DNA encoding a mammalian LPA receptor and uses thereof
This invention provides an isolated nucleic acid encoding a mammalian LPA receptor, a purified mammalian LPA receptor, vectors comprising isolated nucleic acid encoding an mammalian LPA receptor, cells comprising such vectors, antibodies directed to a mammalian LPA receptor, nucleic acid probes useful for detecting nucleic acid encoding a mammalian LPA receptor, antisense oligonucleotides complementary to unique sequences of nucleic acid encoding mammalian LPA receptor, transgenic, nonhuman animals which express DNA encoding a normal or a mutant mammalian LPA receptor, methods of isolating an mammalian LPA receptor, methods of treating an abnormality that is linked to the activity of the mammalian LPA receptor, as well as methods of determining binding of compounds to mammalian LPA receptors.
[0001] Throughout this application, various publications are referenced in parentheses by author and year. Full citations for these references may be found at the end of the specification immediately preceding the sequence listings and the claims. The disclosure of these publications in their entireties are hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.
[0002] Biologically active phospholipids are emerging as important intracellular signaling molecules. Among these, lysophosphatidic acid (LPA; 1-acyl-glycerol-3-phosphate) is the simplest glycerophospholipid (Moolenaar, 1994). LPA produces a wide variety of biological responses such as induction of cell proliferation, stimulation of neurite retraction, platelet aggregation, smooth muscle contraction, tumor cell invasion, neurotransmitter release, chloride efflux and chemotaxis (Moolenaar, 1994; Jalink et al., 1994; Van Corven et al., 1989; Tigyi et al., 1994; Tigyi and Miledi, 1992; Jalink et al., 1993; Tokumura et al., 1994; Piazza et al., 1995). LPA is the product of the blood-clotting process and is therefore present in serum. LPA derived from platelets appears to be an important mediator of wound healing and tissue regeneration (Moolenaar, 1995).
[0003] The known effects of LPA appear to be mediated by G protein-coupled receptor(s). Specific LPA binding sites have been demonstrated in membranes from 3T3 cells and rat brain with Kd values in the low nanomolar range (Thomson et al., 1994). The actions of LPA are mediated by at least four G protein-mediated signaling pathways: stimulation of phospholipase C and phospholipase D, inhibition of adenylyl cyclase, activation of Ras and Raf/MAP kinase pathway, and tyrosine phosphorylation of focal adhesion proteins (Moolenaar, 1995).
[0004] We describe here the isolation and characterization of a novel mammalian LPA receptor, specifically a human LPA receptor. This receptor can serve as a tool for the drug design of novel therapeutic agents for various indications, including cancer (preferably tumor reduction or prevention), platelet aggregation (as an anti-coagulant), vascular restenosis, arthritis (as an anti-inflammatory), wound healing, tissue regeneration (preferably skin and nerve regeneration), blood coagulation, osteoporosis (bone regeneration), and cosmetic uses (preferably the prevention of abnormal growths or scarring or for augmentation).
[0005] Independently, the identification of a complementary DNA from Xenopus that encodes a functional high-affinity receptor for LPA has recently been reported (Guo, et al., 1996). Analysis of this receptor revealed that it is a G-protein-coupled-receptor.
SUMMARY OF THE INVENTION[0006] This invention provides an isolated nucleic acid encoding a mammalian LPA receptor. In an embodiment, the mammalian LPA receptor is a human LPA receptor.
[0007] This invention provides a purified mammalian LPA receptor protein.
[0008] This invention provides a vector comprising a nucleic acid encoding a mammalian LPA receptor. This invention also provides for a vector comprising a nucleic acid encoding a human LPA receptor. Such a vector may be adapted for expression of the mammalian LPA receptor in mammalian or non-mammalian cells.
[0009] This invention provides a plasmid designated HL-18a (ATCC Accession No. 209448). This invention also provides a membrane preparation isolated from the cells.
[0010] This invention provides a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a mammalian LPA receptor, wherein the probe has a unique sequence corresponding to a sequence present within one of the two strands of the nucleic acid which encode the mammalian LPA receptor contained in plasmid HL-18a (ATCC Accession No. 209448).
[0011] This invention also provides a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a mammalian LPA receptor, wherein the probe has a unique sequence corresponding to a sequence present within (a) the nucleic acid sequence of shown in FIG. 2 (Seq. I.D. No. 2) or (b) the reverse complement thereto.
[0012] This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a unique fragment of the sequence of a nucleic acid molecule encoding a mammalian LPA receptor.
[0013] This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to the antisense sequence of a unique fragment of the sequence of a nucleic acid molecule encoding a mammalian LPA receptor.
[0014] This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the RNA of the mammalian LPA receptor, so as to prevent translation of the RNA. This invention also provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the genomic DNA encoding a mammalian LPA receptor.
[0015] This invention further provides an antibody capable of binding to a mammalian LPA receptor. This invention also provides for an antibody capable of competitively inhibiting the binding of the antibody to a mammalian LPA receptor.
[0016] This invention provides a pharmaceutical composition comprising (a) an amount of the oligonucleotide capable of passing through a cell membrane and effective to reduce expression of a mammalian LPA receptor and (b) a pharmaceutically acceptable carrier capable of passing through a cell membrane.
[0017] This invention provides a transgenic, nonhuman mammal expressing DNA encoding a mammalian LPA receptor. This invention also provides a transgenic, nonhuman mammal comprising a homologous recombination knockout of the native mammalian LPA receptor. This invention further provides a transgenic, nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a mammalian LPA receptor so placed within the genome as to be transcribed into antisense mRNA which is complementary to mRNA encoding a mammalian LPA receptor and which hybridizes to mRNA encoding a mammalian LPA receptor, thereby reducing its translation.
[0018] This invention provides for a process for identifying a chemical compound which specifically binds to a mammalian LPA receptor which comprises contacting cells containing DNA encoding and expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian LPA receptor.
[0019] This invention provides for a process for identifying a chemical compound which specifically binds to a mammalian LPA receptor which comprises contacting a membrane fragment from a cell extract of cells containing DNA encoding and expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian LPA receptor.
[0020] This invention provides for a process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian LPA receptor which comprises separately contacting cells expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the mammalian LPA receptor, a decrease in the binding of the second chemical compound to the mammalian LPA receptor in the presence of the chemical compound indicating that the chemical compound binds to the mammalian LPA receptor.
[0021] This invention provides for a process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian LPA receptor which comprises separately contacting a membrane fraction from a cell extract of cells expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the mammalian LPA receptor, a decrease in the binding of the second chemical compound to the mammalian LPA receptor in the presence of the chemical compound indicating that the chemical compound binds to the mammalian LPA receptor.
[0022] This invention provides for a method of screening a plurality of chemical compounds not known to bind to a mammalian LPA receptor to identify a compound which specifically binds to the mammalian LPA receptor, which comprises (a) contacting cells transfected with and expressing DNA encoding the mammalian LPA receptor with a compound known to bind specifically to the mammalian LPA receptor; (b) contacting the preparation of step (a) with the plurality of compounds not known to bind specifically to the mammalian LPA receptor, under conditions permitting binding of compounds known to bind the mammalian LPA receptor; (c) determining whether the binding of the compound known to bind to the mammalian LPA receptor is reduced in the presence of the compounds within the plurality of compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so,
[0023] (d) separately determining the binding to the mammalian LPA receptor of compounds, so as to thereby identify the compound which specifically binds to the mammalian LPA receptor.
[0024] This invention provides for a method of screening a plurality of chemical compounds not known to bind to a mammalian LPA receptor to identify a compound which specifically binds to the mammalian LPA receptor, which comprises (a) preparing a cell extract from cells transfected with and expressing DNA encoding the mammalian LPA receptor, isolating a membrane fraction from the cell extract, contacting the membrane fraction with a compound known to bind specifically to the mammalian LPA receptor; (b) contacting the preparation of step (a) with the plurality of compounds not known to bind specifically to the mammalian LPA receptor, under conditions permitting binding of compounds known to bind the receptor; (c) determining whether the binding of the compound known to bind to the mammalian LPA receptor is reduced in the presence of the compounds within the plurality of compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so (d) separately determining the binding to the mammalian LPA receptor of compounds included in the plurality of compounds, so as to thereby identify the compound which specifically binds to the mammalian LPA receptor.
[0025] This invention provides for a method of detecting expression of a mammalian LPA receptor by detecting the presence of mRNA coding for the mammalian LPA receptor which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained from the nucleic acid probe under hybridizing conditions, detecting the presence of mRNA hybridizing to the probe, and thereby detecting the expression of the mammalian LPA receptor by the cell.
[0026] This invention provides for a method of detecting the presence of a mammalian LPA receptor on the surface of a cell which comprises contacting the cell with the antibody under conditions permitting binding of the antibody to the receptor, detecting the presence of the antibody bound to the cell, and thereby detecting the presence of a mammalian LPA receptor on the surface of the cell.
[0027] This invention provides for a method of determining the physiological effects of varying levels of activity of mammalian LPA receptors which comprises producing a transgenic, nonhuman mammal whose levels of mammalian LPA receptor activity are varied by use of an inducible promoter which regulates mammalian LPA receptor expression.
[0028] This invention provides for a method of determining the physiological effects of varying levels of activity of mammalian LPA receptors which comprises producing a panel of transgenic, nonhuman mammals each expressing a different amount of mammalian LPA receptor.
[0029] This invention provides for a method for identifying an antagonist capable of alleviating an abnormality wherein the abnormality is alleviated by decreasing the activity of a mammalian LPA receptor comprising administering a compound to a transgenic, nonhuman mammal, and determining whether the compound alleviates the physical and behavioral abnormalities displayed by the transgenic, nonhuman mammal as a result of overactivity of a mammalian LPA receptor, the alleviation of the abnormality identifying the compound as an antagonist. This invention also provides an antagonist identified by this method. This invention also provides for a pharmaceutical composition comprising an antagonist identified by this method and a pharmaceutically acceptable carrier.
[0030] This invention further provides for a method of treating an abnormality in a subject wherein the abnormality is alleviated by decreasing the activity of a mammalian LPA receptor which comprises administering to the subject an effective amount of this pharmaceutical composition, thereby treating the abnormality.
[0031] This invention provides for a method for identifying an agonist capable of alleviating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian LPA receptor comprising administering a compound to the transgenic, nonhuman mammal, and determining whether the compound alleviates the physical and behavioral abnormalities displayed by the transgenic, nonhuman mammal, the alleviation of the abnormality identifying the compound as an agonist. This invention also provides for an agonist identified by this method. This invention also provides for a pharmaceutical composition comprising an agonist identified by this method and a pharmaceutically acceptable carrier. This invention further provides for a method of treating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian LPA receptor which comprises administering to the subject an effective amount of this pharmaceutical composition, thereby treating the abnormality.
[0032] This invention provides for a method for diagnosing a predisposition to a disorder associated with the activity of a specific mammalian allele which comprises: (a) obtaining DNA of subjects suffering from the disorder; (b) performing a restriction digest of the DNA with a panel of restriction enzymes; (c) electrophoretically separating the resulting DNA fragments on a sizing gel; (d) contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a mammalian LPA receptor and labeled with a detectable marker; (e) detecting labeled bands which have hybridized to the DNA encoding a mammalian LPA receptor labeled with a detectable marker to create a unique band pattern specific to the DNA of subjects suffering from the disorder; (f) preparing DNA obtained for diagnosis by steps (a)-(e); and (g) comparing the unique band pattern specific to the DNA of subjects suffering from the disorder from step (e) and the DNA obtained for diagnosis from step (f) to determine whether the patterns are the same or different and to diagnose thereby predisposition to the disorder if the patterns are the same.
[0033] This invention provides for a method of preparing the purified mammalian LPA receptor which comprises: (a) inducing cells to express the mammalian LPA receptor; (b) recovering the mammalian LPA receptor from the induced cells; and (c) purifying the mammalian LPA receptor so recovered.
[0034] This invention provides a method of preparing the purified mammalian LPA receptor which comprises: (a) inserting nucleic acid encoding the mammalian LPA receptor in a suitable vector; (b) introducing the resulting vector in a suitable host cell; (c) placing the resulting cell in suitable condition permitting the production of the isolated mammalian LPA receptor; (d) recovering the mammalian LPA receptor produced by the resulting cell; and (e) purifying the mammalian LPA receptor so recovered.
[0035] This invention provides for process for determining whether a chemical compound is an mammalian LPA receptor agonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian LPA receptor with the compound under conditions permitting the activation of the mammalian LPA receptor, and detecting an increase in mammalian LPA receptor activity, so as to thereby determine whether the compound is a mammalian LPA receptor agonist. This invention further provides for a pharmaceutical composition which comprises an amount of mammalian LPA receptor agonist determined by the above-described process effective to increase activity of a mammalian LPA receptor and a pharmaceutically acceptable carrier.
[0036] This invention provides for a process for determining whether a chemical compound is a mammalian LPA receptor antagonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian LPA receptor with the compound in the presence of a known mammalian LPA receptor agonist, under conditions permitting the activation of the mammalian LPA receptor, and detecting a decrease in mammalian LPA receptor activity, so as to thereby determine whether the compound is a mammalian LPA receptor antagonist.
[0037] This invention further provides for a pharmaceutical composition which comprises an amount of mammalian LPA receptor antagonist determined by the above-described process effective to reduce activity of a mammalian LPA receptor and a pharmaceutically acceptable carrier.
