 ml of medium was added to each well (DEM and 10% FSB, 1% glutamax, 0.5% penicillin, 0.5% streptomycin).
- After 2 days of incubation at 37°C the cell medium was removed and the cells were washed with 2 ml PBS. The cells were then incubated for 4 hours with 2ml of a coelentrazine solution (DMEM with 1 % glutamax, 0.5% penicillin, 0.5% streptomycin, 0.1 % FBS, 5μM coelentrazine) at 37°C in the dark. After removal of the coelentrazine solution, the cells were resuspended by treatment with PBS Dulbecco's medium without calcium and magnesium, with 2mM EDTA for 15 min. After brief centrifugation and one wash they were resuspended in room-temperature ECB (extracellular buffer, 140 mM NaCl, 20 mM KCl, 20 mM HEPES, 1 mM MgC12, 1 mM CaC12, 5mM glucose, 0.1 mg/ml BSA) at a concentration of approximately 400,000 cells/ml. Example 5: Cellular Calcium Measurements
The activity of the cloned cDNA was assayed using recombinant cells expressing the LTXR protein. More specifically, changes in cellular calcium flow and resulting cellular calcium levels were measured using a LUMIstar luminometer from Lab Vision, using a 96-well format. The assay is based on the fact that, when cellular calcium levels are elevated, for example as a consequence of receptor activation, aequorin together with the substance coelentrazine, emits a transient light signal that can be detected in a luminometer.
For the assay, one hundred μl of cells were pumped into a well containing 100 μl of ligand in ECB buffer and the relative luminescence was measured and recorded. It takes approximately five to ten seconds after the addition of ligand until the LTXR- mediated calcium response is seen in these HeLa cells. This is true also for the previously cloned BLTR receptor (data not shown).
A representative experiment using LTXR is shown in Fig. 3. Different substances were tested and it was found that LTXR was activated by leukotriene B4. The nature of the response in this system resembled that of the previously cloned receptor of leukotriene B4, BLTR. However, the response differed in that the activation by LTB4 was exclusive with LTXR, whereas the receptor BLTR could be activated also by LTC4 and LTD4 (Fig. 4). As a negative control, HeLa cells that had been mock transfected with an EGFP-pcDNA3 construct together with aequorin-pcDNA3 were also analyzed (Figs. 3 and 4). In each experiment, every ligand was tested in triplicate to avoid experimental artifacts. Thus, this invention provides a new heptahelix receptor of the chemoattractant receptor family, which is capable of binding ligands, including LTB4. Example 6: Phylogenetic Analysis
Protein sequences of related human GPCRs and other known leukotriene receptors of different species, were obtained from GenBank using BLAST (Altshul et al., 1990) and aligned using ClustalX version 1.8. The maximum parsimony (PROTPARS) method was applied to the alignment using the PHYLIP package to produce the phylogenetic tree (with bootstrap resampling of 100 subreplicates for statistical significance evaluation).
In a BLAST search of the sequence databases, LTXR was clearly the receptor that presented the highest degree of similarity with BLTR, although there is only 37% overall amino acid sequence identity between the two receptors (Fig. 5). However, the transmembrane regions (l-VII in Fig. 5) are very similar, a few of them almost identical. Typically, subtypes within the same receptor family would exhibit 50-65% sequence identity, and higher over the transmembrane regions. The NPY receptor family, for example, also includes subtypes that are considerably more divergent, with only 31-34% overall sequence identity (Grundemar et al., 1997). The most highly conserved motif in the heptahelix receptor superfamily is the DRY motif at the end of the third transmembrane region, the arginine residue being particularly well conserved. The corresponding QRC motif of LTXR is very unusual. Though not clearly being a subtype of the BLTR, the present data (Fig. 6) indicate a close evolutionary relationship of the LTXR to the BLTR, whereas the other known leukotriene receptor, CYSLT1 (Lynch et al., 1999) shows a more significant relationship to the formyl peptide receptor-like group. Example 7: Expression Analysis Using mRNA Assays
Expression analysis of LTXR was performed by hybridization in an mRNA array blot (Fig. 7). More specifically, a 5'- TCCAGTTTTGCCCAGATGTGCTA-3' (SEQ ID NO:5) and a 5'- TTCCAGCTCAGCAGTGTCTCGTT-3' (SEQ ID NO:6) primer pair was used to amplify a 422 bp PCR fragment (Probe A). The sequence of the probe is mainly from the LTXR untranslated leader sequence, but has an 142 bp overlap with the open reading frame. A second probe, Probe B, was amplified using the primers, 5'-GAGAGGGCTGCTTCTTAGTATGT-3' (SEQ ID NO:7) and 5'-TACTCCTGTCCTGTGCCTATCA-3 (SEQ ID NO:8), which yielded a 673 bp product located within the leader sequence only. Subsequently the PCR fragments were labelled with [α-32 PjdCTP using the Mega-prime labelling kit (Amersham) according to standard procedures (Sambrook et al., 1989) and used in an mRNA multiple tissue expression array blot (Clontech) according to the protocol supplied by the manufacturer. The blot was exposed to Kodak BioMax autoradiograph film for four days.
The mRNA array showed low expression of LTXR in most tissues, but slightly higher in liver, kidney, adrenal gland, stomach, heart, cerebellum, placenta and bladder (Fig. 7). The amount of mRNA dotted onto the array membrane was varied slightly depending on the transcriptional activity of the tissue. It was normalized to give equally strong signals of housekeeping genes. Similar array experiments made with BLTR probes (data not shown) indicate similarly a low level of expression in all cells, but with higher expression in adrenal gland, kidney, liver, heart and skeletal muscle. Example 8: Expression Analysis Using Northern Blots
Expression analysis of LTXR was performed by hybridization in Northern blots (Fig. 8). The probes disclosed in Example 7 were used for the Northem blotting experiment. The Northern blot was performed according to the protocol supplied by the manufacturer (Invitrogen). The blot was exposed to Kodak BioMax autoradiograph film for four days.
In the Northern blot experiment (Fig. 8), expression of LTXR was detected in liver, lung, and pancreas. Traces of expression were also detected in heart and placenta. In the Northern blot, equal amounts of mRNA were loaded in all lanes.
The results of the mRNA array (Example 7) can be used to give a rough estimate of the expression pattern of the LTXR gene, but the results of the Northern blot should give a more reliable answer because it is more sensitive to subtle differences in expression level. Previous Northern blots with BLTR (Owman et al., 1996a) showed high expression in leukocytes, bone marrow, lymph node, spleen, and thymus, but also in pancreas, skeletal muscle, and heart. The fact that LTXR is expressed in lung tissue, along with the location of its gene in the "asthma locus" of chromosome 14, indicates that this heptahelix receptor might play a significant role in asthma, and that it can be used to develop compositions and treatments for this important and costly disorder.
REFERENCES
The entire disclosure of each of the following publications is relied upon and incorporated by reference herein.
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Claims

