WO1989012687A1 - Recepteur d'acide retinoique - Google Patents

Recepteur d'acide retinoique Download PDF

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Publication number
WO1989012687A1
WO1989012687A1 PCT/US1989/002636 US8902636W WO8912687A1 WO 1989012687 A1 WO1989012687 A1 WO 1989012687A1 US 8902636 W US8902636 W US 8902636W WO 8912687 A1 WO8912687 A1 WO 8912687A1
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rar
dna
receptor
binding domain
polypeptide
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PCT/US1989/002636
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English (en)
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Magnus Pfahl
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La Jolla Cancer Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to cell biology and more specifically to a novel receptor for retinoic acid.
  • Receptors of the steroid-thyroid hormone family are intracellular proteins which consist of discrete DNA-binding regions and ligand-binding regions.
  • the DNA-binding domain of steroid hormone receptors and related proteins consists of a cystein-rich region that has been highly conserved and that has the potential to form two zinc-binding fingers.
  • the ligand binding region is specific to the cognate ligand.
  • Retinoids are known to be essential for the normal function of many epithelial tissues.
  • tissue-specific expression of RAR ⁇ is uncharacteristic for a molecule expected to be a major mediator of retinoid action in epithelial tissues. Because of their critical role in mediating metabolism and growth, it is important to determine other receptors mediating retinoid action. Knowledge of retinoid receptors permits the manipulation and control of metabolic effects resulting from retinoid action.
  • This invention relates to substantially purified Retinoic Acid Receptor e (RAR ⁇ ), having the amino acid sequence given in Figure 1, and to the isolated nucleic acid encoding RAR ⁇ .
  • RAR ⁇ Retinoic Acid Receptor e
  • polypeptides comprising, and nucleic acids encoding, the DNA-binding and hormone-binding domain of the receptor are provided.
  • methods of detecting RAR ⁇ and nucleic acids encoding RAR ⁇ and its domains are provided.
  • Fig. 1 shows a comparison of the amino acid sequence encoded by the HBV-1 clone with amino acid sequences of other members of the nuclear receptor family, as described in Example I.
  • Fig. 2 shows an analysis of in-vitro-synthesized RAR ⁇ protein, as described in Example 2.
  • Fig. 3 shows "Finger Swap” from ER to RAR ⁇ as described in Example III.
  • Fig. 4 shows RAR ⁇ chimaeric protein activation of the transcription of an ERE-CAT reporter gene in the presence of retinoic acid as described in Example IV.
  • Fig. 5 shows tissue specific expression of RAR ⁇ .
  • Fig. 6 shows RAR ⁇ and the endogenous F9 cell RAR activate transcription from thyroid hormone responsive elements.
  • Fig. 7 shows a RAR-E 2 hybrid receptor confers estradiol inducibility to a TRE-CAT gene.
  • Fig. 8 shows RAR ⁇ activates transcription from a thyroid hormone responsive element in CV-1 cells.
  • Fig. 9 shows thyroid hormone receptors repress the activity of RAR ⁇ in F9 and CV-1 cells.
  • Fig. 10 shows RAR regions sensitive to TR repression by hybrid receptor analysis.
  • RAR ⁇ A new retinoic acid receptor, herein termed RAR ⁇ , which is encoded by a cDNA clone from a human placental library, has been identified and characterized.
  • RAR ⁇ is a member of the steroid/thyroid hormone receptor family.
  • a polypeptide having substantially the same function and amino acid sequence as RAR ⁇ is also provided.
  • having substantially the same function means modifications of the amino acid sequence which are neutral, improve, or lessen the function of RAR ⁇ so long as the essential function is maintained.
  • polypeptide analogs which have the activity of any portion of RAR ⁇ , but which differ in amino acid content, are contemplated.
  • Polypeptides comprising the DNA-binding domain of RAR ⁇ are provided. Polypeptides having substantially the same function and amino acid sequence as the DNA-binding domain are also provided. These polypeptides can include not only the DNA-binding domain of RAR ⁇ alone but also any portion of RAR ⁇ which includes the DNA-binding domain.
  • the polypeptides can be attached to other markers or polypeptides to utilize the DNA-binding characteristic. For example, the hormone binding domain of RAR ⁇ receptor can be attached to the DNA-binding domain and transfected into cells. The cognate ligand of that particular receptor can then be added or limited to control transcription of a target gene.
  • Polypeptides comprising the hormone-binding domain of RAR ⁇ or polypeptides having substantially the same function and amino acid sequence are also provided.
  • the ability to bind a ligand, for example retionic acid, is a contemplated function.
  • the polypeptide does not include RAR ⁇ .
  • the hormone-binding domain can include not only the hormone-binding domain of RAR ⁇ alone but also any portion of RAR ⁇ which includes the hormone-binding domain of RAR ⁇ .
  • the polypeptide can be attached to other markers or polypeptides to utilize the hormone-binding characteristic.
  • the hormone-binding receptor can be attached to a known DNA-binding domain of another polypeptide and transfected into cells.
  • a cognate ligand of the hormone-binding receptor for example, retinoic acid, can then be added or limited to control transcription of a target gene.
  • Nucleic acids for example, DNA, RNA, or cDNA
  • Vectors comprising these nucleic acids are also provided.
  • Recombinant host cells can be transformed with such a vector and used to express recombinant polypeptides. Such methods of recombinant expression are well known in the art, see Maniatis et al., Molecular Cloning: A Laboratory Manual (1982), which is herein incorporated by reference. Thus, recombinant polypeptides and the method of their production are also provided.
  • RAR ⁇ is expressed at high levels in a number of epithelial-type tissues. It is also implicated in hepatocellular carcinoma development when its gene is activated by hepatitis B virus (HBV) integration in liver cells, where it is normally not expressed. A determination of its presence in malignant tissue is useful in elucidating the pathology of the tumor and in determining appropriate strategies of therapy.
  • HBV hepatitis B virus
  • the invention provides a method of diagnosing a tumor in a subject comprising detecting in a sample from the subject increased or decreased levels of RAR ⁇ , increased or decreased levels of RAR ⁇ indicating the presence of a tumor.
  • hepatocellular carcinoma can be detected since RAR ⁇ expression can be altered in liver cells of a subject with hepatocellular carcinoma.
  • a method of diagnosing a tumor in a subject comprises detecting in a sample from the subject a mutated form of RAR ⁇ , the mutated RAR ⁇ indicating the presence of a tumor.
  • Any method of detecting polypeptides may be utilized, for example an immunoassay.
  • mutated is meant any variation of naturally occurring RAR ⁇ . The mutation can result, for example, from the integration of a virus, a point mutation, a gene rearrangement, or gene amplification.
  • the suggested open reading frame encodes a 448 amino acid protein of relative molecular mass (M r ) 50,000 (50 K), that contains the domains typical of members of the steroidthyroid hormone receptor family.
  • the cysteine-rich DNA-binding domain (C) is connected through a putative hinge region (domain D) with a ligand or hormone-binding domain (E).
  • the amino-terminal region is short and contains only the (B) domain but no (A) domain.
  • the (A) domain is typical for steroid hormone receptors but is not found in thyroid hormone receptors.
  • the hormone-binding domain (E) of the receptor of the present invention is 88% homologous with the previously described human retinoic acid receptor (RAR ⁇ ), Petkovich et al., supra; Giguere et al., supra. Domain D is also highly conserved and shows a 74% homology with RAR ⁇ .
  • the DNA-binding domain exhibits a 97% identity with RAR ⁇ .
  • a situation in which three colinear domains have been highly conserved has so far only been observed between the two human thyroid hormone receptors encoded by the erbA- ⁇ and the erbA-T genes. In that case, it was shown that an 86% homology in the hormone-binding domain was sufficient to conserve ligand specificity, Benbrook et al., Science 238:788-791 (1987) which is incorporated herein by reference.
  • RNA made in vitro containing the complete 5' untranslated region of the HBV-1 clone was efficiently translated in a rabbit reticulocyte lysate system.
  • the HBV-1 RNA was of a single size, and so the smaller proteins seen in the XR lane of Fig. 2 may represent translation products initiating at AUG codons within the coding sequence.
  • HRE human epidermal growth factor receptor
  • the hybrid receptor was then transfected into susceptible tissue-culture cells together with a reporter gene containing the relevant HRE.
  • the effect of various potential activators of the novel receptor was measured from cells transiently transfected with both genes and grown in the presence and absence of the various ligands.
  • the hER DNA-binding domain was used as the donor domain as outlined in Figure 3.
  • the resulting construct was confirmed by DNA sequencing.
  • RNA of the new hybrid gene was translated in vitro. The major band migrated very similarly to the largest protein made from the wild-type gene, whereas other constructs which did not encode complete receptor proteins gave rise to smaller sized bands (see Fig. 2b).
  • Results from transient co-transfection experiments using a CAT (chloramphenicol acetyl transferase) reporter gene linked to a promoter containing the estrogen-response element (ERE) are shown in Figure 4. Only retinoic acid induced CAT gene expression strongly at a physiological concentration (2.5 x 10 -8 M). The hybrid receptor was inactive in the presence of other unrelated ligands (estradiol and dexamethasone). Other retinoids (retinol, retinal and retinyl-acetate) showed some inducing effect only at high concentrations (3 x 10 -6 M). The results indicate that the gene activated by HBV integration in a human hepatocellular carcinoma, with similarity to steroid-thyroid hormone-like receptors, encodes a retinoic acid receptor.
  • CAT chloramphenicol acetyl transferase
  • Rat tissue was chosen because this species has been extensively used as a model system to establish tissue-responsiveness to retinoic acid. Strong expression was observed in brain (including pineal gland but not pituitary), pituitary gland, kidney, colon, uterus, ovary, testis, prostate gland, adrenal gland, and eye. The size of the transcript appears to vary ranging from 2.4 to
  • Vitamin A and other retinoids have been considered to be, and are used therapeutically as, anti-tumor drugs.
  • RAR ⁇ might contribute to tumor development when expressed erroneously in liver tissue, where it is normally silent (Fig. 5).
  • Retinoids are known to maintain the proliferative state of epithelial cells. This type of cell proliferation, when induced in other types of tissue by erroneous RAR ⁇ expression and the presence of retinoic acid, can lead to tumor development.
  • Gene probes which can hybridize with RAR ⁇ nucleic acid can be prepared.