[0038] This invention provides for a process for determining whether a chemical compound specifically binds to and activates a mammalian LPA receptor, which comprises contacting cells producing a second messenger response and expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with the chemical compound under conditions suitable for activation of the mammalian LPA receptor, and measuring the second messenger response in the presence and in the absence of the chemical compound, a change in the second messenger response in the presence of the chemical compound indicating that the compound activates the mammalian LPA receptor. This invention further provides for a pharmaceutical composition which comprises an amount of mammalian LPA receptor agonist determined by the above-described process effective to increase activity of a mammalian LPA receptor and a pharmaceutically acceptable carrier.
[0039] This invention provides for a process for determining whether a chemical compound specifically binds to and inhibits activation of a mammalian LPA receptor, which comprises separately contacting cells producing a second messenger response and expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with both the chemical compound and a second chemical compound known to activate the mammalian LPA receptor, and with only the second chemical compound, under conditions suitable for activation of the mammalian LPA receptor, and measuring the second messenger response in the presence of only the second chemical compound and in the presence of both the second chemical compound and the chemical compound, a smaller change in the second messenger response in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound indicating that the chemical compound inhibits activation of the mammalian LPA receptor. This invention further provides for a pharmaceutical composition which comprises an amount of mammalian LPA receptor antagonist determined by the above-described process effective to reduce activity of a mammalian LPA receptor and a pharmaceutically acceptable carrier.
[0040] This invention provides for a method of screening a plurality of chemical compounds not known to activate a mammalian LPA receptor to identify a compound which activates the mammalian LPA receptor which comprises: (a) contacting cells transfected with and expressing the mammalian LPA receptor with the plurality of compounds not known to activate the mammalian LPA receptor, under conditions permitting activation of the mammalian LPA receptor; (b) determining whether the activity of the mammalian LPA receptor is increased in the presence of the compounds; and if so (c) separately determining whether the activation of the mammalian LPA receptor is increased by each compound included in the plurality of compounds, so as to thereby identify the compound which activates the mammalian LPA receptor. This invention further provides for a pharmaceutical composition which comprises an amount of mammalian LPA receptor agonist determined by the above-described process effective to increase activity of a mammalian LPA receptor and a pharmaceutically acceptable carrier.
[0041] This invention provides for a method of screening a plurality of chemical compounds not known to inhibit the activation of a mammalian LPA receptor to identify a compound which inhibits the activation of the mammalian LPA receptor, which comprises: (a) contacting cells transfected with and expressing the mammalian LPA receptor with the plurality of compounds in the presence of a known mammalian LPA receptor agonist, under conditions permitting activation of the mammalian LPA receptor; (b) determining whether the activation of the mammalian LPA receptor is reduced in the presence of the plurality of compounds, relative to the activation of the mammalian LPA receptor in the absence of the plurality of compounds; and if so (c) separately determining the inhibition of activation of the mammalian LPA receptor for each compound included in the plurality of compounds, so as to thereby identify the compound which inhibits the activation of the mammalian LPA receptor. This invention further provides for a pharmaceutical composition which comprises an amount of mammalian LPA receptor antagonist determined by the above-described process effective to decrease activity of a mammalian LPA receptor and a pharmaceutically acceptable carrier.
[0042] This invention provides for a method of treating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian LPA receptor which comprises administering to the subject an amount of compound which is a mammalian LPA receptor agonist effective to treat the abnormality.
[0043] This invention provides for a method of treating an abnormality in a subject wherein the abnormality is alleviated by decreasing the activity of a mammalian LPA receptor which comprises administering to the subject an amount of compound which is a mammalian LPA receptor antagonist effective to treat the abnormality.
BRIEF DESCRIPTION OF THE FIGURES[0044] FIGS. 1A-1C
[0045] Nucleotide sequences encoding two human LPA receptors are shown. The smaller of these sequences is included within the larger. In addition, partial 5′ and 3′ untranslated sequences are shown. This entire sequence is designated as Seq. I.D. No. 1. In FIGS. 1A-1C, two start (ATG) codons (at positions 167-169 and 350-352) and one stop (TAG) codon (at position 1466-1468) are underlined. Using the downstream ATG (at position 350-352), with a better Kozak consensus site (Kozak, 1991), results in an open reading frame of 1116 nucleotides. The upstream ATG (at position 167-169) results in a reading frame containing 1299 nucleotides.
[0046] FIGS. 2A-2B
[0047] Nucleotide sequence (Seq. I.D. No. 2) encoding a human LPA receptor. The coding sequence starts from the start codon at positions 350-352 of the smaller reading frame shown in FIGS. 1A-1C and ends at the termination codon at positions 1466-1468 shown in FIGS. 1A-1C.
[0048] FIGS. 3A-3B
[0049] Deduced amino acid sequence (Seq. I.D. No. 3) of the human LPA receptor encoded by the nucleotide sequence shown in FIGS. 2A-2B (Seq. I.D. No. 2). Seven solid lines designated I-VII located above portions of the sequence indicate the seven putative transmembrane (TM) spanning regions.
[0050] FIGS. 4A-4D
[0051] Sequence alignment comprising the nucleotide coding sequence (Seq. I.D. No. 2) of the human LPA receptor and the corresponding nucleotide coding sequence of the Xenopus LPA receptor (Seq. I.D. No. 4) (Guo, 1996). The nucleotide coding sequence of the human LPA receptor (first top line) is aligned with the corresponding nucleotide coding sequence of the Xenopus LPA receptor.
DETAILED DESCRIPTION OF THE INVENTION[0052] Throughout this application, the following standard abbreviations are used to indicate specific nucleotide bases: 1 A = adenine G = guanine C = cytosine T = thymine U = uracil M = adenine or cytosine R = adenine or guanine W = adenine, thymine, or uracil S = cytosine or guanine Y = cytosine, thymine, or uracil K = guanine, thymine, or uracil V = adenine, cytosine, guanine (not thymine or uracil) H = adenine, cytosine, thymine, or uracil (not guanine) D = adenine, guanine, thymine, or uracil (not cytosine) B = cytosine, guanine, thymine, or uracil (not adenine) N = adenine, cytosine, guanine, thymine, or uracil (or other modified base such as inosine) i = inosine
[0053] Furthermore, the term “agonist” is used throughout this application to indicate any peptide or non-peptidyl compound which increases the activity of any of the polypeptides of the subject invention. The term “antagonist” is used throughout this application to indicate any peptide or non-peptidyl compound which decreases the activity of any of the polypeptides of the subject invention.
[0054] The activity of a G-protein coupled receptor such as the polypeptides disclosed herein may be measured using any of a variety of functional assays in which activation of the receptor in question results in an observable change in the level of some second messenger system, including but not limited to adenylate cyclase, calcium mobilization, arachidonic acid release, ion channel activity, inositol phospholipid hydrolysis or guanylyl cyclase. Heterologous expression systems utilizing appropriate host cells to express the nucleic acid of the subject invention are used to obtain the desired second messenger coupling. Receptor activity may also be assayed in an oocyte expression system.
[0055] It is possible that the mammalian LPA receptor contains introns and furthermore, the possibility exists that additional introns could exist in coding or non-coding regions. In addition, spliced form(s) of mRNA may encode additional amino acids either upstream of the currently defined starting methionine or within the coding region. Further, the existence and use of alternative exons is possible, whereby the mRNA may encode different amino acids within the region comprising the exon. In addition, single amino acid substitutions may arise via the mechanism of RNA editing such that the amino acid sequence of the expressed protein is different than that encoded by the original gene (Burns et al., 1996; Chu et al., 1996). Such variants may exhibit pharmacologic properties differing from the polypeptide encoded by the original gene.
[0056] This invention provides a splice variant of the mammalian LPA receptor disclosed herein. This invention further provides for alternate translation initiation sites and alternately spliced or edited variants of nucleic acids encoding mammalian LPA receptors of this invention.
[0057] This invention provides the above-described isolated nucleic acid, wherein the nucleic acid is DNA. In an embodiment, the DNA is cDNA. In another embodiment, the DNA is genomic DNA. In still another embodiment, the nucleic acid molecule is RNA. Methods for production and manipulation of nucleic acid molecules are well known in the art.
[0058] This invention further provides nucleic acid which is degenerate with respect to the DNA comprising the coding sequence of the plasmid HL-18a.
[0059] This invention also encompasses DNAs and cDNAs which encode amino acid sequences which differ from those of the polypeptides of this invention, but which should not produce phenotypic changes. Alternatively, this invention also encompasses DNAs, cDNAs, and RNAs which hybridize to the DNA, cDNA, and RNA of the subject invention. Hybridization methods are well known to those of skill in the art.
[0060] The nucleic acids of the subject invention also include nucleic acid molecules coding for polypeptide analogs, fragments or derivatives of antigenic polypeptides which differ from naturally-occurring forms in terms of the identity or location of one or more amino acid residues (deletion analogs containing less than all of the residues specified for the protein, substitution analogs wherein one or more residues specified are replaced by other residues and addition analogs where in one or more amino acid residues is added to a terminal or medial portion of the polypeptides) and which share some or all properties of naturally-occurring forms. These molecules include: the incorporation of codons “preferred” for expression by selected non-mammalian hosts; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate DNA sequences that facilitate construction of readily expressed vectors.
[0061] The modified polypeptides of this invention may be transfected into cells either transiently or stably using methods well-known in the art, examples of which are disclosed herein. This invention also provides for binding assays using the modified polypeptides, in which the polypeptide is expressed either transiently or in stable cell lines. This invention further provides for a compound identified using a modified polypeptide in a binding assay such as the binding assays described herein.
[0062] The nucleic acids described and claimed herein are useful for the information which they provide concerning the amino acid sequence of the polypeptide and as products for the large scale synthesis of the polypeptide by a variety of recombinant techniques. The nucleic acid molecule is useful for generating new cloning and expression vectors, transformed and transfected prokaryotic and eukaryotic host cells, and new and useful methods for cultured growth of such host cells capable of expression of the polypeptide and related products.
[0063] This invention provides for an isolated nucleic acid encoding a mammalian LPA receptor. In one embodiment, the nucleic acid is DNA. In another embodiment, the DNA in cDNA. In another embodiment, the DNA is genomic DNA. In another embodiment, the nucleic acid is RNA.
[0064] In an embodiment of the present invention, the mammalian LPA receptor is a human LPA receptor.
[0065] In an embodiment, the nucleic acid encodes a mammalian LPA receptor which has substantially the same amino acid sequence as does the mammalian LPA receptor encoded by the plasmid HL-18a (ATCC Accession No. 209448). In another embodiment, the nucleic acid encodes a human LPA receptor which has an amino acid sequence identical to that encoded by the plasmid HL-18a (ATCC Accession No. 209448). In another embodiment, the human LPA receptor has an amino acid sequence, substantially the same as the amino acid sequence shown in FIG. 3 (Seq. I.D. No. 3). In another embodiment, the human LPA receptor has an amino acid sequence identical to the amino acid sequence shown in FIG. 3 (Seq. I.D. No. 3).
[0066] This invention provides an isolated nucleic acid encoding a modified mammalian LPA receptor, which differs from a mammalian LPA receptor by having an amino acid(s) deletion, replacement, or addition in the third intracellular domain.
[0067] This invention provides for a purified mammalian LPA receptor protein.
[0068] This invention provides a vector comprising the nucleic acid of a mammalian LPA receptor. In a further embodiment, the mammalian LPA receptor is a human LPA receptor.
[0069] In an embodiment, the vector is adapted for expression in a bacterial cell which comprises the regulatory elements necessary for expression of the nucleic acid in the bacterial cell operatively linked to the nucleic acid encoding a mammalian LPA receptor as to permit expression thereof. In another embodiment, the vector is adapted for expression in an amphibian cell which comprises the regulatory elements necessary for expression of the nucleic acid in the amphibian cell operatively linked to the nucleic acid encoding a mammalian LPA receptor as to permit expression thereof. In another embodiment, the vector is adapted for expression in a yeast cell which comprises the regulatory elements necessary for expression of the nucleic acid in the yeast cell operatively linked to the nucleic acid encoding a mammalian LPA receptor so as to permit expression thereof. In another embodiment, the vector is adapted for expression in an insect cell which comprises the regulatory elements necessary for expression of the nucleic acid in the insect cell operatively linked to the nucleic acid encoding a mammalian LPA receptor so as to permit expression thereof. In another embodiment, the vector is a baculovirus. In another embodiment, the vector is adapted for expression in a mammalian cell which comprises the regulatory elements necessary for expression of the nucleic acid in the mammalian cell operatively linked to the nucleic acid encoding a mammalian LPA receptor so as to permit expression thereof. In a further embodiment, the vector is a plasmid.
[0070] This invention provides a plasmid designated HL-18a (ATCC Accession No. 209448). This plasmid comprises the regulatory elements necessary for expression of DNA in a mammalian cell operatively linked to DNA encoding the mammalian LPA receptor so as to permit expression thereof.
[0071] This plasmid (HL-18a) was deposited on Nov. 11, 1997, with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and was accorded ATCC Accession No. 209448.
[0072] This invention further provides for any vector or plasmid which comprises modified untranslated sequences, which are beneficial for expression in desired host cells or for use in binding or functional assays. For example, a vector or plasmid with untranslated sequences of varying lengths may express differing amounts of the polypeptide depending upon the host cell used. In an embodiment, the vector or plasmid comprises the coding sequence of the polypeptide and the regulatory elements necessary for expression in the host cell.