WHAT IS CLAIMED IS:
1. A purified nucleic acid having a nucleotide sequence comprising SEQ ID NO:1 or a fragment thereof.
2. The purified nucleic acid of claim 1 , wherein said fragment comprises the sequence (I) in Figure 1.
3. The purified nucleic acid of claim 1 , wherein said fragment comprises the sequence (II) in Figure 1.
4. The purified nucleic acid of claim 1 , wherein said fragment comprises the sequence (III) in Figure 1.
5. The purified nucleic acid of claim 1 , wherein said fragment comprises the sequence (IV) in Figure 1.
6. The purified nucleic acid of claim 1 , wherein said fragment comprises the sequence (V) in Figure 1.
7. The purified nucleic acid of claim 1 , wherein said fragment comprises the sequence (VI) in Figure 1.
8. The purified nucleic acid of claim 1 , wherein said fragment comprises the sequence (VII) in Figure 1.
9. The purified nucleic acid of claim 1 , which encodes a receptor that binds a leukotriene.
10. A purified nucleic acid that binds under moderately stringent conditions to the nucleic acid sequence of SEQ ID NO:1.
11. A vector comprising the nucleic acid of claim 1.
12. The vector of claim 11 , wherein the vector is an expression vector.
13. A recombinant cell containing the vector of claim 11.
14. The recombinant cell of claim 13, wherein the cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell.
15. The recombinant cell of claim 14, wherein the cell is a prokaryotic cell.
16. The recombinant cell of claim 14, wherein the cell is a eukaryotic cell selected from the group consisting of a yeast cell, an insect cell, and a mammalian cell.
17. A purified nucleic acid encoding an amino acid sequence comprising the amino acid sequence of SEQ ID NO:2 or a fragment thereof.
18. A vector comprising the purified nucleic acid of claim 17.
19. A recombinant cell containing the vector of claim 18.
20. A polypeptide having an amino acid sequence comprising SEQ ID NO:2, or a fragment thereof.
21. The polypeptide of claim 20, wherein said fragment comprises an amino acid sequence selected from the group consisting of sequences (I), (II), (III), (IV), (V), (VI), and (VII) of Figure 1.
22. A composition comprising the polypeptide of claim 20 and a second substance, wherein said second substance is biologically active or biologically inactive.
23. An antibody that specifically binds to the polypeptide of claim 20.
24. A method for lowering the level of active leukotriene B4 in a mammal in need thereof, wherein said method comprises administering to said mammal a leukotriene B4-lowering amount of the polypeptide, or fragment thereof, of claim 20.
25. The method of claim 24, wherein said mammal is suffering from inflammation, bronchioconstriction, arthritis, or elevated leukocyte activity.
26. A method of treating asthma, wherein said method comprises administering a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a fragment thereof, to an individual in an amount sufficient to reduce lung tissue inflammation in the individual.
27. The method of claim 26, wherein the method eliminates lung tissue inflammation.
28. The method of claim 25, wherein said inflammation is dues to leukocyte activity induced by LTB4.
29. The method of claim 28, wherein the LTB4 is produced in response to an inflammatory stimulus in the mammal.
30. The method of claim 24, wherein the mammal is a human.
31. A method for assaying a ligand or an antagonist or agonist for said ligand, wherein the method comprises: (A) providing a heptahelix receptor comprising the sequence of SEQ ID NO:2, or a fragment thereof that comprises a binding domain for the ligand, antagonist, or agonist;
(B) incubating the receptor or fragment thereof with a test sample suspected of containing the ligand, antagonist, or agonist; and
(C) determining whether the receptor bound the ligand, antagonist, or agonist, wherein said determining is accomplished by either detecting binding or failing to detect binding.
32. The method of claim 31 , wherein the receptor is expressed in a recombinant cell.
33. The method of claim 31 , wherein binding is detected by the intracellular calcium level in the host cell.
34. The method of claim 31 , wherein the method is a high- throughput screening method.
PCT/IB2000/001682 2000-05-15 2000-11-15 Molecular cloning and identification of a human leukotriene-like receptor ltxr WO2001088130A1 (en)

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WO1999042484A1 (en) * 1998-02-20 1999-08-26 Smithkline Beecham Corporation Fishboy, a g-protein coupled receptor
WO2000029423A1 (en) * 1998-11-12 2000-05-25 Merck & Co., Inc. G protein-coupled receptor resembling the leukotriene b4 receptor

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WO2000029423A1 (en) * 1998-11-12 2000-05-25 Merck & Co., Inc. G protein-coupled receptor resembling the leukotriene b4 receptor

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