  • Basic techniques for preparing DNA oligonucleotide probes, as well as their screening by nucleic acid hybridization, are well known to those of ordinary skill in the art. See, for example, DNA CLONING: VOLUME I (D.M. Glover, ed. 1985); NUCLEIC ACID HYBRIDIZATION (B.D. Hames and S.J. Higgins eds. 1985): OLIGONUCLEOTIDE SYNTHESIS (M. J .
  • oligonucleotides or cDNA or riboprobes derived from the cDNA can be used to probe the library and isolate the desired gene.
  • the oligonucleotides are synthesized by any appropriate method, such as by the use of an automated DNA synthesizer.
  • the particular nucleotide sequences selected are chosen so as to correspond to the codons encoding a known amino acid sequence from the protein. Since the genetic code is redundant, it will often be necessary to synthesize several oligonucleotides to cover all, or a reasonable number, of the possible nucleotide sequences which encode a particular region of the protein.
  • the region it is generally preferred in selecting a region upon which to base the probes, that the region not contain amino acids whose codons are highly degenerate.
  • the region may find it desirable to prepare probes that are fairly long, and/or encompass regions of the amino acid sequence which would have a high degree of redundancy in corresponding nucleic acid sequences, particularly if this lengthy and/or redundant region is highly characteristic of the receptor protein.
  • Probes covering the complete gene, or a substantial part of the genome may also be appropriate, depending upon the expected degree of homology. It may also be desirable to use two or more probes, or sets of probes, each to different regions of the gene, in a single hybridization experiment.
  • the selected oligonucleotide probes are labeled with a marker, such as a radionucleotide or biotin, using standard procedures.
  • the labeled set of probes is then used in the screening step, which consists of allowing the single-stranded probe to hybridize to isolated denatured DNA from the library, according to standard techniques. Either stringent or permissive hybridization conditions can be appropriate, depending upon several factors, such as the length of the probes and whether the probes are derived from the same species as the library or an evolutionarily close or distant species. The selection of the appropriate conditions is within the skill of the art. See generally, NUCLEIC ACID HYBRIDIZATION, supra.
  • hybridization conditions be of sufficient stringency so that selective hybridization occurs; i.e., hybridization is due to a sufficient degree of nucleic acid homology (for example, at least about 75%) , as opposed to nonspecific binding.
  • a DNA coding sequence for the desired protein can be prepared synthetically from overlapping oligonucleotides whose sequence contains codons for the amino acid sequence of the protein.
  • oligonucleotides are prepared by standard methods and assembled into a complete coding sequence. See, for example. Edge, (1981) Nature 292:756; Nambair et al., (1984) Science 223:1299; Jay et al., (1984) J. Biol. Chem. 259:6311, all of which are incorporated herein by reference.
  • RAR ⁇ can be used to produce antibodies, either polyclonal or monoclonal.
  • purified RAR ⁇ protein is used to immunize a selected mammal (for example, mouse, rabbit, goat, horse, etc.) and serum from the immunized animal is later collected and treated according to known procedures.
  • Antisera containing polyclonal antibodies to a variety of antigens in addition to the RAR ⁇ can be made substantially free of antibodies which are not anti-RAR ⁇ specific by passing the composition through a column to which non-RAR ⁇ protein has been bound. After washing, antibodies to the non-RAR ⁇ proteins will bind to the column, whereas anti-RAR ⁇ antibodies elute in the flow through.
  • Monoclonal anti-RAR ⁇ antibodies can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by fusing myelomas and lymphocytes to form hybridomas is well known. Such cells are screened to determine whether they secrete the desired antibodies, and can then be grown either in culture or in the peritoneal cavity of a mammal.
  • Antibodies can also be created by techniques other than fusion, such as direct transformation of ⁇ -lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus, See, for example, M.
  • Antibodies specific to RAR ⁇ have a number of uses. For example, they may be employed in an immunoassay to detect the presence of RAR ⁇ . Detection of increased or decreased levels of RAR ⁇ or a mutant form of RAR ⁇ can be useful in diagnosing a tumor, for example, hepatocellular carcinoma. Various appropriate immunoassay formats are well known to those skilled in the art.
  • the invention also provides a method of detecting a ligand reactive with RAR ⁇ or a polypeptide comprising the ligand-binding domain of RAR ⁇ .
  • the method comprises contacting a sample suspected of containing the ligand with RAR ⁇ , or a polypeptide comprising the ligand-binding domain of RAR ⁇ , or functional equivalents thereof, and detecting the binding to the ligand.
  • functional equivalent is meant any polypeptide, except RAR ⁇ , having the ability to bind ligands reactive with RAR ⁇ .
  • minor changes in the amino acids or changes which increase or decrease the binding function are contemplated. Such changes would be expected to be found on the equivalent receptor in different species.
  • the ligand detected is retinoic acid.
  • any immunoassay-type assay format can be used, substituting RAR ⁇ or purified ligand-receptor for one or more of the antibodies. Since the ligands for the receptor are small molecules, preferably the assay is of the competitive type.
  • the analyte being assayed is allowed to complex with the receptor by incubating the solid phase with a fluid sample suspected of containing the analyte.
  • Radiolabeled analyte (other labels, such as fluorescent labeling are possible) is added to the sample.
  • the receptor-analyte complex is separated from the free analyte by binding the complex to a solid phase such as a Millipore filter.
  • the filter solid phase is washed and analyte receptor complex bound to it is measured.
  • the receptor can be coupled to a solid phase prior to contacting with the sample.
  • the receptor is coupled to a solid phase by means well known in the art.
  • the solid phase may comprise any of a variety of porous materials, including various natural or synthetic materials, alone or in combination. Included among these are polysaccharides, for example, cellulose materials, such as paper and cellulose acetate; silica; inorganic materials such as deactivated alumina diatomaceous earth
  • MgSO 4 or other inorganic finely divided material conveniently substantially uniformly dispersed in a porous polymer matrix with polymers such as vinyl chloride, vinyl chloride-propylene copolymer, and vinyl chloride-vinyl acetate copolymer; cloth, both naturally occurring, for example, cotton, and synthetic, for example, nylon cloth; porous gels, for example, silica gel, agarose, dextran, and gelatin; polymeric films, for example, polyacrylamide or the like.
  • Another alternative is to remove the receptor-analyte complex from solution by allowing it to bind to an anti-receptor antibody which may itself be coupled to a solid support.
  • a method of detecting DNA reactive with RAR ⁇ or a polypeptide comprising the DNA-binding domain of RAR ⁇ comprises contacting DNA with RAR ⁇ or a polypeptide comprising the DNA-binding domain of RAR ⁇ , or a functional equivalents thereof, and detecting the binding to the DNA.
  • specific DNA molecules such as thyroid hormone responsive element (TRE)
  • TRE thyroid hormone responsive element
  • a DNA sequence attached to RAR ⁇ is also provided.
  • the DNA sequence being a sequence which is responsive with RAR ⁇ .
  • attached is meant chemically bound, for example, covalent or ionic.
  • the sequence can occur in the promotor region just upstream from the promotor where the sequence, when properly attached by RAR ⁇ , controls transcription of the coding regions downstream of the promoter.
  • An example of a sequence responsive with RAR ⁇ is thyroid hormone responsive element.
  • a second DNA sequence can be under the control of the promoter.
  • the second DNA sequence can encode RNA which is translated into a desired polypeptide.
  • Such expression can be carried out by placing a vector containing the second DNA sequence under the control of the promoter and the DNA sequence responsive with RAR ⁇ in proper orientation into an appropriate host under polypeptide producing conditions.
  • Appropriate vectors, hosts, DNA orientations, and polypeptide producing conditions are all well known to those skilled in the art, see for example, Maniatis et al., supra.
  • the recombinant host cells can be induced to produce the desired polypeptide by the introduction of RAR ⁇ .
  • the RAR ⁇ can be endogenously produced by the host or may be produced by a transfected vector.
  • transcription of DNA sequences normally activated by a retinoic acid receptor can be inhibited by adding nonactivated thyroid receptor to the cell.
  • the thyroid receptor for RAR ⁇ can be endogenously produced by the host or may be produced by a transfected vector.
  • the retinoic acid receptor can be RAR ⁇ or RAR ⁇ and the thyroid receptor can be TR ⁇ or TR ⁇ .
  • the invention provides a method of screening ligands for ligands which stimulate or inhibit RAR ⁇ comprising adding the ligand to a recombinant host cell of the invention and observing the stimulation or inhibition of the polypeptide encoded by the second DNA sequence, an increase in the polypeptide indicating the stimulation of RAR ⁇ and a decrease in the polypeptide indicating the inhibition of RAR ⁇ .
  • the conditions of the screening can be ascertained without undue experimentation by one skilled in the art.
  • RA Fig. 6a and b
  • TR ⁇ Similar levels of induction were obtained with TR ⁇ .
  • F9 cells have been established as a cellular model for RA dependent differentiation and to contain RAR transcripts. Whether the endogenous F9 RAR can also activate the TRE-CAT genes, by transfecting the receptor genes into F9 cells alone was investigated. A 12 fold induction in the presence of RA is observed for the G17-2 construct and no induction is seen in the presence of T 3 , suggesting that F9 cells do not contain TR.
  • the RA induction is, however, considerably lower than that observed in the presence of cotransfected RAR ⁇ indicating that the endogenous RAR is limiting in concentration.
  • a hybrid receptor which contains the RAR ⁇ DNA binding domain and the estrogen receptor hormone binding domain (Fig. 7a). It has been shown above that individual receptor domains can be exchanged to create receptors with novel specificities.
  • the hybrid receptor RAR-E 2 was cotransfected (Fig. 7a) with the TRE-CAT genes into F9 cells, induction of CAT activity in the presence of the hormone estradiol (E 2 ) was observed (Fig. 7b).
  • Other hybrid receptors containing the ER DNA binding domain and the RAR ⁇ hormone binding domain were unable to stimulate transcription from TRE-CAT genes. These data therefore define the DNA binding region of RAR ⁇ as necessary for activating transcription from TREs in F9 cells.
  • RA and T 3 likely modulate gene transcription via their nuclear receptors from a common family of responsive elements.
  • T 3 hormone
  • RA vitamin
  • both receptors were cotransfected into the same cells together with a TRE-CAT gene. High CAT activity is observed in the presence of both ligands (T 3 + RA). Approximately 70% of the maximal activity is observed when only T 3 is added. Surprisingly, no activity (or very low activity) is observed when only RA is added.
  • F9 cells is observed between RAR ⁇ : TR ⁇ ratios of 1:1 to 4:1.