[0073] This invention provides a cell comprising the vector comprising a nucleic acid encoding the mammalian LPA receptor. In an embodiment, the cell is a non-mammalian cell. In another embodiment, the non-mammalian cell is a Xenopus oocyte cell or a Xenopus melanophore cell. In another embodiment, the cell is a mammalian cell. In a further embodiment, the mammalian cell is a COS-7 cell, a 293 human embryonic kidney cell, a NIH-3T3 cell, a LM(tk−) cell, a mouse Y1 cell, or a CHO cell.
[0074] This invention provides for an insect cell comprising a vector adapted for expression in an insect cell comprising a nucleic acid encoding a mammalian LPA receptor. In a further embodiment, the insect cell is an Sf9 cell, an Sf21 cell or a HighFive cell.
[0075] This invention provides for a membrane preparation isolated from any of the cells described above.
[0076] This invention provides a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a mammalian LPA receptor, wherein the probe has a unique sequence corresponding to a sequence present within one of the two strands of the nucleic acid which encode the mammalian LPA receptor contained in plasmid HL-18a. This invention also provides a nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a mammalian LPA receptor, wherein the probe has a unique sequence corresponding to a sequence present within (a) the nucleic acid sequence shown in FIG. 2 (Seq. I.D. No. 2) or (b) the reverse complement thereto. In an embodiment, the nucleic acid is DNA. In another embodiment, the nucleic acid is RNA.
[0077] This invention provides for a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a unique fragment of the sequence of a nucleic acid molecule encoding a mammalian LPA receptor. This invention also provides for a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to the antisense sequence of a unique fragment of the sequence of a nucleic acid molecule encoding a mammalian LPA receptor.
[0078] As used herein, the phrase “specifically hybridizing” means the ability of a nucleic acid molecule to recognize a nucleic acid sequence complementary to its own and to form double-helical segments through hydrogen bonding between complementary base pairs.
[0079] Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary greatly in length and may be labeled with a detectable label, such as a radioisotope or fluorescent dye, to facilitate detection of the probe. DNA probe molecules may be produced by insertion of a DNA molecule which encodes the polypeptides of this invention into suitable vectors, such as plasmids or bacteriophages, followed by transforming into suitable bacterial host cells, replication in the transformed bacterial host cells and harvesting of the DNA probes, using methods well known in the art. Alternatively, probes may be generated chemically from DNA synthesizers.
[0080] RNA probes may be generated by inserting the DNA molecule which encodes the polypeptides of this invention downstream of a bacteriophage promoter such as T3, T7 or SP6. Large amounts of RNA probe may be produced by incubating the labeled nucleotides with the linearized fragment where it contains an upstream promoter in the presence of the appropriate RNA polymerase.
[0081] This invention provides for an antisense oligonucleotide having a sequence capable of specifically hybridizing to the RNA encoding a mammalian LPA receptor, so as to prevent translation of the RNA. This invention also provides for an antisense oligonucleotide having a sequence capable of specifically hybridizing to the genomic DNA encoding a mammalian LPA receptor. In an embodiment, the antisense oligonucleotide comprises chemically modified nucleotides or nucleotide analogues.
[0082] This invention provides an antibody capable of binding to a mammalian LPA receptor. In an embodiment, the mammalian LPA receptor is a human LPA receptor. This invention also provides a agent capable of competitively inhibiting the binding of the antibody to a mammalian LPA receptor. In a further embodiment, the antibody is a monoclonal antibody or antisera.
[0083] This invention provides for a pharmaceutical composition comprising (a) an amount of an oligonucleotide capable of passing through a cell membrane and effective to reduce expression of a mammalian LPA receptor and (b) a pharmaceutically acceptable carrier capable of passing through a cell membrane. In an embodiment, the oligonucleotide is coupled to a substance which inactivates mRNA. In a further embodiment, the substance which inactivates mRNA is a ribozyme. In another embodiment, the pharmaceutically acceptable carrier comprises a structure which binds to a mammalian LPA receptor on a cell capable of being taken up by the cells after binding to the structure. In a further embodiment, the pharmaceutically acceptable carrier is capable of binding to a mammalian LPA receptor which is specific for a selected cell type.
[0084] This invention provides a pharmaceutical composition which comprises an amount of the antibody effective to block binding of a ligand to a human LPA receptor and a pharmaceutically acceptable carrier.
[0085] As used herein, “pharmaceutically acceptable carriers” means any of the standard pharmaceutically acceptable carriers. Examples include, but are not limited to, phosphate buffered saline, physiological saline, water and emulsions, such as oil/water emulsions.
[0086] This invention provides for a transgenic, nonhuman mammal expressing the DNA encoding a mammalian LPA receptor. This invention also provides for a transgenic, nonhuman mammal comprising a homologous recombination knockout of the native mammalian LPA receptor. This invention further provides for a transgenic, nonhuman mammal whose genome comprises antisense DNA complementary to the DNA encoding a mammalian LPA receptor so placed within the genome as to be transcribed into antisense mRNA which is complementary to mRNA encoding a mammalian LPA receptor and which hybridizes to mRNA encoding a mammalian LPA receptor, thereby reducing its translation. In an embodiment, the DNA encoding a mammalian LPA receptor additionally comprises an inducible promoter. In a further embodiment, the DNA encoding a mammalian LPA receptor additionally comprises tissue specific regulatory elements. In another embodiment, the transgenic, nonhuman mammal is a mouse.
[0087] Animal model systems which elucidate the physiological and behavioral roles of the polypeptides of this invention are produced by creating transgenic animals in which the activity of the polypeptide is either increased or decreased, or the amino acid sequence of the expressed polypeptide is altered, by a variety of techniques. Examples of these techniques include, but are not limited to: 1) Insertion of normal or mutant versions of DNA encoding the polypeptide, by microinjection, electroporation, retroviral transfection or other means well known to those skilled in the art, into appropriate fertilized embryos in order to produce a transgenic animal or 2) Homologous recombination of mutant or normal, human or animal versions of these genes with the native gene locus in transgenic animals to alter the regulation of expression or the structure of these polypeptide sequences. The technique of homologous recombination is well known in the art. It replaces the native gene with the inserted gene and so is useful for producing an animal that cannot express native polypeptides but does express, for example, an inserted mutant polypeptide, which has replaced the native polypeptide in the animal's genome by recombination, resulting in underexpression of the transporter. Microinjection adds genes to the genome, but does not remove them, and so is useful for producing an animal which expresses its own and added polypeptides, resulting in overexpression of the polypeptides.
[0088] One means available for producing a transgenic animal, with a mouse as an example, is as follows: Female mice are mated, and the resulting fertilized eggs are dissected out of their oviducts. The eggs are stored in an appropriate medium such as M2 medium. DNA or cDNA encoding a polypeptide of this invention is purified from a vector by methods well known in the art. Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the trans-gene. Alternatively, or in addition, tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the trans-gene. The DNA, in an appropriately buffered solution, is put into a microinjection needle (which may be made from capillary tubing using a pipet puller) and the egg to be injected is put in a depression slide. The needle is inserted into the pronucleus of the egg, and the DNA solution is injected. The injected egg is then transferred into the oviduct of a pseudopregnant mouse (a mouse stimulated by the appropriate hormones to maintain pregnancy but which is not actually pregnant), where it proceeds to the uterus, implants, and develops to term. As noted above, microinjection is not the only method for inserting DNA into the egg cell, and is used here only for exemplary purposes.
[0089] This invention provides for a process for identifying a chemical compound which specifically binds to a mammalian LPA receptor which comprises contacting cells containing DNA encoding and expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian LPA receptor. This invention also provides a process for identifying a chemical compound which specifically binds to a mammalian LPA receptor which comprises contacting a membrane fragment from a cell extract of cells containing DNA encoding and expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian LPA receptor. In an embodiment, the mammalian LPA receptor is a human LPA receptor. In a further embodiment, the mammalian LPA receptor has substantially the same amino acid sequence as encoded by plasmid HL-18a (ATCC Accession No. 209448). In a further embodiment, the mammalian LPA receptor has substantially the same amino acid sequence as shown in FIG. 3 (Seq. I.D. No. 3). In another embodiment, the mammalian LPA receptor has the amino acid sequence shown in FIG. 3 (Seq. I.D. No. 3). In another embodiment, the compound is a compound not previously known to bind to a mammalian LPA receptor. This invention further provides a compound determined by the above-described process.
[0090] In a further embodiment the cell is an insect cell. In another embodiment, the cell is a mammalian cell. In another embodiment, the cell is non-neuronal in origin. In a further embodiment, the non-neuronal cell is a COS-7 cell, 293 human embryonic kidney cell, a CHO cell, a NIH-3T3 cell, a mouse Y1 cell, or a LM(tk−) cell. In another embodiment, the compound is a compound not previously known to bind to a mammalian LPA receptor. In a further embodiment, the compound is determined by the above-described process.
[0091] This invention provides process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian LPA receptor which comprises separately contacting cells expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the mammalian LPA receptor, a decrease in the binding of the second chemical compound to the mammalian LPA receptor in the presence of the chemical compound indicating that the chemical compound binds to the mammalian LPA receptor.
[0092] This invention also provides a process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian LPA receptor which comprises separately contacting a membrane fraction from a cell extract of cells expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the mammalian LPA receptor, a decrease in the binding of the second chemical compound to the mammalian LPA receptor in the presence of the chemical compound indicating that the chemical compound binds to the mammalian LPA receptor.
[0093] In an embodiment, the mammalian LPA receptor is a human LPA receptor. In another embodiment, the human LPA receptor has substantially the same amino acid sequence as encoded by plasmid HL-18a (ATCC Accession No. 209448). In another embodiment, the mammalian LPA receptor has substantially the same amino acid sequence as shown in FIG. 3 (Seq. I.D. No. 3). In another embodiment, the mammalian LPA receptor has the amino acid sequence shown in FIG. 3 (Seq. I.D. No. 3).
[0094] In another embodiment, the cell is an insect cell. In another embodiment, the cell is a mammalian cell. In another embodiment, the cell is non-neuronal in origin. In a further embodiment, the non-neuronal cell is a COS-7 cell, 293 human embryonic kidney cell, a CHO cell, a NIH-3T3 cell, a mouse Y1 cell, or a LM(tk−) cell. In another embodiment, the compound is a compound not previously known to bind to a mammalian LPA receptor. This invention also provides for a compound determined by the above-described process.
[0095] This invention provides a method of screening a plurality of chemical compounds not known to bind to a mammalian LPA receptor to identify a compound which specifically binds to the mammalian LPA receptor, which comprises (a) contacting cells transfected with and expressing DNA encoding the mammalian LPA receptor with a compound known to bind specifically to the mammalian LPA receptor; (b) contacting the preparation of step (a) with the plurality of compounds not known to bind specifically to the mammalian LPA receptor, under conditions permitting binding of compounds known to bind the mammalian LPA receptor; (c) determining whether the binding of the compound known to bind to the mammalian LPA receptor is reduced in the presence of the compounds within the plurality of compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so (d) separately determining the binding to the mammalian LPA receptor of compounds included in the plurality of compounds, so as to thereby identify the compound which specifically binds to the mammalian LPA receptor.
[0096] This invention provides for a method of screening a plurality of chemical compounds not known to bind to a mammalian LPA receptor to identify a compound which specifically binds to the mammalian LPA receptor, which comprises (a) preparing a cell extract from cells transfected with and expressing DNA encoding the mammalian LPA receptor, isolating a membrane fraction from the cell extract, contacting the membrane fraction with a compound known to bind specifically to the mammalian LPA receptor; (b) contacting the preparation of step (a) with the plurality of compounds not known to bind specifically to the mammalian LPA receptor, under conditions permitting binding of compounds known to bind the mammalian LPA receptor; (c) determining whether the binding of the compound known to bind to the mammalian LPA receptor is reduced in the presence of the compounds within the plurality of compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so (d) separately determining the binding to the mammalian LPA receptor of compounds included in the plurality of compounds, so as to thereby identify the compound which specifically binds to the mammalian LPA receptor.
[0097] In an embodiment, the mammalian LPA receptor is a human LPA receptor. In another embodiment, the cell is a mammalian cell. In another embodiment, the mammalian cell is non-neuronal in origin. In a further embodiment, the non-neuronal cell is a COS-7 cell, a 293 human embryonic kidney cell, a LM(tk−) cell, a CHO cell, a mouse Y1 cell, or an NIH-3T3 cell.
[0098] This invention provides a method of detecting expression of a mammalian LPA receptor by detecting the presence of mRNA coding for the mammalian LPA receptor which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained from the nucleic acid probe under hybridizing conditions, detecting the presence of mRNA hybridizing to the probe, and thereby detecting the expression of the mammalian LPA receptor by the cell.
[0099] This invention provides for a method of detecting the presence of a mammalian LPA receptor on the surface of a cell which comprises contacting the cell with the antibody under conditions permitting binding of the antibody to the receptor, detecting the presence of the antibody bound to the cell, and thereby detecting the presence of a mammalian LPA receptor on the surface of the cell.
[0100] This invention provides a method of determining the physiological effects of varying levels of activity of mammalian LPA receptors which comprises producing a transgenic, nonhuman mammal whose levels of mammalian LPA receptor activity are varied by use of an inducible promoter which regulates mammalian LPA receptor expression.
[0101] This invention provides a method of determining the physiological effects of varying levels of activity of mammalian LPA receptors which comprises producing a panel of transgenic, nonhuman mammals each expressing a different amount of mammalian LPA receptor.