  • TR repressor mechanism still need to be elucidated; in particular, whether the receptors compete independently for the DNA binding site or whether an additional level of regulatory control by mutual protein-protein interaction exists. If the competition model applies, one would predict that TR ⁇ and TR ⁇ both bind to TREs in the absence of ligand. This would be in agreement with reported findings that several steroid hormone receptors can bind specific DNA sequences in the absence of hormone. See, for example, Willmann, T., et al., Nature, 324, 688-691 (1986), incorporated herein by reference. Our observed repression is highly specific for RAR since the hybrid receptors activating an ERE-CAT gene are not significantly inhibited (Fig. 10c).
  • NUCLEOTIDE SEQUENCE OF RAR ⁇ cDNA AND AMINO ACID
  • the amino acid sequences of the DNA- and hormone-binding domains are compared with the same domains of other human proteins of this family: RAR, retinoic acid receptor Petkovich, et al., supra; and Giguere et al., supra; hTR and hTR£, thyroid hormone receptor, Benbrook et al., supra; and Weinberger et al., Nature 324, 642-649 (1986); hER, estrogen receptor Green et al., Nature 320, 134-139 (1986) and Green et al., Science 231, 1150-1154 (1986); hGR, glucocorticoid receptor Weinberger et al., Nature 318, 610-612 (1985); hMR, mineralocorticoid receptor Arriza et al., Science 237, 268-275 (1987); hPR, progesterone receptor Misrahi et al., Comm.
  • RNAs from clone HBV-1 were used to translate 35 S-labeled RAR protein (lane XR); as controls, human estrogen receptor (lane ER), and human thyroid hormone receptor (lane T-1) were also synthesized using the same in vitro system, as shown in Fig. 2.
  • FIG. 2 Analysis of the protein from the RAR-ER hybrid gene is shown in Fig. 2.
  • An in vitro transcription-translation system was used to analyze the protein encoded by the RAR-ER hybrid gene (lane XER) shown in Figure 3.
  • Fig. 3 presents a schematic representation given to illustrate the construction of an RAR ⁇ -ER chimaeric gene.
  • the brackets indicate the portions of the RAR ⁇ (hXR) and ER DNA-binding domains used in the construct. These exchange regions contain the potential zinc-fingers of the DNA-binding domain.
  • Example I The ⁇ 1-RAR clone of Example I was cut with HindIII and Apal, the Apal ends were blunt-ended with mung-bean nuclease.
  • the plasmid was then religated which resulted in a clone
  • RAR-9 in which the XhoI and HincII sites of the cloning box were deleted.
  • RAR-9 hXR
  • RAR-9 hXR
  • XhoI and HincII which cut at unique sites.
  • the staggered end of the XhoI sites was filled in.
  • the ⁇ 1-ER fragment encodes amino acid residues 168 through 240 and contains also the first nucleotide of the Arg-241 codon of hER. Arg-137 of RAR and Arg-241 of ER are at the same positions in the two DNA-binding domains. The 5' end of the ER fragment encodes 13 amino acids more than were present on the excised RAR fragment. Constructs were analyzed by DNA sequencing. The clone with the described features is designated ⁇ 1-RAER (XER). The major protein obtained from this clone by in vitro transcription-translation is indistinguishable in size from the largest RAR ⁇ wild-type translation product (Fig. 4) .
  • the RAER gene was cut out with BamHI and KpnI (both sides in the cloning box) and ligated into the BglII and KpnI sites of the PECE eukaryotic expression vector, Ellis et al., Cell 45:721-732 (1986), incorporated herein by reference, which yielded clone PECE-RAER.
  • BamHI and KpnI both sides in the cloning box
  • HeLa cells were co-transfected with the PECE-RAER expression vector (RAER) and the oestrogen-responsive CAT gene (ERE-CAT) and grown in the presence of either 5 x 10 -9 M oestradiol (Est) or 5 x 10 -7 M dexamethasone (Dex) or the indicated molar concentrations of various retinoids (Ret) : 1, retinoic acid; 2,retinol; 3,retinal; 4,retinylacetate (see Fig. 4).
  • RAER PECE-RAER expression vector
  • ERE-CAT oestrogen-responsive CAT gene
  • HeLa cells (1.5 x 10 6 per dish) were transfected using calcium phosphate, Wigler et al., Cell 11, 223-232 (1977) which is incorporated herein by reference, with 5 ⁇ g of the expression vectors PECE-RAER or pckR2ER and 20 ⁇ g of the ERE-CAT vector or the pSV2CAT vector. After 5 hours, cells were shocked with 10% glycerol for 2 min. Eighteen hours after the start of transfection, steroid hormones or retinoids were added to the medium and cells were incubated for a further 24 hours before harvesting. CAT activity in cell extracts was determined as described by Gormon et al., Biol 2, 1044-1051 (1982), herein incorporated by reference.
  • RNA (20 ⁇ g per lane) extracted from different rat tissues.
  • Lane 1 brain; lane 2, pituitary; lane 3, atrium; lane 4, lung; lane 5, diaphragm; lane 6, kidney; lane 7, liver; lane 8, spleen; lane 9, colon; lane 10, uterus; lane 11, ovary; lane 12, testis; lane 13, prostate; lane 14, seminal vesicle; lane 15, adrenal; lane 16, eye (see Fig. 5) .
  • Markers on the side are in kb.
  • RNA was isolated from tissues extracted from adult BK1: (SD) rats.
  • Northern blots were prepared from 1% formaldehyde-agarose gels containing 20 ⁇ g RNA per lane, prehybridized at 42oC overnight in 50% formamide, 5x SSPE (1x SSPE is 0.18 M NaCl, 10 mM NaPO 4 , pH 7.7 1 mM EDTA), 5x Denhardt's reagent, 0.1% SDS, and 250 ⁇ g/ml -1 sheared salmon sperm DNA.
  • Hybridizations were at 42oC in 50% formamide, 5x SSPE, lx Denhardt's reagent, 0.1% SDS, 100 ⁇ g/ml -1 sheared salmon sperm DNA, and 20 ng/ml -1 of probe 1 for all lanes except 10, 12, 13, and 14 for which 2 x 10 6 cpm/ml of probe 2 was used.
  • Probe I was labeled by nick-translating the ⁇ 1-RAR whole plasmid (10 8 cpm/ ⁇ g -1 ) and probe 2 consisted of an EcoRI-SphI fragment of the 5' end of RAR labeled with Klenow using random oligonucleotides as primers (10 9 cpm/ ⁇ g -1 ).
  • F9 cells were cotransfected with 5 ⁇ g of the expression vectors PECE-RAR ⁇ or PECE-TR ⁇ and 20 ⁇ g of the reporter plasmids G17-2 CAT or MHC-CAT. As a control, cells were also transfected with the reporter plasmids only.
  • the G17-2 CAT construct contains three copies of a synthetic TRE derived from the rat GH gene. Glass, C. K., et al., supra, incorporated herein by reference.
  • the MHC construct contains nucleotides -163 to -81 upstream of the CAP site of the rat ⁇ MHC gene, Izumo, S., et al., supra.
  • Fold activation is expressed as the ratio of relative CAT activity after hormone induction to relative CAT activity without hormone induction.
  • the mean of at least three experiments is given in Figure 6(a).
  • Ligand dependant activation of RAR ⁇ and the endogenous F9 cell RAR is shown in Figure 6(b).
  • F9 cells were cotransfected with 5 ⁇ g of PECE-RAR ⁇ and 20 ⁇ g of G17-2 CAT ( ⁇ - ⁇ ) or with 20 ⁇ g of the expression vector alone ( ⁇ - ⁇ ) . Indicated amounts of hormones were added 24 hours after transfection.
  • F9 cells were cultured as monolayer on gelatin-coated dishes in alpha-MEM (GIBCO, Santa Clara, CA), supplemented by 10% fetal calf serum (FCS), glutamine and nucleosides as described in Grover, A., et al., J. Cell Biol. 96, 1690-1996 (1983) which is incorporated herein by reference.
  • FCS fetal calf serum
  • FCS fetal calf serum
  • nucleosides as described in Grover, A., et al., J. Cell Biol. 96, 1690-1996 (1983) which is incorporated herein by reference.
  • FCS fetal calf serum
  • Two to five hours before transfection cells were fed the above medium containing charcoal-treated FCS. Twenty-four hours after transfection, cells were fed with charcoal treated medium together with the appropriate amount of hormone (final hormone concentrations 5 x 10 -8 M). Forty-eight hours after transfection, cells were harvested, lysed and as
  • Bl-RAR clone was digested with Apal and HincII to remove a 1 kB fragment with the hormone-binding domain and the COOH-terminus of the RAR ⁇ .
  • a 1.15 kb Nael - Apal fragment of the human ER clone B1-ER, Benbrook, D. and Pfahl, M., Science, 238, 788-791 (1987) was ligated to the RAR ⁇ fragment which encodes the aminoterminal and DNA binding domains.
  • DNA sequencing confirmed the in-frame connection between the RAR DNA binding domain and the ER hinge region.
  • a 1.8 kb BamHI-SalI fragment from B1-RAR-E 2 was cloned into the BgII & SalI sites of PECE.
  • F9 cells were cotransfected with 5 ⁇ g of PECE-RAR-E 2 and 20 ⁇ g of G17-2 CAT or MHC-CAT.
  • Estradiol (E 2 ) was added 24 hours after transfection (final concentration 5 x 10 -7 M).
  • CAT activity was determined as described in Example VI. In the presence of E 2 , MHC-CAT is induced twofold by RAR-E 2 and G17-2 CAT is induced 20 fold (fold induction is calculated from 3 independent experiments) (See Figure 7b).
  • CV-1 cells were cotransfected with 5 ⁇ g of PECE-RAR ⁇ , PECE-TR ⁇ or PECE-TR ⁇ as indicated and 20 ⁇ g of G17-2 CAT.
  • Final concentrations of hormone were 6 x 10 -7 (RA) and 10 -7 (T 3 ) (See, Figure 8). Fold activation was determined as the mean of 3 separate experiments.
  • RAER-II contains amino acids 1 -287 of the human ER (which includes the DNA binding domain up to the hinge region) and amino acids 169 - 448 of RAR (which includes a portion of the hinge region and the hormone binding domain).
  • Figure 10(c) shows RAER-I and RAER-II activate transcription from an ERE-TK-CAT reporter gene in the presence of TR ⁇ .
  • CV-1 cells were cotransfected with 2.5 ⁇ g of PECE-RAER-I or PECE-RAER-II and 2.5 ⁇ g PECE-TR ⁇ , together with 20 ⁇ g of ERE-TK-CAT reporter gene as described above.
  • the ERE-TK-CAT contains an estrogen responsive element (ERE), Klein-Hitpass, L., et al., Cell 46, 1053-1061 (1986), incorporated herein by reference. Transfected cells were treated with 6 x 10 -7 M RA, 10 -7 M T 3 , or RA + T 3 as indicated.
  • ERP estrogen responsive element
  • This invention relates to cell biology and more specifically to a novel receptor for retinoic acid.
  • Receptors of the steroid-thyroid hormone family are intracellular proteins which consist of discrete DNA-binding regions and ligand-binding regions.
  • the DNA-binding domain of steroid hormone receptors and related proteins consists of a cystein-rich region that has been highly conserved and that has the potential to form two zinc-binding fingers.
  • the ligand binding region is specific to the cognate ligand.
  • Retinoids are known to be essential for the normal function of many epithelial tissues.
  • tissue-specific expression of RAR ⁇ is uncharacteristic for a molecule expected to be a major mediator of retinoid action in epithelial tissues. Because of their critical role in mediating metabolism and growth, it is important to determine other receptors mediating retinoid action. Knowledge of retinoid receptors permits the manipulation and control of metabolic effects resulting from retinoid action.
  • This invention relates to substantially purified Retinoic Acid Receptor ⁇ (RAR ⁇ ), having the amino acid sequence given in Figure 1, and to the isolated nucleic acid encoding RAR ⁇ .
  • RAR ⁇ Retinoic Acid Receptor ⁇
  • polypeptides comprising, and nucleic acids encoding, the DNA-binding and hormone-binding domain of the receptor are provided.
  • methods of detecting RAR ⁇ and nucleic acids encoding RAR ⁇ and its domains are provided.
  • Fig. 1 shows a comparison of the amino acid sequence encoded by the HBV-1 clone with amino acid sequences of other members of the nuclear receptor family, as described in Example I.
  • Fig. 2 shows an analysis of in-vitro-synthesized RAR ⁇ protein, as described in Example 2.
  • Fig. 3 shows "Finger Swap” from ER to RAR ⁇ as described in Example III.
  • Fig. 4 shows RAR ⁇ chimaeric protein activation of the transcription of an ERE-CAT reporter gene in the presence of retinoic acid as described in Example IV.
  • Fig. 5 shows tissue specific expression of RAR ⁇ .
  • Fig. 6 shows RAR ⁇ and the endogenous F9 cell RAR activate transcription from thyroid hormone responsive elements.
  • Fig. 7 shows a RAR-E 2 hybrid receptor confers estradiol inducibility to a TRE-CAT gene.
  • Fig. 8 shows RAR ⁇ activates transcription from a thyroid hormone responsive element in CV-1 cells.
  • Fig. 9 shows thyroid hormone receptors repress the activity of RAR ⁇ in F9 and CV-1 cells.
  • Fig. 10 shows RAR regions sensitive to TR repression by hybrid receptor analysis.
  • RAR ⁇ A new retinoic acid receptor, herein termed RAR ⁇ , which is encoded by a cDNA clone from a human placental library, has been identified and characterized.
  • RAR ⁇ is a member of the steroid/thyroid hormone receptor family.
  • a polypeptide having substantially the same function and amino acid sequence as RAR ⁇ is also provided.
  • having substantially the same function means modifications of the amino acid sequence which are neutral, improve, or lessen the function of RAR ⁇ so long as the essential function is maintained.
  • polypeptide analogs which have the activity of any portion of RAR ⁇ , but which differ in amino acid content, are contemplated.
  • Polypeptides comprising the DNA-binding domain of RAR ⁇ are provided. Polypeptides having substantially the same function and amino acid sequence as the DNA-binding domain are also provided. These polypeptides can include not only the DNA-binding domain of RAR ⁇ alone but also any portion of RAR ⁇ which includes the DNA-binding domain.
  • the polypeptides can be attached to other markers or polypeptides to utilize the DNA-binding characteristic. For example, the hormone binding domain of RAR ⁇ receptor can be attached to the DNA-binding domain and transfected into cells. The cognate ligand of that particular receptor can then be added or limited to control transcription of a target gene.
  • Polypeptides comprising the hormone-binding domain of RARe or polypeptides having substantially the same function and amino acid sequence are also provided.
  • the ability to bind a ligand, for example retionic acid, is a contemplated function.
  • the polypeptide does not include RAR ⁇ .
  • the hormone-binding domain can include not only the hormone-binding domain of RAR ⁇ alone but also any portion of RAR ⁇ which includes the hormone-binding domain of RAR ⁇ .
  • the polypeptide can be attached to other markers or polypeptides to utilize the hormone-binding characteristic.
  • the hormone-binding receptor can be attached to a known DNA-binding domain of another polypeptide and transfected into cells.
  • a cognate ligand of the hormone-binding receptor for example, retinoic acid, can then be added or limited to control transcription of a target gene.
  • Nucleic acids for example, DNA, RNA, or cDNA
  • Vectors comprising these nucleic acids are also provided.
  • Recombinant host cells can be transformed with such a vector and used to express recombinant polypeptides. Such methods of recombinant expression are well known in the art, see Maniatis et al., Molecular Cloning: A Laboratory Manual (1982), which is herein incorporated by reference. Thus, recombinant polypeptides and the method of their production are also provided.
  • RAR ⁇ is expressed at high levels in a number of epithelial-type tissues. It is also implicated in hepatocellular carcinoma development when its gene is activated by hepatitis B virus (HBV) integration in liver cells, where it is normally not expressed. A determination of its presence in malignant tissue is useful in elucidating the pathology of the tumor and in determining appropriate strategies of therapy.
  • HBV hepatitis B virus
  • the invention provides a method of diagnosing a tumor in a subject comprising detecting in a sample from the subject increased or decreased levels of RAR ⁇ , increased or decreased levels of RAR ⁇ indicating the presence of a tumor.
  • hepatocellular carcinoma can be detected since RAR ⁇ expression can be altered in liver cells of a subject with hepatocellular carcinoma.
  • a method of diagnosing a tumor in a subject comprises detecting in a sample from the subject a mutated form of RAR ⁇ , the mutated RAR ⁇ indicating the presence of a tumor.
  • Any method of detecting polypeptides may be utilized, for example an immunoassay.
  • mutated is meant any variation of naturally occurring RAR ⁇ . The mutation can result, for example, from the integration of a virus, a point mutation, a gene rearrangement, or gene amplification.
  • the suggested open reading frame encodes a 448 amino acid protein of relative molecular mass (M r ) 50,000 (50 K), that contains the domains typical of members of the steroid- thyroid hormone receptor family.
  • the cysteine-rich DNA-binding domain (C) is connected through a putative hinge region (domain D) with a ligand or hormone-binding domain (E).
  • the amino-terminal region is short and contains only the (B) domain but no (A) domain.
  • the (A) domain is typical for steroid hormone receptors but is not found in thyroid hormone receptors.
  • the hormone-binding domain (E) of the receptor of the present invention is 88% homologous with the previously described human retinoic acid receptor (RAR ⁇ ), Petkovich et al., supra; Giguere et al., supra. Domain D is also highly conserved and shows a 74% homology with RAR ⁇ .
  • the DNA-binding domain exhibits a 97% identity with RAR ⁇ .
  • a situation in which three colinear domains have been highly conserved has so far only been observed between the two human thyroid hormone receptors encoded by the erbA- ⁇ and the erbA-T genes. In that case, it was shown that an 86% homology in the hormone-binding domain was sufficient to conserve ligand specificity, Benbrook et al., Science 238:788-791 (1987) which is incorporated herein by reference.
  • RNA made in vitro containing the complete 5' untranslated region of the HBV-l clone was efficiently translated in a rabbit reticulocyte lysate system.
  • the HBV-1 RNA was of a single size, and so the smaller proteins seen in the XR lane of Fig. 2 may represent translation products initiating at AUG codons within the coding sequence.
  • HRE human epidermal growth factor receptor
  • the hybrid receptor was then transfected into susceptible tissue-culture cells together with a reporter gene containing the relevant HRE.
  • the effect of various potential activators of the novel receptor was measured from cells transiently transfected with both genes and grown in the presence and absence of the various ligands.
  • the hER DNA-binding domain was used as the donor domain as outlined in Figure 3.
  • the resulting construct was confirmed by DNA sequencing.
  • RNA of the new hybrid gene was translated in vitro. The major band migrated very similarly to the largest protein made from the wild-type gene, whereas other constructs which did not encode complete receptor proteins gave rise to smaller sized bands (see Fig. 2b).
  • Results from transient co-transfeetion experiments using a CAT (chloramphenicol acetyl transferase) reporter gene linked to a promoter containing the estrogen-response element (ERE) are shown in Figure 4. Only retinoic acid induced CAT gene expression strongly at a physiological concentration (2.5 x 10 -8 M). The hybrid receptor was inactive in the presence of other unrelated ligands (estradiol and dexamethasone). Other retinoids (retinol, retinal and retinyl-acetate) showed some inducing effect only at high concentrations (3 x 10 -6 M). The results indicate that the gene activated by HBV integration in a human hepatocellular carcinoma, with similarity to steroid-thyroid hormone-like receptors, encodes a retinoic acid receptor.
  • CAT chloramphenicol acetyl transferase
  • Rat tissue was chosen because this species has been extensively used as a model system to establish tissue-responsiveness to retinoic acid. Strong expression was observed in brain (including pineal gland but not pituitary), pituitary gland, kidney, colon, uterus, ovary, testis, prostate gland, adrenal gland, and eye. The size of the transcript appears to vary ranging from 2.4 to
  • Vitamin A and other retinoids have been considered to be, and are used therapeutically as, anti-tumor drugs.
  • RAR ⁇ might contribute to tumor development when expressed erroneously in liver tissue, where it is normally silent (Fig. 5).
  • Retinoids are known to maintain the proliferative state of epithelial cells. This type of cell proliferation, when induced in other types of tissue by erroneous RAR ⁇ expression and the presence of retinoic acid, can lead to tumor development.
  • Gene probes which can hybridize with RAR ⁇ nucleic acid can be prepared.
  • Basic techniques for preparing DNA oligonucleotide probes, as well as their screening by nucleic acid hybridization, are well known to those of ordinary skill in the art. See, for example, DNA CLONING: VOLUME I (D.M. Glover, ed. 1985); NUCLEIC ACID HYBRIDIZATION (B.D. Hames and S.J. Higgins eds. 1985): OLIGONUCLEOTIDE SYNTHESIS (M.J.