[0102] This invention provides a method for identifying an antagonist capable of alleviating an abnormality wherein the abnormality is alleviated by decreasing the activity of a mammalian LPA receptor comprising administering a compound to the transgenic, nonhuman mammal and determining whether the compound alleviates the physical and behavioral abnormalities displayed by the transgenic, nonhuman mammal as a result of overactivity of a mammalian LPA receptor, the alleviation of the abnormality identifying the compound as an antagonist. This invention provides an antagonist identified by the above-described method. This invention further provides a pharmaceutical composition comprising an antagonist identified by the above-described method and a pharmaceutically acceptable carrier. This invention further provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by decreasing the activity of a mammalian LPA receptor which comprises administering to the subject an effective amount of the pharmaceutical composition, thereby treating the abnormality.
[0103] This invention provides a method for identifying an agonist capable of alleviating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian LPA receptor comprising administering a compound to the transgenic, nonhuman mammal, and determining whether the compound alleviates the physical and behavioral abnormalities displayed by the transgenic, nonhuman mammal, the alleviation of the abnormality identifying the compound as an agonist. This invention also provides an agonist identified by the above-described method. This invention further provides a pharmaceutical composition comprising an agonist identified by the above-described method and a pharmaceutically acceptable carrier. This invention further provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian LPA receptor which comprises administering to the subject an effective amount of the pharmaceutical composition, thereby treating the abnormality.
[0104] This invention provides for a method for diagnosing a predisposition to a disorder associated with the activity of a specific mammalian allele which comprises: (a) obtaining DNA of subjects suffering from the disorder; (b) performing a restriction digest of the DNA with a panel of restriction enzymes; (c) electrophoretically separating the resulting DNA fragments on a sizing gel; (d) contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a mammalian LPA receptor and labeled with a detectable marker; (e) detecting labeled bands which have hybridized to the DNA encoding a mammalian LPA receptor labeled with a detectable marker to create a unique band pattern specific to the DNA of subjects suffering from the disorder; (f) preparing DNA obtained for diagnosis by steps (a)-(e); and (g) comparing the unique band pattern specific to the DNA of subjects suffering from the disorder from step (e) and the DNA obtained for diagnosis from step (f) to determine whether the patterns are the same or different and to diagnose thereby predisposition to the disorder if the patterns are the same. In an embodiment, the disorder associated with the activity of a specific mammalian allele is diagnosed.
[0105] This invention provides a method of preparing the purified mammalian LPA receptor which comprises: (a) inducing cells to express the mammalian LPA receptor; (b) recovering the mammalian LPA receptor from the induced cells; and (c) purifying the mammalian LPA receptor so recovered.
[0106] This invention provides a method of preparing the purified mammalian LPA receptor which comprises: (a) inserting nucleic acid encoding the mammalian LPA receptor in a suitable vector; (b) introducing the resulting vector in a suitable host cell; (c) placing the resulting cell in suitable condition permitting the production of the isolated mammalian LPA receptor; (d) recovering the mammalian LPA receptor produced by the resulting cell; and (e) purifying the mammalian LPA receptor so recovered.
[0107] This invention provides a process for determining whether a chemical compound is an mammalian LPA receptor agonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian LPA receptor with the compound under conditions permitting the activation of the mammalian LPA receptor, and detecting an increase in mammalian LPA receptor activity, so as to thereby determine whether the compound is a mammalian LPA receptor agonist. This invention also provides a process for determining whether a chemical compound is a mammalian LPA receptor antagonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian LPA receptor with the compound in the presence of a known mammalian LPA receptor agonist, under conditions permitting the activation of the mammalian LPA receptor, and detecting a decrease in mammalian LPA receptor activity, so as to thereby determine whether the compound is a mammalian LPA receptor antagonist. In an embodiment, the mammalian LPA receptor is a human LPA receptor.
[0108] This invention provides a pharmaceutical composition which comprises an amount of a mammalian LPA receptor agonist determined by the above-described process effective to increase activity of a mammalian LPA receptor and a pharmaceutically acceptable carrier. In a further embodiment, the mammalian LPA receptor agonist is not previously known.
[0109] This invention provides for a pharmaceutical composition which comprises an amount of a mammalian LPA receptor antagonist determined by the above-described process effective to reduce activity of a mammalian LPA receptor and a pharmaceutically acceptable carrier. In a further embodiment, the mammalian LPA receptor antagonist is not previously known.
[0110] This invention provides a process for determining whether a chemical compound specifically binds to and activates a mammalian LPA receptor, which comprises contacting cells producing a second messenger response and expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with the chemical compound under conditions suitable for activation of the mammalian LPA receptor, and measuring the second messenger response in the presence and in the absence of the chemical compound, a change in the second messenger response in the presence of the chemical compound indicating that the compound activates the mammalian LPA receptor. In an embodiment, the second messenger response comprises chloride channel activation and the change in second messenger is an increase in the level of inward chloride current.
[0111] This invention provides a process for determining whether a chemical compound specifically binds to and inhibits activation of a mammalian LPA receptor, which comprises separately contacting cells producing a second messenger response and expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with both the chemical compound and a second chemical compound known to activate the mammalian LPA receptor, and with only the second chemical compound, under conditions suitable for activation of the mammalian LPA receptor, and measuring the second messenger response in the presence of only the second chemical compound and in the presence of both the second chemical compound and the chemical compound, a smaller change in the second messenger response in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound indicating that the chemical compound inhibits activation of the mammalian LPA receptor. In a further embodiment, the second messenger response comprises chloride channel activation and the change in second messenger response is a smaller increase in the level of inward chloride current in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound.
[0112] In an embodiment, the mammalian LPA receptor is a human LPA receptor. In another embodiment, the human LPA receptor has substantially the same amino acid sequence as encoded by the plasmid HL-18a (ATCC Accession No. 209448). In another embodiment, the human LPA receptor has substantially the same amino acid sequence as that shown in FIG. 3 (Seq. I.D. No. 3). In another embodiment, the human LPA receptor has an amino acid sequence, identical to the amino acid sequence shown in FIG. 3 (Seq. I.D. No. 3).
[0113] In another embodiment, the cell is an insect cell. In another embodiment, the cell is a mammalian cell. In another embodiment, the mammalian cell is non-neuronal in origin. In a further embodiment, the non-neuronal cell is a COS-7 cell, CHO cell, 293 human embryonic kidney cell, NIH-3T3 cell or LM(tk−) cell. In another embodiment, the compound is not previously known to bind to a mammalian LPA receptor. In another embodiment, the compound is determined by the above-described process.
[0114] This invention provides for a pharmaceutical composition which comprises an amount of a mammalian LPA receptor agonist determined by the above-described process effective to increase activity of a mammalian LPA receptor and a pharmaceutically acceptable carrier.
[0115] In a further embodiment, the mammalian LPA receptor agonist is not previously known.
[0116] This invention provides for a pharmaceutical composition which comprises an amount of a mammalian LPA receptor antagonist determined by the above-described process effective to reduce activity of a mammalian LPA receptor and a pharmaceutically acceptable carrier. In a further embodiment, the mammalian LPA receptor antagonist is not previously known.
[0117] This invention provides a method of screening a plurality of chemical compounds not known to activate a mammalian LPA receptor to identify a compound which activates the mammalian LPA receptor which comprises: (a) contacting cells transfected with and expressing the mammalian LPA receptor with the plurality of compounds not known to activate the mammalian LPA receptor, under conditions permitting activation of the mammalian LPA receptor; (b) determining whether the activity of the mammalian LPA receptor is increased in the presence of the compounds; and if so (c) separately determining whether the activation of the mammalian LPA receptor is increased by each compound included in the plurality of compounds, so as to thereby identify the compound which activates the mammalian LPA receptor. In a further embodiment, the mammalian LPA receptor is a human LPA receptor.
[0118] This invention provides a method of screening a plurality of chemical compounds not known to inhibit the activation of a mammalian LPA receptor to identify a compound which inhibits the activation of the mammalian LPA receptor, which comprises: (a) contacting cells transfected with and expressing the mammalian LPA receptor with the plurality of compounds in the presence of a known mammalian LPA receptor agonist, under conditions permitting activation of the mammalian LPA receptor; (b) determining whether the activation of the mammalian LPA receptor is reduced in the presence of the plurality of compounds, relative to the activation of the mammalian LPA receptor in the absence of the plurality of compounds; and if so (c) separately determining the inhibition of activation of the mammalian LPA receptor for each compound included in the plurality of compounds, so as to thereby identify the compound which inhibits the activation of the mammalian LPA receptor. In an embodiment, the mammalian LPA receptor is a human LPA receptor.
[0119] In another embodiment, the cell is a mammalian cell. In another embodiment, the mammalian cell is non-neuronal in origin. In another embodiment, the non-neuronal cell is a COS-7 cell, a 293 human embryonic kidney cell, a LM(tk−) cell or an NIH-3T3 cell.
[0120] This invention also provides a pharmaceutical composition comprising a compound identified by the above-described method effective to increase mammalian LPA receptor activity and a pharmaceutically acceptable carrier.
[0121] This invention also provides a pharmaceutical composition comprising a compound identified by the above-described methods effective to decrease mammalian LPA receptor activity and a pharmaceutically acceptable carrier.
[0122] This invention further provides a method of measuring polypeptide activation in an oocyte expression system such as a Xenopus oocyte or melanophore. In an embodiment, polypeptide activation is determined by measurement of ion channel activity. In another embodiment, polypeptide activation is measured by aequorin luminescence.
[0123] Expression of genes in Xenopus oocytes is well known in the art (Coleman, A., 1984; Masu, Y. et al., 1994) and is performed using microinjection of native mRNA or in vitro synthesized mRNA into frog oocytes. The preparation of in vitro synthesized mRNA can be performed by various standard techniques (Sambrook et al., 1989) including using T7 polymerase with the mCAP RNA capping kit (Stratagene).
[0124] This invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian LPA receptor which comprises administering to the subject an amount of compound which is a mammalian LPA receptor agonist effective to treat the abnormality.
[0125] In an embodiment, the abnormality is a wound or tissue or cosmetic defect in which the agonist would activate wound healing, tissue regeneration (preferably skin or nerve regeneration), or cosmetic augmentation. In a another embodiment, the abnormality is osteoporosis in which the agonist would activate bone regeneration. In still another embodiment, the abnormality is a blood disorder in which the agonist would activate blood coagulation.
[0126] This invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by decreasing the activity of a mammalian LPA receptor which comprises administering to the subject an amount of compound which is a mammalian LPA receptor antagonist effective to treat the abnormality.
[0127] In a further embodiment, the abnormality is cancer in which the antagonist preferably reduces or prevents tumors, platelet aggregation in which the antagonist preferably acts as an anti-coagulant, vascular restenosis, arthritis in which the antagonist acts as an anti-inflammatory, and cosmetic uses in which the antagonist preferably prevents abnormal growths or scarring.
[0128] This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.
[0129] Experimental Details
[0130] Materials and Methods
[0131] Cloning and Sequencing a Novel Mammalian LPA Receptor
[0132] Isolation and Sequencing of a Partial Mammalian LPA Receptor
[0133] A human lymphocyte genomic phage library (Stratagene, LaJolla, Calif.) was screened using degenerate transmembrane (TM) oligonucleotide probes, from TM 2 and 7, derived from the human Y1 (Larhammar et al., 1992; GenBank accession No. Z11504), human Y2 (Gerald et al., 1995; GenBank accession No. U36269), human Y4 (Bard et al., 1995; GenBank accession No. U35232) and human Y5 (Gerald et al., 1996; GenBank accession No. U56079) neuropeptide receptor genes.
[0134] The following degenerate oligomers from TM2 were used: 2 A 5′-(A/G)(C/T)I(A/G)(C/T)(A/C)AAC(A/C/G/T)Ti (C/T)TiATiG(A/C/C/T)(G/C)AA(C/T)CT(G/T)(G/T)C(C/ T)(G/T)Ti(G/T)C (A/T)GA(C/T)(A/C/G/T)Ti(C/T)TC- 3′; B 5′-GA(C/T)(A/C/G/T)Ti(C/T)Ti(A/G)T(G/T)T(A/C/G/T) (G/C) (A/C)(C/T)I(A/C/G/T)TiTG(C/T)(C/T) (A/C/G/T)iCCi(C/T)TiACii(C/T)I(A/C)(C/T)(G/C)T( A/C)T-3′; C 5′-GT(G/C)ACiAA(C/T)(A/T)T(C/T)(C/T)T(G/C)AT (C/T)G(C/T) (C/T)AA(C/T)CT(G/C)(G/T)C(C/T)(G/T)T (C/T)(G/T)C(C/T)GA(C/T)CT(G/C)CTC-3′; and D 5′-GA(C/T)CT(G/C)CT(G/C)CT(G/C)(A/G)(A/C)(C/T)A (C/T)I(A/C)T(G/C)TG(C/T)CT(G/C)CC(A/T)TT(C/T)AC i(C/T)T(G/C)GC (G/C)TAT-3′.