  • oligonucleotides or cDNA or riboprobes derived from the cDNA can be used to probe the library and isolate the desired gene.
  • the oligonucleotides are synthesized by any appropriate method, such as by the use of an automated DNA synthesizer.
  • the particular nucleotide sequences selected are chosen so as to correspond to the codons encoding a known amino acid sequence from the protein. Since the genetic code is redundant, it will often be necessary to synthesize several oligonucleotides to cover all, or a reasonable number, of the possible nucleotide sequences which encode a particular region of the protein.
  • the region it is generally preferred in selecting a region upon which to base the probes, that the region not contain amino acids whose codons are highly degenerate.
  • the region may find it desirable to prepare probes that are fairly long, and/or encompass regions of the amino acid sequence which would have a high degree of redundancy in corresponding nucleic acid sequences, particularly if this lengthy and/or redundant region is highly characteristic of the receptor protein.
  • Probes covering the complete gene, or a substantial part of the genome may also be appropriate, depending upon the expected degree of homology. It may also be desirable to use two or more probes, or sets of probes, each to different regions of the gene, in a single hybridization experiment.
  • the selected oligonucleotide probes are labeled with a marker, such as a radionucleotide or biotin, using standard procedures.
  • the labeled set of probes is then used in the screening step, which consists of allowing the single-stranded probe to hybridize to isolated denatured DNA from the library, according to standard techniques. Either stringent or permissive hybridization conditions can be appropriate, depending upon several factors, such as the length of the probes and whether the probes are derived from the same species as the library or an evolutionarily close or distant species. The selection of the appropriate conditions is within the skill of the art. See generally, NUCLEIC ACID HYBRIDIZATION, supra.
  • hybridization conditions be of sufficient stringency so that selective hybridization occurs; i.e., hybridization is due to a sufficient degree of nucleic acid homology (for example, at least about 75%), as opposed to nonspecific binding.
  • a DNA coding sequence for the desired protein can be prepared synthetically from overlapping oligonucleotides whose sequence contains codons for the amino acid sequence of the protein.
  • oligonucleotides are prepared by standard methods and assembled into a complete coding sequence. See, for example, Edge, (1981) Nature 292:756; Nambair et al., (1984) Science 223:1299; Jay et al., (1984) J. Biol. Chem. 259:6311, all of which are incorporated herein by reference.
  • RAR ⁇ can be used to produce antibodies, either polyclonal or monoclonal.
  • purified RAR ⁇ protein is used to immunize a selected mammal (for example, mouse, rabbit, goat, horse, etc.) and serum from the immunized animal is later collected and treated according to known procedures.
  • Antisera containing polyclonal antibodies to a variety of antigens in addition to the RAR ⁇ can be made substantially free of antibodies which are not anti-RAR ⁇ specific by passing the composition through a column to which non-RAR ⁇ protein has been bound. After washing, antibodies to the non-RAR ⁇ proteins will bind to the column, whereas anti-RAR ⁇ antibodies elute in the flow through.
  • Monoclonal anti-RAR ⁇ antibodies can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by fusing myelomas and lymphocytes to form hybridomas is well known. Such cells are screened to determine whether they secrete the desired antibodies, and can then be grown either in culture or in the peritoneal cavity of a mammal.
  • Antibodies can also be created by techniques other than fusion, such as direct transformation of ⁇ -lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, for example, M.
  • Antibodies specific to RAR ⁇ have a number of uses. For example, they may be employed in an immunoassay to detect the presence of RAR ⁇ . Detection of increased or decreased levels of RAR ⁇ or a mutant form of RAR ⁇ can be useful in diagnosing a tumor, for example, hepatocellular carcinoma. Various appropriate immunoassay formats are well known to those skilled in the art.
  • the invention also provides a method of detecting a ligand reactive with RAR ⁇ or a polypeptide comprising the ligand-binding domain of RAR ⁇ .
  • the method comprises contacting a sample suspected of containing the ligand with RAR ⁇ , or a polypeptide comprising the ligand-binding domain of RAR ⁇ , or functional equivalents thereof, and detecting the binding to the ligand.
  • functional equivalent is meant any polypeptide, except RAR ⁇ , having the ability to bind ligands reactive with RAR ⁇ .
  • minor changes in the amino acids or changes which increase or decrease the binding function are contemplated. Such changes would be expected to be found on the equivalent receptor in different species.
  • the ligand detected is retinoic acid.
  • any immunoassay-type assay format can be used, substituting RAR ⁇ or purified ligand-receptor for one or more of the antibodies. Since the ligands for the receptor are small molecules, preferably the assay is of the competitive type.
  • the analyte being assayed is allowed to complex with the receptor by incubating the solid phase with a fluid sample suspected of containing the analyte.
  • Radiolabeled analyte (other labels, such as fluorescent labeling are possible) is added to the sample.
  • the receptor-analyte complex is separated from the free analyte by binding the complex to a solid phase such as a Millipore filter.
  • the filter solid phase is washed and analyte receptor complex bound to it is measured.
  • the receptor can be coupled to a solid phase prior to contacting with the sample.
  • the receptor is coupled to a solid phase by means well known in the art.
  • the solid phase may comprise any of a variety of porous materials, including various natural or synthetic materials, alone or in combination. Included among these are polysaccharides, for example, cellulose materials, such as paper and cellulose acetate; silica; inorganic materials such as deactivated alumina diatomaceous earth
  • MgSO 4 or other inorganic finely divided material conveniently substantially uniformly dispersed in a porous polymer matrix with polymers such as vinyl chloride, vinyl chloride-propylene copolymer, and vinyl chloride-vinyl acetate copolymer; cloth, both naturally occurring, for example, cotton, and synthetic, for example, nylon cloth; porous gels, for example, silica gel, agarose, dextran, and gelatin; polymeric films, for example, polyacrylamide or the like.
  • Another alternative is to remove the receptor-analyte complex from solution by allowing it to bind to an anti-receptor antibody which may itself be coupled to a solid support.
  • a method of detecting DNA reactive with RAR ⁇ or a polypeptide comprising the DNA-binding domain of RAR ⁇ comprises contacting DNA with RAR ⁇ or a polypeptide comprising the DNA-binding domain of RAR ⁇ , or a functional equivalents thereof, and detecting the binding to the DNA.
  • specific DNA molecules such as thyroid hormone responsive element (TRE)
  • TRE thyroid hormone responsive element
  • This detection can be useful, for example, in diagnosing genetic defects in receptor binding DNA sequences or in detecting DNA sequences which can be used in conjunction with RAR ⁇ to regulate transcription.
  • a DNA sequence attached to RAR ⁇ is also provided.
  • the DNA sequence being a sequence which is responsive with RAR ⁇ .
  • attached is meant chemically bound, for example, covalent or ionic.
  • the sequence can occur in the promotor region just upstream from the promotor where the sequence, when properly attached by RAR ⁇ , controls transcription of the coding regions downstream of the promoter.
  • An example of a sequence responsive with RAR ⁇ is thyroid hormone responsive element.
  • a second DNA sequence can be under the control of the promoter.
  • the second DNA sequence can encode RNA which is translated into a desired polypeptide.
  • Such expression can be carried out by placing a vector containing the second DNA sequence under the control of the promoter and the DNA sequence responsive with RAR ⁇ in proper orientation into an appropriate host under polypeptide producing conditions.
  • Appropriate vectors, hosts, DNA orientations, and polypeptide producing conditions are all well known to those skilled in the art, see for example, Maniatis et al., supra.
  • the recombinant host cells can be induced to produce the desired polypeptide by the introduction of RAR ⁇ .
  • the RAR ⁇ can be endogenously produced by the host or may be produced by a transfected vector.
  • transcription of DNA sequences normally activated by a retinoic acid receptor can be inhibited by adding nonactivated thyroid receptor to the cell.
  • the thyroid receptor for RAR ⁇ can be endogenously produced by the host or may be produced by a transfected vector.
  • the retinoic acid receptor can be RAR ⁇ or RAR ⁇ and the thyroid receptor can be TR ⁇ or TR ⁇ .
  • the invention provides a method of screening ligands for ligands which stimulate or inhibit RAR ⁇ comprising adding the ligand to a recombinant host cell of the invention and observing the stimulation or inhibition of the polypeptide encoded by the second DNA sequence, an increase in the polypeptide indicating the stimulation of RAR ⁇ and a decrease in the polypeptide indicating the inhibition of RAR ⁇ .
  • the conditions of "fehe screening can be ascertained without undue experimentation by one skilled in the art.
  • MHC and G17-2-TRE constructs yield very similar levels of CAT activity.
  • the MHC-TRE-CAT gene is highly constitutive (high basal level activity) whereas the G17-2 construct functions as an ideal RA inducible gene in that we observe a low basal activity and a more than 30 fold induction of CAT activity by
  • RA (Fig. 6a and b) . Similar levels of induction were obtained with TR ⁇ . F9 cells have been established as a cellular model for RA dependent differentiation and to contain RAR transcripts. Whether the endogenous F9 RAR can also activate the TRE-CAT genes, by transfecting the receptor genes into F9 cells alone was investigated. A 12 fold induction in the presence of RA is observed for the G17-2 construct and no induction is seen in the presence of T 3 , suggesting that F9 cells do not contain TR. The RA induction is, however, considerably lower than that observed in the presence of cotransfected RAR ⁇ indicating that the endogenous RAR is limiting in concentration.
  • a hybrid receptor which contains the RAR ⁇ DNA binding domain and the estrogen receptor hormone binding domain (Fig. 7a). It has been shown above that individual receptor domains can be exchanged to create receptors with novel specificities.
  • the hybrid receptor RAR-E 2 was cotransfected (Fig. 7a) with the TRE-CAT genes into F9 cells, induction of CAT activity in the presence of the hormone estradiol (E 2 ) was observed (Fig. 7b).
  • Other hybrid receptors containing the ER DNA binding domain and the RAR ⁇ hormone binding domain were unable to stimulate transcription from TRE-CAT genes. These data therefore define the DNA binding region of RAR ⁇ as necessary for activating transcription from TREs in F9 cells.
  • RA and T 3 likely modulate gene transcription via their nuclear receptors from a common family of responsive elements.
  • T 3 hormone
  • RA vitamin
  • both receptors were cotransfected into the same cells together with a TRE-CAT gene. High CAT activity is observed in the presence of both ligands (T 3 + RA). Approximately 70% of the maximal activity is observed when only T 3 is added. Surprisingly, no activity (or very low activity) is observed when only RA is added.