[0135] The sequences of the preceding oligomers may be represented as follows: 3 A 5′-RYNRYMAACNTNYTNATNGNSAAYCTKKCYKTNKCWGAYNTNYTC-3′; (Sequence I.D. No. 5) B 5′-GAYNTNYTNRTKNNSMYNNTNTGYYNNCCNYTNACNNYNRYSTMT-3′; (Sequence I.D. No. 6) C 5′-GTSACNAAYWTYYTSATYGYYAAYCTSKCYKTYKCYGAYCTSCTC-3′; (Sequence I.D. No. 7) and D 5′-GAYCTSCTSGTSRNYAYNMTSTGYCTSCCWTTYACNYTSGCSTAT-3′, (Sequence I.D. No. 8) respectively.
[0136] The following degenerate oligomers from TM7 were used: 4 E 5′-(A/T/C)(A/C/G/T)I(G/A/C)TiT(G/T)(C/T)CA(C/T) (A/C/G/T)T(G/C)(A/T/C)(C/T)iG(Gb)(A/C)ATG(G/A/ T)iiTCC(A/T)(G/C)(C/T)T(G/T)(C/T)(G/C)(C/T)iAA( C/T)CCA-3′; F 5′-ATG(G/A/T)(A/C/G/T)iTCC(A/T)(G/C)(C/T)T(G/T) (C/T)(G/C)(C/T)(C/T)AA(C/T)CC(A/C)(A/C/C/T)Ti (A/C/G/T)TiTATGG(C/C)T(C/T)I(A/C)TiAAC-3′; G 5′-AC(C/T)(A/G)T(G/C)TT(C/T)CA(C/T)AT(C/T)AT (C/T)G(G/C)(C/T)ATG(A/T)(G/T)iTC(C/T)AC(C/T)T(G/ T)(C/T)G(C/T)(C/T)AA(C/T)CCA-3′; and H 5′-ATG(A/T)(G/T)GTC(C/T)TG(C/T)T(G/T)(C/T)CT(G/C) AA(C/T)CCA(A/C)T(C/T)CT(G/C)TA(C/T)GG(A/C)T(G/T) (C/T)CT(G/C)AAT-3′.
[0137] The sequences of the preceding oligomers may be represented as follows: 5 E 5′-HNNVTNTKYCAYNTSHYNGSMATGDNNTCCWSYTKYSYNAAYCCA-3′; (Sequence I.D. No. 9) F 5′-ATGDNNTCCWSYTKYSYYAAYCCIVINTNKTNTATGGSTKNNTNAAC -3′; (Sequence I.D. No. 10) G 5′-ACYRTSTTYCAYATYATYGSYATGWKNTCYACYTKYGYYAAYCCA-3′; (Sequence I.D. No. 11) and H 5′-ATGWKGTCYTGYTKYCTSAAYCCAMTYCTSTAYGGMTKYCTSAAT -3′. (Sequence I.D. No. 12)
[0138] Degenerate oligomers from TM2 and TM7 were labeled with [32P]-ATP using T4 polynucleotide kinase (Boehringer Mannheim). Hybridization was performed at low stringency conditions: 40° C. in a solution containing 5×SSC (1×SSC is 0.15 M sodium chloride, 0.015 M sodium citrate), 1× Denhardt's solution (0.02% polyvinylpyrrolindone, 0.02% Ficoll, 0.02% bovine serum albumin), 7 mM Tris, 7% sodium dodecyl sulfate and 25 &mgr;g/ml sonicated salmon sperm DNA. The filters were washed at 40° C. in 0.1×SSC containing 0.5% sodium dodecyl sulfate and exposed at −70° C. to Kodak XAR film in the presence of an intensifying screen. Lambda phage clones hybridizing with the probes were plaque purified and DNA was prepared for Southern blot analysis (Sambrook et al., 1989). A genomic clone hybridizing with the TM2 probes, designated hl18a, was isolated using this method. For subcloning and further Southern blot analysis, the hl18a DNA was cloned into pUC18 (Pharmacia, Piscataway, N.J.). Nucleotide sequence analysis was accomplished by the Sanger dideoxy nucleotide chain termination method on denatured double-stranded plasmid templates, using Sequenase (US Biochemical Corp., Cleveland, Ohio).
[0139] Isolation and Sequencing of a Full-Length Mammalian LPA Receptor
[0140] To isolate a full-length hl18a receptor, a human hippocampal library (Gerald, 1995) was screened by polymerase chain reaction (PCR) using primers designed against hl18a DNA (sense primer and reverse primer). 6 sense primer: 5′-CCCCTCAGGATCTCCTTGGCCAT-3′ (Sequence I.D. No. 13) reverse primer: 5′GCGCTGGACGATGATGAGGAAGC-3′ (Sequence I.D. No. 14)
[0141] 1 &mgr;l of bacterial stock from each of 37 superpools of 50,000 independent clones was amplified using the Expand Long Template PCR System (Boehringer Mannheim), and the resulting products analyzed by agarose gel electrophoresis. The conditions for PCR were 94° C. for 2 min. and 68° C. for 4 min. for 40 cycles with a 5 min. preincubation at 95° C. and a 10 min. postincubation at 68° C. One positive superpool, H. Hippo. B3, was analyzed further and a single cDNA clone, H. Hippo. 220-74-49, was isolated by sib selection. By PCR, this clone (renamed H. Hippo hl18a) was determined to be in the wrong orientation for expression. To obtain a construct in the correct orientation, an EcoRI fragment of H. Hippo hl18a was ligated into the pEXJ expression vector. Plasmid DNA was prepared and was named #775 (also known as HL-18a).
[0142] Nucleotide sequence analysis was accomplished by the Sanger dideoxy nucleotide chain termination method on denatured double-stranded plasmid templates, using Sequenase (US Biochemical Corp., Cleveland, Ohio).
[0143] Cell Culture
[0144] COS-7 cells are grown on 150 mm plates in DMEM with supplements (Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 &mgr;g/ml streptomycin) at 37° C., 5% CO2. Stock plates of COS-7 cells are trypsinized and split 1:6 every 3-4 days.
[0145] Human embryonic kidney 293 cells are grown on 150 mm plates in DMEM with supplements (10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 &mgr;g/ml streptomycin) at 37° C., 5% CO2. Stock plates of 293 cells are trypsinized and split 1:6 every 3-4 days.
[0146] Mouse fibroblast LM(tk−) cells are grown on 150 mm plates in D-MEM with supplements (Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 &mgr;g/ml streptomycin) at 37° C., 5% CO2. Stock plates of LM(tk−) cells are trypsinized and split 1:10 every 3-4 days.
[0147] Chinese hamster ovary (CHO) cells were grown on 150 mm plates in HAM's F-12 medium with supplements (10% bovine calf serum, 4 mM L-glutamine and 100 units/ml penicillin/100 ug/ml streptomycin) at 37° C., 5% CO2. Stock plates of CHO cells are trypsinized and split 1:8 every 3-4 days.
[0148] Mouse embryonic fibroblast NIH-3T3 cells are grown on 150 mm plates in Dulbecco's Modified Eagle Medium (DMEM) with supplements (10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 &mgr;g/ml streptomycin) at 37° C., 5% CO2. Stock plates of NIH-3T3 cells are trypsinized and split 1:15 every 3-4 days.
[0149] Sf9 and Sf21 cells are grown in monolayers on 150 mm tissue culture dishes in TMN-FH media supplemented with 10% fetal calf serum, at 27° C., no CO2. High Five insect cells are grown on 150 mm tissue culture dishes in Ex-Cell 400™ medium supplemented with L-Glutamine, also at 27° C., no CO2.
[0150] Transfection
[0151] Receptors studied may be transiently transfected into COS-7 cells by the DEAE-dextran method, using 1 &mgr;g of DNA/106 cells (Cullen, 1987). In addition, Schneider 2 Drosophila cells may be cotransfected with vectors containing the receptor gene, under control of a promoter which is active in insect cells, and a selectable resistance gene, eg., the G418 resistant neomycin gene, for expression of the polypeptides disclosed herein.
[0152] Stable Transfection
[0153] DNA encoding the polypeptides disclosed herein may be co-transfected with a G-418 resistant gene into the human embryonic kidney 293 cell line by a calcium phosphate transfection method (Cullen, 1987). Stably transfected cells are selected with G-418.
[0154] Membrane Preparations
[0155] LM(tk−) cells stably transfected with the DNA encoding the mammalian LPA receptor disclosed herein may be routinely converted from an adherent monolayer to a viable suspension. Adherent cells are harvested with trypsin at the point of confluence, resuspended in a minimal volume of complete DMEM for a cell count, and further diluted to a concentration of 106 cells/ml in suspension media (10% bovine calf serum, 10% 10× Medium 199 (Gibco), 9 mM NaHCO3, 25 mM glucose, 2 mM L-glutamine, 100 units/ml penicillin/100 &mgr;g/ml streptomycin, and 0.05% methyl cellulose). Cell suspensions are maintained in a shaking incubator at 37° C., 5% CO2 for 24 hours. Membranes harvested from cells grown in this manner may be stored as large, uniform batches in liquid nitrogen. Alternatively, cells may be returned to adherent cell culture in complete DMEM by distribution into 96-well microtiter plates coated with poly-D-lysine (0.01 mg/ml) followed by incubation at 37° C., 5% CO2 for 24 hours.
[0156] Generation of Baculovirus
[0157] The coding region of DNA encoding the mammalian LPA receptor disclosed herein may be subcloned into pBlueBacIII into existing restriction sites, or sites engineered into sequences 5′ and 3′ to the coding region of the polypeptides. To generate baculovirus, 0.5 &mgr;g of viral DNA (BaculoGold) and 3 &mgr;g of DNA construct encoding a polypeptide may be co-transfected into 2×106 Spodoptera frugiperda insect Sf9 cells by the calcium phosphate co-precipitation method, as outlined in by Pharmingen (in “Baculovirus Expression Vector System: Procedures and Methods Manual”). The cells then are incubated for 5 days at 27° C.
[0158] The supernatant of the co-transfection plate may be collected by centrifugation and the recombinant virus plaque purified. The procedure to infect cells with virus, to prepare stocks of virus and to titer the virus stocks are as described in Pharmingen's manual.
[0159] Radioligand Binding Assays
[0160] Cells may be screened for the presence of endogenous mammalian LPA receptor using radioligand binding or functional assays (described in detail in the following experimental description). Cells with either no or a low level of endogenous LPA receptor present may be transfected with the mammalian LPA receptor.
[0161] Transfected cells from culture flasks are scraped into 5 ml of Tris-HCl, 5 mM EDTA, pH 7.5, and lysed by sonication. The cell lysates are centrifuged at 1000 rpm for 5 min. at 4° C., and the supernatant is centrifuged at 30,000×g for 20 min. at 4° C. The pellet is suspended in binding buffer (50 mM Tris-HCl, 5 mM MgSO4, 1 mM EDTA at pH 7.5 supplemented with 0.1% BSA, 2 &mgr;g/ml aprotinin, 0.5 mg/ml leupeptin, and 10 &mgr;g/ml phosphoramidon). Optimal membrane suspension dilutions, defined as the protein concentration required to bind less than 10% of the added radioligand, are added to 96-well polpropylene microtiter plates containing 3H-labeled compound, unlabeled compounds, and binding buffer to a final volume of 250 &mgr;l. In equilibrium saturation binding assays membrane preparations are incubated in the presence of increasing concentrations of [3H]-labeled compound. The binding affinities of the different compounds are determined in equilibrium competition binding assays, using [3H]-labeled compound in the presence of ten to twelve different concentrations of the displacing ligands. Binding reaction mixtures are incubated for 1 hr at 30° C., and the reaction stopped by filtration through GF/B filters treated with 0.5% polyethyleneimine, using a cell harvester. Radioactivity may be measured by scintillation counting and data are analyzed by a computerized non-linear regression program. Non-specific binding is defined as the amount of radioactivity remaining after incubation of membrane protein in the presence of unlabeled. Protein concentration may be measured by the Bradford method using Bio-Rad Reagent, with bovine serum albumin as a standard.
[0162] Functional Assays
[0163] Cells may be screened for the presence of endogenous mammalian LPA receptor using radioligand binding or functional assays (described in detail in the above or following experimental description, respectively). Cells with no or a low level of endogenous LPA receptor present may be transfected with the mammalian LPA receptor for use in the following functional assays.
[0164] Cyclic AMP (cAMP) Formation Assay
[0165] The receptor-mediated inhibition of cyclic AMP (cAMP) formation may be assayed in transfected cells expressing the mammalian LPA receptors. Cells are plated in 96-well plates and incubated in Dulbecco's phosphate buffered saline (PBS) supplemented with 10 mM HEPES, 5 mM theophylline, 2 &mgr;g/ml aprotinin, 0.5 mg/ml leupeptin, and 10 &mgr;g/ml phosphoramidon for 20 min at 37° C., in 5% CO2. Test compounds are added and incubated for an additional 10 min at 37° C. The medium is then aspirated and the reaction stopped by the addition of 100 mM HCl. The plates are stored at 4° C. for 15 min, and the cAMP content in the stopping solution measured by radioimmunoassay. Radioactivity may be quantified using a gamma counter equipped with data reduction software.
[0166] Arachidonic Acid Release Assay
[0167] Stably transfected cells with the mammalian LPA receptor are seeded into 96 well plates and grown for 3 days in HAM's F-12 with supplements. 3H-arachidonic acid (specific activity=0.75 &mgr;Ci/ml) is delivered as a 100 &mgr;L aliquot to each well and samples were incubated at 37° C., 5% CO2 for 18 hours. The labeled cells are washed three times with 200 &mgr;L HAM's F-12. The wells are then filled with medium (200 &mgr;L) and the assay is initiated with the addition of peptides or buffer (22 &mgr;L). Cells are incubated for 30 min at 37° C., 5% CO2. Supernatants are transferred to a microtiter plate and evaporated to dryness at 75° C. in a vacuum oven. Samples are then dissolved and resuspended in 25 AL distilled water. Scintillant (300 &mgr;L) is added to each well and samples are counted for 3H in a Trilux plate reader. Data are analyzed using non-linear regression and statistical techniques available in the GraphPAD Prism package (San Diego, Calif.).
[0168] Intracellular Calcium Mobilization Assay
[0169] The intracellular free calcium concentration may be measured by microspectroflourometry using the fluorescent indicator dye Fura-2/AM (Bush et al, 1991). Stably transfected cells are seeded onto a 35 mm culture dish containing a glass coverslip insert. Cells are washed with HBS and loaded with 100 &mgr;L of Fura-2/AM (10 &mgr;M) for 20 to 40 min. After washing with HBS to remove the Fura-2/AM solution, cells are equilibrated in HBS for 10 to 20 min. Cells are then visualized under the 40× objective of a Leitz Fluovert FS microscope and fluorescence emission is determined at 510 nM with excitation wavelengths alternating between 340 nM and 380 nM. Raw fluorescence data are converted to calcium concentrations using standard calcium concentration curves and software analysis techniques.
[0170] Phosphoinositide Metabolism Assay
[0171] Cells stably expressing the mammalian LPA receptor cDNA are plated in 96-well plates and grown to confluence. The day before the assay the growth medium is changed to 100 &mgr;l of medium containing 1% serum and 0.5 &mgr;Ci [3H]myo-inositol, and the plates are incubated overnight in a CO2 incubator (5% CO2 at 37° C.). Alternatively, arachidonic acid release may be measured if [3H]arachidonic acid is substituted for the [3H]myo-inositol. Immediately before the assay, the medium is removed and replaced by 200 &mgr;L of PBS containing 10 mM LiCl, and the cells are equilibrated with the new medium for 20 min. During this interval cells are also equilibrated with the antagonist, added as a 10 &mgr;L aliquot of a 20-fold concentrated solution in PBS. The [3H]inositol-phosphates accumulation from inositol phospholipid metabolism may be started by adding 10 &mgr;L of a solution containing the agonist. To the first well 10 &mgr;L may be added to measure basal accumulation, and 11 different concentrations of agonist are assayed in the following 11 wells of each plate row. All assays are performed in duplicate by repeating the same additions in two consecutive plate rows. The plates are incubated in a CO2 incubator for 1 hr. The reaction may be terminated by adding 15 &mgr;L of 50% v/v trichloroacetic acid (TCA), followed by a 40 min. incubation at 4° C. After neutralizing TCA with 40 &mgr;L of 1 M Tris, the content of the wells may be transferred to a Multiscreen HV filter plate (Millipore) containing Dowex AG1-X8 (200-400 mesh, formate form). The filter plates are prepared adding 200 &mgr;L of Dowex AG1-X8 suspension (50% v/v, water: resin) to each well. The filter plates are placed on a vacuum manifold to wash or elute the resin bed. Each well is washed 2 times with 200 &mgr;L of water, followed by 2×200 &mgr;L of 5 mM sodium tetraborate/60 mM ammonium formate. The [3H]IPs are eluted into empty 96-well plates with 200 &mgr;L of 1.2 M ammonium formate/0.1 formic acid. The content of the wells is added to 3 ml of scintillation cocktail, and the radioactivity is determined by liquid scintillation counting.
[0172] GTP&ggr;S Functional Assay
[0173] Membranes from cells transfected with the mammalian LPA receptors are suspended in assay buffer (50 mM Tris, 100 mM NaCl, 5 mM MgCl2, pH 7.4) supplemented with 0.1% BSA, 0.1% bacitracin and 10 &mgr;M GDP. Membranes are incubated on ice for 20 minutes, transferred to a 96-well Millipore microtiter GF/C filter plate and mixed with GTP&ggr;35S (e.g., 250,000 cpm/sample, specific activity ˜1000 Ci/mmol) plus or minus GTP&ggr;S (final concentration=100 &mgr;M). Final membrane protein concentration≈90 &mgr;g/ml. Samples are incubated in the presence or absence of test compound (final concentration=1 &mgr;M) for 30 min. at room temperature, then filtered on a Millipore vacuum manifold and washed three times with cold assay buffer. Samples collected in the filter plate are treated with scintillant and counted for 35S in a Trilux (Wallac) liquid scintillation counter. It is expected that optimal results are obtained when the mammalian LPA receptor membrane preparation is derived from an appropriately engineered heterologous expression system, i.e., an expression system resulting in high levels of expression of the mammalian LPA receptor and/or expressing G-proteins having high turnover rates (for the exchange of GDP for GTP). GTP&ggr;S assays are well-known in the art, and it is expected that variations on the method described above, such as are described by e.g., Tian et al. (1994) or Lazareno and Birdsall (1993), may be used by one of ordinary skill in the art.
[0174] MAP Kinase Assay
[0175] MAP kinase (mitogen activated kinase) may be monitored to evaluate receptor activation. MAP kinase is activated by multiple pathways in the cell. A primary mode of activation involves the ras/raf/MEK/MAP kinase pathway. Growth factor (tyrosine kinase) receptors feed into this pathway via SHC/Grb-2/SOS/ras. Gi coupled receptors are also known to activate ras and subsequently produce an activation of MAP kinase. Receptors that activate phospholipase C (Gq and G11) produce diacylglycerol (DAG) as a consequence of phosphatidyl inositol hydrolysis. DAG activates protein kinase C which in turn phosphorylates MAP kinase.
[0176] MAP kinase activation can be detected by several approaches. One approach is based on an evaluation of the phosphorylation state, either unphosphorylated (inactive) or phosphorylated (active). The phosphorylated protein has a slower mobility in SDS-PAGE and can therefore be compared with the unstimulated protein using Western blotting. Alternatively, antibodies specific for the phosphorylated protein are available (New England Biolabs) which can be used to detect an increase in the phosphorylated kinase. In either method, cells are stimulated with the mitogen and then extracted with Laemmli buffer. The soluble fraction is applied to an SDS-PAGE gel and proteins are transferred electrophoretically to nitrocellulose or Immobilon. Immunoreactive bands are detected by standard Western blotting technique. Visible or chemiluminescent signals are recorded on film and may be quantified by densitometry.
[0177] Another approach is based on evaluation of the MAP kinase activity via a phosphorylation assay. Cells are stimulated with the mitogen and a soluble extract is prepared. The extract is incubated at 30° C. for 10 min with gamma-32-ATP, an ATP regenerating system, and a specific substrate for MAP kinase such as phosphorylated heat and acid stable protein regulated by insulin, or PHAS-I. The reaction is terminated by the addition of H3PO4 and samples are transferred to ice. An aliquot is spotted onto Whatman P81 chromatography paper, which retains the phosphorylated protein. The chromatrography paper is washed and counted for 32P in a liquid scintillation counter. Alternatively, the cell extract is incubated with gamma-32-ATP, an ATP regenerating system, and biotinylated myelin basic protein bound by streptavidin to a filter support. The myelin basic protein is a substrate for activated MAP kinase. The phosphorylation reaction is carried out for 10 min at 30° C. The extract can then by aspirated through the filter, which retains the phosphorylated myelin basic protein. The filter is washed and counted for 32P by liquid scintillation counting.
[0178] Cell Proliferation Assay
[0179] Receptor activation of a G protein coupled receptor may lead to a mitogenic or proliferative response which can be monitored via 3H-thymidine uptake. When cultured cells are incubated with 3H-thymidine, the thymidine translocates into the nuclei where it is phosphorylated to thymidine triphosphate. The nucleotide triphosphate is then incorporated into the cellular DNA at a rate that is proportional to the rate of cell growth. Typically, cells are grown in culture for 1-3 days. Cells are forced into quiescence by the removal of serum for 24 hrs. A mitogenic agent is then added to the media. 24 hrs later, the cells are incubated with 3H-thymidine at specific activities ranging from 1 to 10 uCi/ml for 2-6 hrs. Harvesting procedures may involve trypsinization and trapping of cells by filtration over GF/C filters with or without a prior incubation in TCA to extract soluble thymidine. The filters are processed with scintillant and counted for 3H by liquid scintillation counting. Alternatively, adherant cells are fixed in MeOH or TCA, washed in water, and solubilized in 0.05% deoxycholate/0.1 N NaOH. The soluble extract is transferred to scintillation vials and counted for 3H by liquid scintillation counting.
[0180] It is to be understood that the cell lines described herein are merely illustrative of the methods used to evaluate the binding and function of the mammalian LPA receptors of the present invention, and that other suitable cells may be used in the assays described herein.
[0181] Methods for Recording Currents in Xenopus Oocytes
[0182] Female Xenopus laevis (Xenopus-1, Ann Arbor, Mich.) are anesthetized in 0.2% tricain (3-aminobenzoic acid ethyl ester, Sigma Chemical Corp.) and a portion of ovary is removed using aseptic technique (Quick and Lester, 1994). Oocytes are defolliculated using 3 mg/ml collagenase (Worthington Biochemical Corp., Freehold, N.J.) in a solution containing 87.5 mM NaCl, 2 mM KCl, 2 mM MgCl2 and 5 mM HEPES, pH 7.5. Oocytes are injected (Nanoject, Drummond Scientific, Broomall, Pa.) with 2-2.5 ng cRNA transcribed from mammalian cDNA using T7 polymerase (Ambion). Alternatively, mRNA may be translated from a template generated by PCR, incorporating a T7 promoter and a poly A+ tail. After injection of mRNA, oocytes are incubated at 16° on a rotating platform for 3-8 days.
[0183] Dual electrode voltage clamp (“GeneClamp”, Axon Instruments Inc., Foster City, Calif.) is performed using 3 M KCl-filled glass microelectrodes having resistances of 1-3 Mohms. Unless otherwise specified, oocytes are voltage clamped at a holding potential of −80 mV. During recordings, oocytes are bathed in continuously flowing (2-5 ml/min) medium containing 96 mM NaCl, 2 mM KCl, 2 mM CaCl2, 2 mM MgCl2, and 5 mM HEPES, pH 7.5 (“ND96”). Drugs are applied by switching from a series of gravity fed perfusion lines.
[0184] Heterologous expression of GPCRs in Xenopus oocytes has been widely used to determine the identity of signaling pathways activated by agonist stimulation (Gundersen et al., 1983; Takahashi et al., 1987). Activation of the phospholipase C (PLC) pathway is assayed by applying test compound in ND96 solution to oocytes previously injected with mRNA for the mammalian LPA receptor and observing inward currents at a holding potential of −80 mV. The appearance of currents that reverse at −25 mV and display other properties of the Ca++-activated Cl− (chloride) channel is indicative of mammalian LPA receptor-activation of PLC and release of IP3 and intracellular Ca++. Such activity is exhibited by GPCRs that couple to Gq.
[0185] Measurement of G-protein inwardly rectifying K+ (potassium) channel (GIRK) activity is monitored in oocytes that have been co-injected with mRNAs encoding the mammalian LPA receptor, GIRK1, and GIRK4. The two GIRK gene products co-assemble to form a G-protein activated potassium channel known to be activated (i.e., stimulated) by a number of GPCRs that couple to Gi or Go (Kubo et al., 1993; Dascal et al., 1993). Genes encoding GIRK1 and GIRK4 are obtained by PCR using the published sequences (Kubo et al., 1993; Dascal et al., 1993; Krapivinsky et al., 1995 and 1995b) to derive appropriate 5′ and 3′ primers. Human heart cDNA was used as template together with the primers 7 5′-CGCGGATCCATTATGTCTGCACTCCCAAGGAAATTTG-3′ and (Seq. I. ID. No. 15) 5′-CGCGAATTCTTATGTGAAGCGATCAGAGTTCATTTTTC -3′for GIRK1 and (Seq. I.D. No. 16) 5′-GCGGGATCCGCTATGGCTGGTGATTCTAGGAATG-3′ and (Seq. I. D. No. 17) 5′-CCGGAATTCCCCTCACACCGAGCCCCTGG-3′ for GIRK4. (Seq. I.D. No. 18)
[0186] In each primer pair, the upstream primer contained a BamHI site and the downstream primer contained an EcoRI site to facilitate cloning of the PCR product into pcDNA1-Amp (Invitrogen). mRNAs are prepared from separate DNA plasmids containing the complete coding regions of the mammalian LPA receptor, GIRK1, and GIRK4. Plasmids are linearized and transcribed using the T7 polymerase (“Message Machine”, Ambion). Oocytes (prepared as described above) are injected with mammalian LPA receptor mRNA (2-2.5 ng) plus the two GIRK subunits (2 ng each). After injection of mRNA, oocytes are incubated at 16° on a rotating platform for 3-8 days.
[0187] Dual electrode voltage clamp is performed as described above, except that oocytes are bathed in continuously flowing (2-5 ml/min) medium containing elevated K+ containing 49 mM KCl, 49 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, and 5 mM HEPES, pH 7.5 (“hK”). The oocytes are tested for responsivity to agonists and antagonists by measuring K+ currents in elevated K+ solution (hK). Activation of inwardly rectifying currents that are sensitive to 300 &mgr;M Ba++ signifies the mammalian LPA receptor coupling to a Gi or Go pathway in the oocytes.