  • F9 cells is observed between RAR ⁇ : TR ⁇ ratios of 1:1 to 4:1.
  • TR repressor mechanism still need to be elucidated; in particular, whether the receptors compete independently for the DNA binding site or whether an additional level of regulatory control by mutual protein-protein interaction exists. If the competition model applies, one would predict that TR ⁇ and TR ⁇ both bind to TREs in the absence of ligand. This would be in agreement with reported findings that several steroid hormone receptors can bind specific DNA sequences in the absence of hormone. See, for example, Willmann, T., et al., Nature, 324, 688-691 (1986), incorporated herein by reference. Our observed repression is highly specific for RAR since the hybrid receptors activating an ERE-CAT gene are not significantly inhibited (Fig. 10c).
  • NUCLEOTIDE SEQUENCE OF RAR ⁇ cDNA AND AMINO ACTD
  • the amino acid sequences of the DNA- and hormone-binding domains are compared with the same domains of other human proteins of this family: RAR, retinoic acid receptor Petkovich, et al., supra; and Giguere et al., supra; hTR and hTR ⁇ , thyroid hormone receptor, Benbrook et al., supra; and Weinberger et al., Nature 324, 642-649 (1986); hER, estrogen receptor Green et al., Nature 320, 134-139 (1986) and Green et al., Science 231, 1150-1154 (1986); hGR, glucocorticoid receptor Weinberger et al., Nature 318, 610-612 (1985); hMR, mineralocorticoid receptor Arriza et al., Science 237, 268-275 (1987); hPR, progesterone receptor Misrahi et al., Comm.
  • RNAs from clone HBV-1 were used to translate 35 S-labeled RAR protein (lane XR); as controls, human estrogen receptor (lane ER), and human thyroid hormone receptor (lane T-1) were also synthesized using the same in vitro system, as shown in Fig. 2.
  • FIG. 2 Analysis of the protein from the RAR-ER hybrid gene is shown in Fig. 2.
  • An in vitro transcription-translation system was used to analyze the protein encoded by the RAR-ER hybrid gene (lane XER) shown in Figure 3.
  • Fig. 3 presents a schematic representation given to illustrate the construction of ' an RAR ⁇ -ER chimaeric gene.
  • the brackets indicate the portions of the RAR ⁇ (hXR) and ER DNA-binding domains used in the construct. These exchange regions contain the potential zinc-fingers of the DNA-binding domain.
  • Example I The ⁇ 1-RAR clone of Example I was cut with HindIII and Apal, the Apal ends were blunt-ended with mung-bean nuclease.
  • the plasmid was then religated which resulted in a clone
  • RAR-9 in which the XhoI and HincII sites of the cloning box were deleted.
  • RAR-9 hXR
  • RAR-9 was digested with XhoI and HincII which cut at unique sites. The staggered end of the XhoI sites was filled in. This resulted in a linearized plasmid from which the coding sequences of amino acids 77 through 136 and the first nucleotide of the Arg-137 codon of the RAR ⁇ protein were deleted.
  • the ⁇ 1-ER fragment encodes amino acid residues 168 through 240 and contains also the first nucleotide of the Arg-241 codon of hER. Arg-137 of RAR and Arg-241 of ER are at the same positions in the two DNA-binding domains. The 5' end of the ER fragment encodes 13 amino acids more than were present on the excised RAR fragment. Constructs were analyzed by DNA sequencing. The clone with the described features is designated ⁇ 1-RAER (XER). The major protein obtained from this clone by in vitro transcription-translation is indistinguishable in size from the largest RAR ⁇ wild-type translation product (Fig. 4).
  • the RAER gene was cut out with BamHI and KpnI (both sides in the cloning box) and ligated into the BglII and KpnI sites of the PECE eukaryotic expression vector, Ellis et al., Cell 45:721-732 (1986), incorporated herein by reference, which yielded clone PECE-RAER.
  • BamHI and KpnI both sides in the cloning box
  • HeLa cells were co-transfected with the PECE-RAER expression vector (RAER) and the oestrogen-responsive CAT gene (ERE-CAT) and grown in the presence of either 5 x 10 -9 M oestradiol (Est) or 5 x 10 -7 M dexamethasone (Dex) or the indicated molar concentrations of various retinoids (Ret):1, retinoic acid; 2,retinol; 3,retinal; 4,retinylacetate (see Fig. 4).
  • RAER PECE-RAER expression vector
  • ERE-CAT oestrogen-responsive CAT gene
  • HeLa cells (1.5 x 10 6 per dish) were transfected using calcium phosphate, Wigler et al., Cell 11, 223-232 (1977) which is incorporated herein by reference, with 5 ⁇ g of the expression vectors PECE-RAER or pckR2ER and 20 ⁇ g of the ERE-CAT vector or the pSV2CAT vector. After 5 hours, cells were shocked with 10% glycerol for 2 min. Eighteen hours after the start of transfection, steroid hormones or retinoids were added to the medium and cells were incubated for a further 24 hours before harvesting. CAT activity in cell extracts was determined as described by Gormon et al., Biol 2, 1044-1051 (1982), herein incorporated by reference.
  • RNA (20 ⁇ g per lane) extracted from different rat tissues.
  • Lane 1 brain; lane 2, pituitary; lane 3, atrium; lane 4, lung; lane 5, diaphragm; lane 6, kidney; lane 7, liver; lane 8, spleen; lane 9, colon; lane 10, uterus; lane 11, ovary; lane 12, testis; lane 13, prostate; lane 14, seminal vesicle; lane 15, adrenal; lane 16, eye (see Fig. 5) .
  • Markers on the side are in kb.
  • RNA was isolated from tissues extracted from adult BK1: (SD) rats.
  • Northern blots were prepared from 1% formaldehyde-agarose gels containing 20 ⁇ g RNA per lane, prehybridized at 42 oC overnight in 50% formamide, 5x SSPE (lx SSPE is 0.18 M NaCl, 10 mM NaPO 4 , pH 7.7 1 mM EDTA), 5x Denhardt's reagent, 0.1% SDS, and 250 ⁇ g/ml -1 sheared salmon sperm DNA.
  • Hybridizations were at 42 oC in 50% formamide, 5x SSPE, lx Denhardt's reagent, 0.1% SDS, 100 ⁇ g/ml -1 sheared salmon sperm DNA, and 20 ng/ml -1 of probe 1 for all lanes except 10, 12, 13, and 14 for which 2 x 10 6 cpm/ml of probe 2 was used.
  • Probe I was labeled by nick-translating the 01-RAR whole plasmid (10 8 cpm/ ⁇ g -1 ) and probe 2 consisted of an EcoRI-SphI fragment of the 5' end of RAR labeled with Klenow using random oligonucleotides as primers (10 9 cpm/ ⁇ g -1 ).
  • F9 cells were cotransfected with 5 ⁇ g of the expression vectors PECE-RAR ⁇ or PECE-TR ⁇ and 20 ⁇ g of the reporter plasmids G17-2 CAT or MHC-CAT. As a control, cells were also transfected with the reporter plasmids only.
  • the G17-2 CAT construct contains three copies of a synthetic TRE derived from the rat GH gene. Glass, C. K., et al., supra, incorporated herein by reference.
  • the MHC construct contains nucleotides -163 to -81 upstream of the CAP site of the rat ⁇ MHC gene, Izumo, S., et al., supra.
  • Fold activation is expressed as the ratio of relative CAT activity after hormone induction to relative CAT activity without hormone induction.
  • the mean of at least three experiments is given in Figure 6(a).
  • Ligand dependant activation of RAR ⁇ and the endogenous F9 cell RAR is shown in Figure 6(b).
  • F9 cells were cotransfected with 5 ⁇ g of PECE-RAR ⁇ and 20 ⁇ g of G17-2 CAT ( ⁇ - ⁇ ) or with 20 ⁇ g of the expression vector alone ( ⁇ - ⁇ ). Indicated amounts of hormones were added 24 hours after transfection.
  • F9 cells were cultured as monolayer on gelatin-coated dishes in alpha-MEM (GIBCO, Santa Clara, CA), supplemented by 10% fetal calf serum (FCS), glutamine and nucleosides as described in Grover, A., et al., J. Cell Biol. 96, 1690-1996 (1983) which is incorporated herein by reference.
  • FCS fetal calf serum
  • FCS fetal calf serum
  • nucleosides as described in Grover, A., et al., J. Cell Biol. 96, 1690-1996 (1983) which is incorporated herein by reference.
  • FCS fetal calf serum
  • Two to five hours before transfection cells were fed the above medium containing charcoal-treated FCS. Twenty-four hours after transfection, cells were fed with charcoal treated medium together with the appropriate amount of hormone (final hormone concentrations 5 x 10 -8 M). Forty-eight hours after transfection, cells were harvested, lysed and as
  • Bl-RAR clone was digested with Apal and HincII to remove a 1 kB fragment with the hormone-binding domain and the COOH-terminus of the RAR ⁇ .
  • a 1.15 kb NaeI - ApaI fragment of the human ER clone B1-ER, Benbrook, D. and Pfahl, M., Science, 238, 788-791 (1987) was ligated to the RAR ⁇ fragment which encodes the aminoterminal and DNA binding domains.
  • DNA sequencing confirmed the in-frame connection between the RAR DNA binding domain and the ER hinge region.
  • a 1.8 kb BamHI-SalI fragment from B1-RAR-E 2 was cloned into the BgII & SalI sites of PECE.
  • F9 cells were cotransfected with 5 ⁇ g of PECE-RAR-E 2 and 20 ⁇ g of G17-2 CAT or MHC-CAT.
  • Estradiol (E 2 ) was added 24 hours after transfection (final concentration 5 x 10 -7 M) .
  • CAT activity was determined as described in Example VI. In the presence of E 2 , MHC-CAT is induced two-fold by RAR-E 2 and G17-2 CAT is induced 20 fold (fold induction is calculated from 3 independent experiments) (See Figure 7b).
  • CV-1 cells were cotransfected with 5 ⁇ g of PECE-RAR ⁇ , PECE-TR ⁇ or PECE-TR/3 as indicated and 20 ⁇ g of G17 -2 CAT.
  • Final concentrations of hormone were 6 x 10 -7 (RA) and 10 -7 (T 3 ) (See, Figure 8). Fold activation was determined as the mean of 3 separate experiments.
  • RAER-II contains amino acids 1 -287 of the human ER (which includes the DNA binding domain up to the hinge region) and amino acids 169 - 448 of RAR (which includes a portion of the hinge region and the hormone binding domain).
  • Figure 10(c) shows RAER-I and RAER-II activate transcription from an ERE-TK-CAT reporter gene in the presence of TR ⁇ .
  • CV-1 cells were cotransfected with 2.5 ⁇ g of PECE-RAER-I or PECE-RAER-II and 2.5 ⁇ g PECE-TR ⁇ , together with 20 ⁇ g of ERE-TK-CAT reporter gene as described above.
  • the ERE-TK-CAT contains an estrogen responsive element (ERE), Klein-Hitpass, L., et al., Cell 46, 1053-1061 (1986), incorporated herein by reference. Transfected cells were treated with 6 x 10 -7 M RA, 10 -7 M T 3 , or RA + T 3 as indicated.
  • ERP estrogen responsive element
  • Priority data tent), GB (European patent), HU, IT (European patent),
  • This invention relates to substantially purified Retinoic Acid Receptor ⁇ (RAR ⁇ ), having the amino acid sequence given in ig. 1, and to the isolated nucleic acid encoding RAR ⁇ .
  • RAR ⁇ Retinoic Acid Receptor ⁇
  • polypeptides comprising, and nucleic acids encoding, the DNA-binding and hormone-binding domain of the receptor are provided.
  • methods of detecting RAR ⁇ and nucleic acids encoding RAR ⁇ and its domains are provided.
  • This invention relates to cell biology and more specifically to a novel receptor for retinoic acid.
  • Receptors of the steroid-thyroid hormone family are intracellular proteins which consist of discrete DNA-binding regions and ligand-binding regions.
  • the DNA-binding domain of steroid hormone receptors and related proteins consists of a cystein-rich region that has been highly conserved and that has the potential to form two zinc-binding fingers.
  • the ligand binding region is specific to the cognate ligand.
  • Retinoids are known to be essential for the normal function of many epithelial tissues.
  • tissue-specific expression of RAR ⁇ is uncharacteristic for a molecule expected to be a major mediator of retinoid action in epithelial tissues. Because of their critical role in mediating metabolism and growth, it is important to determine other receptors mediating retinoid action. Knowledge of retinoid receptors permits the manipulation and control of metabolic effects resulting from retinoid action.
  • This invention relates to substantially purified Retinoic Acid Receptor e (RAR ⁇ ), having the amino acid sequence given in Figure 1, and to the isolated nucleic acid encoding RAR ⁇ .
  • RAR ⁇ Retinoic Acid Receptor e
  • polypeptides comprising, and nucleic acids encoding, the DNA-binding and hormone-binding domain of the receptor are provided.
  • methods of detecting RAR ⁇ and nucleic acids encoding RAR ⁇ and its domains are provided.
  • Fig. 1 shows a comparison of the amino acid sequence encoded by the HBV-1 clone with amino acid sequences of other members of the nuclear receptor family, as described in Example I.
  • Fig. 2 shows an analysis of in-vitro-synthesized RAR ⁇ protein, as described in Example 2.
  • Fig. 3 shows "Finger Swap” from ER to RAR ⁇ as described in Example III.
  • Fig. 4 shows RAR ⁇ chimaeric protein activation of the transcription of an ERE-CAT reporter gene in the presence of retinoic acid as described in Example IV.
  • Fig. 5 shows tissue specific expression of RAR ⁇ .
  • Fig. 6 shows RAR ⁇ and the endogenous F9 cell RAR activate transcription from thyroid hormone responsive elements.
  • Fig. 7 shows a RAR-E2 hybrid receptor confers estradiol inducibility to a TRE-CAT gene.
  • Fig. 8 shows RAR ⁇ activates transcription from a thyroid hormone responsive element in CV-1 cells.
  • Fig. 9 shows thyroid hormone receptors repress the activity of RAR ⁇ in F9 and CV-1 cells.
  • Fig. 10 shows RAR regions sensitive to TR repression by hybrid receptor analysis.
  • RAR ⁇ A new retinoic acid receptor, herein termed RAR ⁇ , which is encoded by a cDNA clone from a human placental library, has been identified and characterized.
  • RAR ⁇ is a member of the steroid/thyroid hormone receptor family.
  • a polypeptide having substantially the same function and amino acid sequence as RAR ⁇ is also provided.
  • having substantially the same function means modifications of the amino acid sequence which are neutral, improve, or lessen the function of RAR ⁇ so long as the essential function is maintained.
  • polypeptide analogs which have the activity of any portion of RAR ⁇ , but which differ in amino acid content, are contemplated.
  • Polypeptides comprising the DNA-binding domain of RAR ⁇ are provided. Polypeptides having substantially the same function and amino acid sequence as the DNA-binding domain are also provided. These polypeptides can include not only the DNA-binding domain of RAR ⁇ alone but also any portion of RAR ⁇ which includes the DNA-binding domain.
  • the polypeptides can be attached to other markers or polypeptides to utilize the DNA-binding characteristic. For example, the hormone binding domain of RAR ⁇ -receptor can be attached to the DNA-binding domain and transfected into cells. The cognate ligand of that particular receptor can then be added or limited to control transcription of a target gene.
  • Polypeptides comprising the hormone-binding domain of RAR ⁇ or polypeptides having substantially the same function and amino acid sequence are also provided.
  • the ability to bind a ligand, for example retionic acid, is a contemplated function.
  • the polypeptide does not include RAR ⁇ .
  • the hormone-binding domain can include not only the hormone-binding domain of RAR ⁇ alone but also any portion of RAR ⁇ which includes the hormone-binding domain of RAR ⁇ .
  • the polypeptide can be attached to other markers or polypeptides to utilize the hormone-binding characteristic.
  • the hormone-binding receptor can be attached to a known DNA-binding domain of another polypeptide and transfected into cells.
  • a cognate ligand of the hormone-binding receptor for example, retinoic acid, can then be added or limited to control transcription of a target gene.
  • Nucleic acids for example, DNA, RNA, or cDNA
  • Vectors comprising these nucleic acids are also provided.
  • Recombinant host cells can be transformed with such a vector and used to express recombinant polypeptides. Such methods of recombinant expression are well known in the art, see Maniatis et al., Molecular Cloning: A Laboratory Manual (1982), which is herein incorporated by reference. Thus, recombinant polypeptides and the method of their production are also provided.
  • RAR ⁇ is expressed at high levels in a number of epithelial-type tissues. It is also implicated in hepatocellular carcinoma development when its gene is activated by hepatitis B virus (HBV) integration in liver cells, where it is normally not expressed. A determination of its presence in malignant tissue is useful in elucidating the pathology of the tumor and in determining appropriate strategies of therapy.
  • HBV hepatitis B virus
  • the invention provides a method of diagnosing a tumor in a subject comprising detecting in a sample from the subject increased or decreased levels of RAR ⁇ , increased or decreased levels of RAR ⁇ indicating the presence of a tumor.
  • hepatocellular carcinoma can be detected since RAR ⁇ expression can be altered in liver cells of a subject with hepatocellular carcinoma.
  • a method of diagnosing a tumor in a subject comprises detecting in a sample from the subject a mutated form of RAR ⁇ , the mutated RAR ⁇ indicating the presence of a tumor.
  • Any method of detecting polypeptides may be utilized, for example an immunoassay.
  • mutated is meant any variation of naturally occurring RAR ⁇ . The mutation can result, for example, from the integration of a virus, a point mutation, a gene rearrangement, or gene amplification.
  • the suggested open reading frame encodes a 448 amino acid protein of relative molecular mass (M r ) 50,000 (50 K), that contains the domains typical of members of the steroidthyroid hormone receptor family.
  • the cysteine-rich DNA-binding domain (C) is connected through a putative hinge region (domain D) with a ligand or hormone-binding domain (E).
  • the amino-terminal region is short and contains only the (B) domain but no (A) domain.
  • the (A) domain is typical for steroid hormone receptors but is not found in thyroid hormone receptors.
  • the hormone-binding domain (E) of the receptor of the present invention is 88% homologous with the previously described human retinoic acid receptor (RAR ⁇ ), Petkovich et al., supra; Giguere et al., supra. Domain D is also highly conserved and shows a 74% homology with RAR ⁇ .
  • the DNA-binding domain exhibits a 97% identity with RAR ⁇ .
  • a situation in which three colinear domains have been highly conserved has so far only been observed between the two human thyroid hormone receptors encoded by the erbA- ⁇ and the erbA-T genes. In that case, it was shown that an 86% homology in the hormone-binding domain was sufficient to conserve ligand specificity, Benbrook et al., Science 238:788-791 (1987) which is incorporated herein by reference.
  • RNA made in vitro containing the complete 5' untranslated region of the HBV-1 clone was efficiently translated in a rabbit reticulocyte lysate system.
  • the HBV-1 RNA was of a single size, and so the smaller proteins seen in the XR lane of Fig. 2 may represent translation products initiating at AUG codons within the coding sequence.
  • HRE human epidermal growth factor receptor
  • the hybrid receptor was then transfected into susceptible tissue-culture cells together with -a reporter gene containing the relevant HRE.
  • the effect of various potential activators of the novel receptor was measured from cells transiently transfected with both genes and grown in the presence and absence of the various ligands.
  • the hER DNA-binding domain was used as the donor domain as outlined in Figure 3.
  • the resulting construct was confirmed by DNA sequencing.
  • RNA of the new hybrid gene was translated in vitro. The major band migrated very similarly to the largest protein made from the wild-type gene, whereas other constructs which did not encode complete receptor proteins gave rise to smaller sized bands (see Fig. 2b).
  • Results from transient co-transfeetion experiments using a CAT (chloramphenicol acetyl transferase) reporter gene linked to a promoter containing the estrogen-response element (ERE) are shown in Figure 4. Only retinoic acid induced CAT gene expression strongly at a physiological concentration (2.5 x 10 -8 M). The hybrid receptor was inactive in the presence of other unrelated ligands (estradiol and dexamethasone). Other retinoids (retinol, retinal and retinyl-acetate) showed some inducing effect only at high concentrations (3 x 10 -6 M). The results indicate that the gene activated by HBV integration in a human hepatocellular carcinoma, with similarity to steroid-thyroid hormone-like receptors, encodes a retinoic acid receptor.