[0188] Experimental Results
[0189] A human genomic lymphocyte library was screened, under reduced stringency conditions, with degenerate oligonucleotide probes directed to the second and seventh transmembrane regions of the human Y1 (Larhammar, D. et al., 1992; GenBank accession No. Z11504), human Y2 (Gerald et al., 1995; GenBank accession No. U36269), human Y4 (Bard et al., 1995; GenBank accession No. U35232) and human Y5 (Gerald et al., 1996; GenBank accession No. U56079) neuropeptide receptor genes. Positively-hybridizing clones were isolated, plaque-purified, and characterized by Southern blot analysis and sequencing. One clone, hl18a, contained an 800 bp HindIII/HindIII fragment which hybridized with the rat Y2-derived oligonucleotide probes and was subsequently subcloned into a pUC vector. This clone was a partial gene fragment, encoding a starting methionine through the 3′ end of a putative TM3, with the possibility of an immediate downstream intron.
[0190] In order to obtain a full-length clone, a human hippocampal library was screened by PCR using primers directed against hl18a. One positive superpool, H.Hippo.B3, was successfully subdivided until a single clone, H.Hippo. 220-74-49, was isolated. As this clone was in the wrong orientation for expression, the insert was removed by restriction digestion and re-ligated into the expression vector pEXJ in the correct orientation. The largest open reading frame in this construct, #775 (also known as HL-18a), contains 1299 nucleotides, which is predicted to encode a protein of 433 amino acids. Using a downstream methionine, with a better Kozak consensus site (Kozak, 1991), results in an open frame of 1116 nucleotides and is predicted to encode a protein of 372 amino acids. Hydropathy analysis of the protein is consistent with a putative topography of seven transmembrane domains, indicative of the G protein-coupled receptor family.
[0191] A comparison of nucleotide and peptide sequences of clone #775 (HL-18a) with sequences contained in the Genbank/EMBL databases reveals that the clone is most related to Xenopus laevis high-affinity lysophosphatidic acid receptor (GenBank accession # U76385) (Guo, et al., 1996). There is a 67% nucleotide identity and 70% amino acid identity between clone #775 (HL-18a) and the Xenopus laevis high-affinity lysophosphatidic acid receptor.
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[0223]
Claims
1. An isolated nucleic acid encoding a mammalian LPA receptor.
2. The nucleic acid of claim 1, wherein the nucleic acid is DNA.
3. The DNA of claim 2, wherein the DNA is cDNA.
4. The DNA of claim 2, wherein the DNA is genomic DNA.
5. The nucleic acid of claim 1, wherein the nucleic acid is RNA.
6. The nucleic acid of claim 1, wherein the mammalian LPA receptor is a human LPA receptor.
7. The nucleic acid of claim 1, wherein the nucleic acid encodes a mammalian LPA receptor which has substantially the same amino acid sequence as does the mammalian LPA receptor encoded by the plasmid HL-18a (ATCC Accession No. 209448).
8. The nucleic acid of claim 6, wherein the nucleic acid encodes a human LPA receptor which has an amino acid sequence identical to that encoded by the plasmid HL-18a (ATCC Accession No. 209448).
9. The nucleic acid of claim 6, wherein the human LPA receptor has an amino acid sequence, substantially the same as the amino acid sequence shown in FIG. 3 (Seq. I.D. No. 3).
10. The nucleic acid of claim 9, wherein the human LPA receptor has an amino acid sequence identical to the amino acid sequence shown in FIG. 3 (Seq. I.D. No. 3).
11. A purified mammalian LPA receptor protein.
12. A vector comprising the nucleic acid of claim 1.
13. A vector comprising the nucleic acid of claim 6.
14. A vector of claim 12 adapted for expression in a bacterial cell which comprises the regulatory elements necessary for expression of the nucleic acid in the bacterial cell operatively linked to the nucleic acid encoding the mammalian LPA receptor so as to permit expression thereof.
15. A vector of claim 12 adapted for expression in an amphibian cell which comprises the regulatory elements necessary for expression of the nucleic acid in the amphibian cell operatively linked to the nucleic acid encoding the mammalian LPA receptor so as to permit expression thereof.
16. A vector of claim 12 adapted for expression in a yeast cell which comprises the regulatory elements necessary for expression of the nucleic acid in the yeast cell operatively linked to the nucleic acid encoding the mammalian LPA receptor so as to permit expression thereof.
17. A vector of claim 12 adapted for expression in an insect cell which comprises the regulatory elements necessary for expression of the nucleic acid in the insect cell operatively linked to the nucleic acid encoding the mammalian LPA receptor so as to permit expression thereof.
18. The vector of claim 17 which is a baculovirus.
19. A vector of claim 12 adapted for expression in a mammalian cell which comprises the regulatory elements necessary for expression of the nucleic acid in the mammalian cell operatively linked to the nucleic acid encoding the mammalian LPA receptor so as to permit expression thereof.
20. The vector of claim 12, wherein the vector is a plasmid.
21. The plasmid of claim 20 designated HL-18a (ATCC Accession No. 209448).
22. A cell comprising the vector of claim 12.
23. A cell of claim 22, wherein the cell is a non-mammalian cell.
24. A cell of claim 23, wherein the non-mammalian cell is a Xenopus oocyte cell or a Xenopus melanophore cell.
25. A cell of claim 22, wherein the cell is a mammalian cell.
26. A mammalian cell of claim 25, wherein the cell is a COS-7 cell, a 293 human embryonic kidney cell, a NIH-3T3 cell, a LM(tk−) cell, a mouse Y1 cell, or a CHO cell.
27. An insect cell comprising the vector of claim 17.
28. An insect cell of claim 27, wherein the insect cell is an Sf9 cell, an Sf21 cell or a HighFive cell.
29. A membrane preparation isolated from the cell of any one of claims 22, 23, 25 or 27.
30. A nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a mammalian LPA receptor, wherein the probe has a unique sequence corresponding to a sequence present within one of the two strands of nucleic acid which encode the mammalian LPA receptor and are contained in plasmid HL-18a (ATCC Accession No. 209448).
31. A nucleic acid probe comprising at least 15 nucleotides, which probe specifically hybridizes with a nucleic acid encoding a mammalian LPA receptor, wherein the probe has a unique sequence corresponding to a sequence present within (a) the nucleic acid sequence shown in FIG. 2 (Seq. I.D. No. 2) or (b) the reverse complement thereto.
32. The nucleic acid probe of claim 30 or 31, wherein the nucleic acid is DNA.
33. The nucleic acid probe of claim 30 or 31, wherein the nucleic acid is RNA.
34. A nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to a unique fragment of the sequence of a nucleic acid molecule encoding a mammalian LPA receptor.
35. A nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to the antisense sequence of a unique fragment of the sequence of a nucleic acid molecule encoding a mammalian LPA receptor.
36. An antisense oligonucleotide having a sequence capable of specifically hybridizing to the RNA of claim 5, so as to prevent translation of the RNA.
37. An antisense oligonucleotide having a sequence capable of specifically hybridizing to the genomic DNA of claim 4.
38. An antisense oligonucleotide of claim 36 or 37, wherein the oligonucleotide comprises chemically modified nucleotides or nucleotide analogues.
39. An antibody capable of binding to a mammalian LPA receptor encoded by the nucleic acid of claim 1.
40. An antibody of claim 39, wherein the mammalian LPA receptor is a human LPA receptor.
41. An agent capable of competitively inhibiting the binding of the antibody of claim 39 to a mammalian LPA receptor.
42. The antibody of claim 39, wherein the antibody is a monoclonal antibody or antisera.
43. A pharmaceutical composition comprising (a) an amount of the oligonucleotide of claim 36 capable of passing through a cell membrane and effective to reduce expression of a mammalian LPA receptor and (b) a pharmaceutically acceptable carrier capable of passing through the cell membrane.
44. A pharmaceutical composition of claim 43, wherein the oligonucleotide is coupled to a substance which inactivates mRNA.
45. A pharmaceutical composition of claim 44, wherein the substance which inactivates mRNA is a ribozyme.
46. A pharmaceutical composition of claim 43, wherein the pharmaceutically acceptable carrier comprises a structure which binds to a mammalian LPA receptor on a cell capable of being taken up by the cells after binding to the structure.
47. A pharmaceutical composition of claim 46, wherein the pharmaceutically acceptable carrier is capable of binding to a mammalian LPA receptor which is specific for a selected cell type.
48. A pharmaceutical composition which comprises an amount of the antibody of claim 39 effective to block binding of a ligand to a human LPA receptor and a pharmaceutically acceptable carrier.
49. A transgenic, nonhuman mammal expressing the DNA encoding a mammalian LPA receptor of claim 1.
50. A transgenic, nonhuman mammal comprising a homologous recombination knockout of the native mammalian LPA receptor.
51. A transgenic, nonhuman mammal whose genome comprises antisense DNA complementary to the DNA encoding a mammalian LPA receptor of claim 1 so placed within the genome as to be transcribed into antisense mRNA which is complementary to mRNA encoding the mammalian LPA receptor and which hybridizes to mRNA encoding the mammalian LPA receptor, thereby reducing its translation.
52. The transgenic, nonhuman mammal of either claim 49 or 50, wherein the DNA encoding the mammalian LPA receptor additionally comprises an inducible promoter.
53. The transgenic, nonhuman mammal of claim 49 or 50, wherein the DNA encoding the mammalian LPA receptor additionally comprises tissue specific regulatory elements.
54. A transgenic, nonhuman mammal of claim 49, 50, or 51, wherein the transgenic, nonhuman mammal is a mouse.
55. A process for identifying a chemical compound which specifically binds to a mammalian LPA receptor which comprises contacting cells containing DNA encoding and expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian LPA receptor.
56. A process for identifying a chemical compound which specifically binds to a mammalian LPA receptor which comprises contacting a membrane fragment from a cell extract of cells containing DNA encoding and expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the mammalian LPA receptor.
57. The process of claim 55 or 56, wherein the mammalian LPA receptor is a human LPA receptor.
58. The process of claim 55 or 56, wherein the mammalian LPA receptor has substantially the same amino acid sequence as the mammalian LPA receptor encoded by plasmid HL-18a (ATCC Accession No. 209448).
59. The process of claim 55 or 56, wherein the mammalian LPA receptor has substantially the same amino acid sequence as that shown in FIG. 3 (Seq. I.D. No. 3).
60. The process of claim 55 or 56, wherein the mammalian LPA receptor has the amino acid sequence shown in FIG. 3 (Seq. I.D. No. 3).
61. The process of claim 59, wherein the compound is a compound not previously known to bind to a mammalian LPA receptor.
62. A compound determined by the process of claim 61.
63. A process of claim 59, wherein the cell is an insect cell.
64. The process of claim 59, wherein the cell is a mammalian cell.
65. The process of claim 64, wherein the cell is non-neuronal in origin.
66. The process of claim 65, wherein the non-neuronal cell is a COS-7 cell, 293 human embryonic kidney cell, a CHO cell, a NIH-3T3 cell, a mouse Y1 cell, or a LM(tk−) cell.
67. A process of claim 64, wherein the compound is a compound not previously known to bind to a mammalian LPA receptor.
68. A compound determined by the process of claim 67.
69. A process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian LPA receptor which comprises separately contacting cells expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the mammalian LPA receptor, a decrease in the binding of the second chemical compound to the mammalian LPA receptor in is the presence of the chemical compound indicating that the chemical compound binds to the mammalian LPA receptor.
70. A process involving competitive binding for identifying a chemical compound which specifically binds to a mammalian LPA receptor which comprises separately contacting a membrane fraction from a cell extract of cells expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the mammalian LPA receptor, a decrease in the binding of the second chemical compound to the mammalian LPA receptor in the presence of the chemical compound indicating that the chemical compound binds to the mammalian LPA receptor.
71. A process of claim 69 or 70, wherein the mammalian LPA receptor is a human LPA receptor.
72. The process of claim 71, wherein the human LPA receptor has substantially the same amino acid sequence as the human LPA receptor encoded by plasmid HL-18a (ATCC Accession No. 209448).
73. The process of claim 69 or 70, wherein the mammalian LPA receptor has substantially the same amino acid sequence as that shown in FIG. 3 (Seq. I.D. No. 3).
74. The process of claim 69 or 70, wherein the mammalian LPA receptor has the amino acid sequence shown in FIG. 3 (Seq. I.D. No. 3).
75. The process of claim 73, wherein the cell is an insect cell.
76. The process of claim 73, wherein the cell is a mammalian cell.
77. The process of claim 76, wherein the cell is non-neuronal in origin.
78. The process of claim 77, wherein the non-neuronal cell is a COS-7 cell, 293 human embryonic kidney cell, a CHO cell, a NIH-3T3 cell, a mouse Y1 cell, or a LM(tk−) cell.
79. The process of claim 78, wherein the compound is a compound not previously known to bind to a mammalian LPA receptor.
80. A compound determined by the process of claim 79.
81. A method of screening a plurality of chemical compounds not known to bind to a mammalian LPA receptor to identify a compound which specifically binds to the mammalian LPA receptor, which comprises
- (a) contacting cells transfected with and expressing DNA encoding the mammalian LPA receptor with a compound known to bind specifically to the mammalian LPA receptor;
- (b) contacting the preparation of step (a) with the plurality of compounds not known to bind specifically to the mammalian LPA receptor, under conditions permitting binding of compounds known to bind the mammalian LPA receptor;
- (c) determining whether the binding of the compound known to bind to the mammalian LPA receptor is reduced in the presence of the compounds within the plurality of compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so
- (d) separately determining the binding to the mammalian LPA receptor of compounds included in the plurality of compounds, so as to thereby identify the compound which specifically binds to the mammalian LPA receptor.
82. A method of screening a plurality of chemical compounds not known to bind to a mammalian LPA receptor to identify a compound which specifically binds to the mammalian LPA receptor, which comprises
- (a) preparing a cell extract from cells transfected with and expressing DNA encoding the mammalian LPA receptor, isolating a membrane fraction from the cell extract, contacting the membrane fraction with a compound known to bind specifically to the mammalian LPA receptor;
- (b) contacting the preparation of step (a) with the plurality of compounds not known to bind specifically to the mammalian LPA receptor, under conditions permitting binding of compounds known to bind the mammalian LPA receptor;
- (c) determining whether the binding of the compound known to bind to the mammalian LPA receptor is reduced in the presence of the compounds within the plurality of compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so
- (d) separately determining the binding to the mammalian LPA receptor of compounds included in the plurality of compounds, so as to thereby identify the compound which specifically binds to the mammalian LPA receptor.
83. A method of either of claims 81 or 82, wherein the mammalian LPA receptor is a human LPA receptor.
84. A method of either of claims 81 or 82, wherein the cell is a mammalian cell.
85. A method of claim 84, wherein the mammalian cell is non-neuronal in origin.
86. The method of claim 85 wherein the non-neuronal cell is a COS-7 cell, a 293 human embryonic kidney cell, a LM(tk−) cell, a CHO cell, a mouse Y1 cell, or an NIH-3T3 cell.
87. A method of detecting expression of a mammalian LPA receptor by detecting the presence of mRNA coding for the mammalian LPA receptor which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained from the nucleic acid probe of claim 30 under hybridizing conditions, detecting the presence of mRNA hybridizing to the probe, and thereby detecting the expression of the mammalian LPA receptor by the cell.
88. A method of detecting the presence of a mammalian LPA receptor on the surface of a cell which comprises contacting the cell with the antibody of claim 39 under conditions permitting binding of the antibody to the receptor, detecting the presence of the antibody bound to the cell, and thereby detecting the presence of a mammalian LPA receptor on the surface of the cell.
89. A method of determining the physiological effects of varying levels of activity of mammalian LPA receptors which comprises producing a transgenic, nonhuman mammal of claim 52 whose levels of mammalian LPA receptor activity are varied by use of an inducible promoter which regulates mammalian LPA receptor expression.
90. A method of determining the physiological effects of varying levels of activity of mammalian LPA receptors which comprises producing a panel of transgenic, nonhuman mammals of claim 52 each expressing a different amount of mammalian LPA receptor.
91. A method for identifying an antagonist capable of alleviating an abnormality wherein the abnormality is alleviated by decreasing the activity of a mammalian LPA receptor comprising administering a compound to the transgenic, nonhuman mammal of any one of claims 49, 52, 53, or 54, and determining whether the compound alleviates the physical and behavioral abnormalities displayed by the transgenic, nonhuman mammal as a result of overactivity of a mammalian LPA receptor, the alleviation of the abnormality identifying the compound as an antagonist.
92. An antagonist identified by the method of claim 91.
93. A pharmaceutical composition comprising an antagonist identified by the method of claim 92 and a pharmaceutically acceptable carrier.
94. A method of treating an abnormality in a subject wherein the abnormality is alleviated by decreasing the activity of a mammalian LPA receptor which comprises administering to the subject an effective amount of the pharmaceutical composition of claim 93, thereby treating the abnormality.
95. A method for identifying an agonist capable of alleviating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian LPA receptor comprising administering a compound to the transgenic, nonhuman mammal of any one of claims 49, 52, 53, or 54, and determining whether the compound alleviates the physical and behavioral abnormalities displayed by the transgenic, nonhuman mammal, the alleviation of the abnormality identifying the compound as an agonist.
96. An agonist identified by the method of claim 95.
97. A pharmaceutical composition comprising an agonist identified by the method of claim 95 and a pharmaceutically acceptable carrier.
98. A method of treating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian LPA receptor which comprises administering to the subject an effective amount of the pharmaceutical composition of claim 97, thereby treating the abnormality.
99. A method for diagnosing a predisposition to a disorder associated with the activity of a specific mammalian allele which comprises:
- (a) obtaining DNA of subjects suffering from the disorder;
- (b) performing a restriction digest of the DNA with a panel of restriction enzymes;
- (c) electrophoretically separating the resulting DNA fragments on a sizing gel;
- (d) contacting the resulting gel with a nucleic acid probe capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a mammalian LPA receptor and labeled with a detectable marker;
- (e) detecting labeled bands which have hybridized to the DNA encoding a mammalian LPA receptor of claim 1 labeled with a detectable marker to create a unique band pattern specific to the DNA of subjects suffering from the disorder;
- (f) preparing DNA obtained for diagnosis by steps (a)-(e); and
- (g) comparing the unique band pattern specific to the DNA of subjects suffering from the disorder from step (e) and the DNA obtained for diagnosis from step (f) to determine whether the patterns are the same or different and to diagnose thereby predisposition to the disorder if the patterns are the same.
100. The method of claim 99 wherein a disorder associated with the activity of a specific mammalian allele is diagnosed.
101. A method of preparing the purified mammalian LPA receptor of claim 11 which comprises:
- (a) inducing cells to express the mammalian LPA receptor;
- (b) recovering the mammalian LPA receptor from the induced cells; and
- (c) purifying the mammalian LPA receptor so recovered.
102. A method of preparing the purified mammalian LPA receptor of claim 11 which comprises:
- (a) inserting nucleic acid encoding the mammalian LPA receptor in a suitable vector;
- (b) introducing the resulting vector in a suitable host cell;
- (c) placing the resulting cell in suitable condition permitting the production of the isolated mammalian LPA receptor;
- (d) recovering the mammalian LPA receptor produced by the resulting cell; and
- (e) purifying the mammalian LPA receptor so recovered.
103. A process for determining whether a chemical compound is a mammalian LPA receptor agonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian LPA receptor with the compound under conditions permitting the activation of the mammalian LPA receptor, and detecting an increase in mammalian LPA receptor activity, so as to thereby determine whether the compound is a mammalian LPA receptor agonist.
104. A process for determining whether a chemical compound is a mammalian LPA receptor antagonist which comprises contacting cells transfected with and expressing DNA encoding the mammalian LPA receptor with the compound in the presence of a known mammalian LPA receptor agonist, under conditions permitting the activation of the mammalian LPA receptor, and detecting a decrease in mammalian LPA receptor activity, so as to thereby determine whether the compound is a mammalian LPA receptor antagonist.
105. A process of any one of claims 103 or 104, wherein the mammalian LPA receptor is a human LPA receptor.
106. A pharmaceutical composition which comprises an amount of a mammalian LPA receptor agonist determined by the process of claim 103 effective to increase activity of a mammalian LPA receptor and a pharmaceutically acceptable carrier.
107. A pharmaceutical composition of claim 106, wherein the mammalian LPA receptor agonist is not previously known.
108. A pharmaceutical composition which comprises an amount of a mammalian LPA receptor antagonist determined by the process of claim 104 effective to reduce activity of a mammalian LPA receptor and a pharmaceutically acceptable carrier.
109. A pharmaceutical composition of claim 108, wherein the mammalian LPA receptor antagonist is not previously known.
110. A process for determining whether a chemical compound specifically binds to and activates a mammalian LPA receptor, which comprises contacting cells producing a second messenger response and expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with the chemical compound under conditions suitable for activation of the mammalian LPA receptor, and measuring the second messenger response in the presence and in the absence of the chemical compound, a change in the second messenger response in the presence of the chemical compound indicating that the compound activates the mammalian LPA receptor.
111. The process of claim 110, wherein the second messenger response comprises chloride channel activation and the change in second messenger is an increase in the level of inward chloride current.
112. A process for determining whether a chemical compound specifically binds to and inhibits activation of a mammalian LPA receptor, which comprises separately contacting cells producing a second messenger response and expressing on their cell surface the mammalian LPA receptor, wherein such cells do not normally express the mammalian LPA receptor, with both the chemical compound and a second chemical compound known to activate the mammalian LPA receptor, and with only the second chemical compound, under conditions suitable for activation of the mammalian LPA receptor, and measuring the second messenger response in the presence of only the second chemical compound and in the presence of both the second chemical compound and the chemical compound, a smaller change in the second messenger response in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound indicating that the chemical compound inhibits activation of the mammalian LPA receptor.
113. The process of claim 112, wherein the second messenger response comprises chloride channel activation and the change in second messenger response is a smaller increase in the level of inward chloride current in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound.
114. A process of any one of claims 110, 111, 112 or 113, wherein the mammalian LPA receptor is a human LPA receptor.
115. The process of claim 114, wherein the human LPA receptor has substantially the same amino acid sequence as encoded by the plasmid HL-18a (ATCC Accession No. 209448).
116. The process of claim 114, wherein the human LPA receptor has substantially the same amino acid sequence as that shown in FIG. 3 (Seq. I.D. No. 3).
117. The process of claim 114, wherein the human LPA receptor has an amino acid sequence, identical to the amino acid sequence shown in FIG. 3 (Seq. I.D. No. 3).
118. The process of any one of claims 110, 111, 112, 113, 114, 115, 116, or 117 wherein the cell is an insect cell.
119. The process of any one of claims 110, 111, 112, 113, 114, 115, 116, or 117 wherein the cell is a mammalian cell.
120. The process of claim 119, wherein the mammalian cell is non-neuronal in origin.
121. The process of claim 120, wherein the non-neuronal cell is a COS-7 cell, CHO cell, 293 human embryonic kidney cell, NIH-3T3 cell or LM(tk−) cell.
122. The process of claim 119, wherein the compound is a compound not previously known to bind to a mammalian LPA receptor.
123. A compound determined by the process of claim 122.
124. A pharmaceutical composition which comprises an amount of a mammalian LPA receptor agonist determined by the process of claim 110 or 111 effective to increase activity of a mammalian LPA receptor and a pharmaceutically acceptable carrier.
125. A pharmaceutical composition of claim 124, wherein the mammalian LPA receptor agonist is not previously known.
126. A pharmaceutical composition which comprises an amount of a mammalian LPA receptor antagonist determined by the process of any one of claims 112 or 113 effective to reduce activity of a mammalian LPA receptor and a pharmaceutically acceptable carrier.
127. A pharmaceutical composition of claim 126, wherein the mammalian LPA receptor antagonist is not previously known.
128. A method of screening a plurality of chemical compounds not known to activate a mammalian LPA receptor to identify a compound which activates the mammalian LPA receptor which comprises:
- (a) contacting cells transfected with and expressing the mammalian LPA receptor with the plurality of compounds not known to activate the mammalian LPA receptor, under conditions permitting activation of the mammalian LPA receptor;
- (b) determining whether the activity of the mammalian LPA receptor is increased in the presence of the compounds; and if so
- (c) separately determining whether the activation of the mammalian LPA receptor is increased by each compound included in the plurality of compounds, so as to thereby identify the compound which activates the mammalian LPA receptor.
129. A method of claim 128, wherein the mammalian LPA receptor is a human LPA receptor.
130. A method of screening a plurality of chemical compounds not known to inhibit the activation of a mammalian LPA receptor to identify a compound which inhibits the activation of the mammalian LPA receptor, which comprises:
- (a) contacting cells transfected with and expressing the mammalian LPA receptor with the plurality of compounds in the presence of a known mammalian LPA receptor agonist, under conditions permitting activation of the mammalian LPA receptor;
- (b) determining whether the activation of the mammalian LPA receptor is reduced in the presence of the plurality of compounds, relative to the activation of the mammalian LPA receptor in the absence of the plurality of compounds; and if so
- (c) separately determining the inhibition of activation of the mammalian LPA receptor for each compound included in the plurality of compounds, so as to thereby identify the compound which inhibits the activation of the mammalian LPA receptor.
131. A method of claim 130, wherein the mammalian LPA receptor is a human LPA receptor.
132. A method of any one of claims 128, 129, 130, or 131, wherein the cell is a mammalian cell.
133. A method of claim 132, wherein the mammalian cell is non-neuronal in origin.
134. The method of claim 133, wherein the non-neuronal cell is a COS-7 cell, a 293 human embryonic kidney cell, a LM(tk−) cell or an NIH-3T3 cell.
135. A pharmaceutical composition comprising a compound identified by the method of any one of claims 128 or 129 effective to increase mammalian LPA receptor activity and a pharmaceutically acceptable carrier.
136. A pharmaceutical composition comprising a compound identified by the method of any one of claims 130 or 131 effective to decrease mammalian LPA receptor activity and a pharmaceutically acceptable carrier.
137. A method of treating an abnormality in a subject wherein the abnormality is alleviated by increasing the activity of a mammalian LPA receptor which comprises administering to the subject an amount of compound which is a mammalian LPA receptor agonist effective to treat the abnormality.
138. A method of treating an abnormality in a subject wherein the abnormality is alleviated by decreasing the activity of a mammalian LPA receptor which comprises administering to the subject an amount of compound which is a mammalian LPA receptor antagonist effective to treat the abnormality.
Type: Application
Filed: Jul 26, 2002
Publication Date: Sep 18, 2003
Inventor: Jonathan A Bard (Doylestown, PA)
Application Number: 10205911
International Classification: C07K014/74; C07H021/04; C12P021/02; C12N005/06;