  • CAT chloramphenicol acetyl transferase
  • Rat tissue was chosen because this species has been extensively used as a model system to establish tissue-responsiveness to retinoic acid. Strong expression was observed in brain (including pineal gland but not pituitary), pituitary gland, kidney, colon, uterus, ovary, testis, prostate gland, adrenal gland, and eye. The size of the transcript appears to vary ranging from 2.4 to
  • Vitamin A and other retinoids have been considered to be, and are used therapeutically as, anti-tumor drugs.
  • RAR ⁇ might contribute to tumor development when expressed erroneously in liver tissue, where it is normally silent (Fig. 5).
  • Retinoids are known to maintain the proliferative state of epithelial cells. This type of cell proliferation, when induced in other types of tissue by erroneous RAR ⁇ expression and the presence of retinoic acid, can lead to tumor development.
  • Gene probes which can hybridize with RAR ⁇ nucleic acid can be prepared.
  • Basic techniques for preparing DNA oligonucleotide probes, as well as their screening by nucleic acid hybridization, are well known to those of ordinary skill in the art. See, for example, DNA CLONING: VOLUME I (D.M. Glover, ed. 1985); NUCLEIC ACID HYBRIDIZATION (B.D. Hames and S.J. Higgins eds. 1985): OLIGONUCLEOTIDE SYNTHESIS (M.J.
  • oligonucleotides or cDNA or riboprobes derived from the cDNA can be used to probe the library and isolate the desired gene.
  • the oligonucleotides are synthesized by any appropriate method, such as by the use of an automated DNA synthesizer.
  • the particular nucleotide sequences selected are chosen so as to correspond to the codons encoding a known amino acid sequence from the protein. Since the genetic code is redundant, it will often be necessary to synthesize several oligonucleotides to cover all, or a reasonable number, of the possible nucleotide sequences which encode a particular region of the protein.
  • the region it is generally preferred in selecting a region upon which to base the probes, that the region not contain amino acids whose codons are highly degenerate.
  • the region may find it desirable to prepare probes that are fairly long, and/or encompass regions of the amino acid sequence which would have a high degree of redundancy in corresponding nucleic acid sequences, particularly if this lengthy and/or redundant region is highly characteristic of the receptor protein.
  • Probes covering the complete gene, or a substantial part of the genome may also be appropriate, depending upon the expected degree of homology. It may also be desirable to use two or more probes, or sets of probes, each to different regions of the gene, in a single hybridization experiment.
  • the selected oligonucleotide probes are labeled with a marker, such as a radionucleotide or biotin, using standard procedures.
  • the labeled set of probes is then used in the screening step, which consists of allowing the single- stranded probe to hybridize to isolated denatured DNA from the library, according to standard techniques. Either stringent or permissive hybridization conditions can be appropriate, depending upon several factors, such as the length of the probes and whether the probes are derived from the same species as the library or an evolutionarily close or distant species. The selection of the appropriate conditions is within the skill of the art. See generally, NUCLEIC ACID HYBRIDIZATION, supra.
  • hybridization conditions be of sufficient stringency so that selective hybridization occurs; i.e., hybridization is due to a sufficient degree of nucleic acid homology (for example, at least about 75%), as opposed to nonspecific binding.
  • a DNA coding sequence for the desired protein can be prepared synthetically from overlapping oligonucleotides whose sequence contains codons for the amino acid sequence of the protein.
  • oligonucleotides are prepared by standard methods and assembled into a complete coding sequence. See, for example. Edge, (1981) Nature 292:756; Nambair et al., (1984) Science 223:1299; Jay et al., (1984) J. Biol. Chem. 259:6311, all of which are incorporated herein by reference.
  • RAR ⁇ can be used to produce antibodies, either polyclonal or monoclonal.
  • purified RAR ⁇ protein is used to immunize a selected mammal (for example, mouse, rabbit, goat, horse, etc.) and serum from the immunized animal is later collected and treated according to known procedures.
  • Antisera containing polyclonal antibodies to a variety of antigens in addition to the RAR ⁇ can be made substantially free of antibodies which are not anti-RAR ⁇ specific by passing the composition through a column to which non-RAR ⁇ protein has been bound. After washing, antibodies to the non-RAR ⁇ proteins will bind to the column, whereas anti-RAR ⁇ antibodies elute in the flow through.
  • Monoclonal anti-RAR ⁇ antibodies can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by fusing myelomas and lymphocytes to form hybridomas is well known. Such cells are screened to determine whether they secrete the desired antibodies, and can then be grown either in culture or in the peritoneal cavity of a mammal.
  • Antibodies can also be created by techniques other than fusion, such as direct transformation of ⁇ -lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus, See, for example, M.
  • Antibodies specific to RAR e have a number of uses. For example, they may be employed in an immunoassay to detect the presence of RAR ⁇ . Detection of increased or decreased levels of RAR ⁇ or a mutant form of RAR ⁇ can be useful in diagnosing a tumor, for example, hepatocellular carcinoma. Various appropriate immunoassay formats are well known to those skilled in the art.
  • the invention also provides a method of detecting a ligand reactive with RAR ⁇ or a polypeptide comprising the ligand-binding domain of RAR ⁇ .
  • the method comprises contacting a sample suspected of containing the ligand with RAR ⁇ , or a polypeptide comprising the ligand-binding domain of RAR ⁇ , or functional equivalents thereof, and detecting the binding to the ligand.
  • functional equivalent is meant any polypeptide, except RAR ⁇ , having the ability to bind ligands reactive with RAR ⁇ .
  • minor changes in the amino acids or changes which increase or decrease the binding function are contemplated. Such changes would be expected to be found on the equivalent receptor in different species.
  • the ligand detected is retinoic acid.
  • any immunoassay-type assay format can be used, substituting RAR ⁇ or purified ligand-receptor for one or more of the antibodies. Since the ligands for the receptor are small molecules, preferably the assay is of the competitive type.
  • the analyte being assayed is allowed to complex with the receptor by incubating the solid phase with a fluid sample suspected of containing the analyte.
  • Radiolabeled analyte (other labels, such as fluorescent labeling are possible) is added to the sample.
  • the receptor-analyte complex is separated from the free analyte by binding the complex to a solid phase such as a Millipore filter.
  • the filter solid phase is washed and analyte receptor complex bound to it is measured.
  • the receptor can be coupled to a solid phase prior to contacting with the sample.
  • the receptor is coupled to a solid phase by means well known in the art.
  • the solid phase may comprise any of a variety of porous materials, including various natural or synthetic materials, alone or in combination. Included among these are polysaccharides, for example, cellulose materials, such as paper and cellulose acetate; silica; inorganic materials such as deactivated alumina diatomaceous earth
  • MgSO 4 or other inorganic finely divided material conveniently substantially uniformly dispersed in a porous polymer matrix with polymers such as vinyl chloride, vinyl chloride-propylene copolymer, and vinyl chloride-vinyl acetate copolymer; cloth, both naturally occurring, for example, cotton, and synthetic, for example, nylon cloth; porous gels, for example, silica gel, agarose, dextran, and gelatin; polymeric films, for example, polyacrylamide or the like.
  • Another alternative is to remove the receptor-analyte complex from solution by allowing it to bind to an anti- receptor antibody which may itself be coupled to a solid support.
  • a method of detecting DNA reactive with RAR ⁇ or a polypeptide comprising the DNA-binding domain of RAR ⁇ comprises contacting DNA with RAR ⁇ or a polypeptide comprising the DNA-binding domain of RAR ⁇ , or a functional equivalents thereof, and detecting the binding to the DNA.
  • specific DNA molecules such as thyroid hormone responsive element (TRE)
  • TRE thyroid hormone responsive element
  • This detection can be useful, for example, in diagnosing genetic defects in receptor binding DNA sequences or in detecting DNA sequences which can be used in conjunction with RAR ⁇ to regulate transcription.
  • a DNA sequence attached to RAR ⁇ is also provided.
  • the DNA sequence being a sequence which is responsive with RAR ⁇ .
  • attached is meant chemically bound, for example, covalent or ionic.
  • the sequence can occur in the promotor region just upstream from the promotor where the sequence, when properly attached by RAR ⁇ , controls transcription of the coding regions downstream of the promoter.
  • An example of a sequence responsive with RAR ⁇ is thyroid hormone responsive element.
  • a second DNA sequence can be under the control of the promoter.
  • the second DNA sequence can encode RNA which is translated into a desired polypeptide.
  • Such expression can be carried out by placing a vector containing the second DNA sequence under the control of the promoter and the DNA sequence responsive with RAR ⁇ in proper orientation into an appropriate host under polypeptide producing conditions.
  • Appropriate vectors, hosts, DNA orientations, and polypeptide producing conditions are all well known to those skilled in the art, see for example, Maniatis et al., supra.
  • the recombinant host cells can be induced to produce the desired polypeptide by the introduction of RAR ⁇ .
  • the RAR ⁇ can be endogenously produced by the host or may be produced by a transfected vector.
  • transcription of DNA sequences normally activated by a retinoic acid receptor can be inhibited by adding nonactivated thyroid receptor to the cell.
  • the thyroid receptor for RAR ⁇ can be endogenously produced by the host or may be produced by a transfected vector.
  • the retinoic acid receptor can be RAR ⁇ or RAR ⁇ and the thyroid receptor can be TR ⁇ or TR ⁇ .
  • the invention provides a method of screening ligands for ligands which stimulate or inhibit RAR ⁇ comprising adding the ligand to a recombinant host cell of the invention and observing the stimulation or inhibition of the polypeptide encoded by the second DNA sequence, an increase in the polypeptide indicating the stimulation of RAR ⁇ and a decrease in the polypeptide indicating the inhibition of RAR ⁇ .
  • the conditions of the screening can be ascertained without undue experimentation by one skilled in the art.
  • MHC and G17-2-TRE constructs yield very similar levels of CAT activity.
  • the MHC-TRE-CAT gene is highly constitutive (high basal level activity) whereas the G17-2 construct functions as an ideal RA inducible gene in that we observe a low basal activity and a more than 30 fold induction of CAT activity by
  • RA (Fig. 6a and b) . Similar levels of induction were obtained with TR ⁇ . F9 cells have been established as a cellular model for RA dependent differentiation and to contain RAR transcripts . Whether the endogenous F9 RAR can also activate the TRE-CAT genes, by transfecting the receptor genes into F9 cells alone was investigated. A 12 fold induction in the presence of RA is observed for the G17-2 construct and no induction is seen in the presence of T 3 , suggesting that F9 cells do not contain TR. The RA induction is, however, considerably lower than that observed in the presence of cotransfected RAR ⁇ indicating that the endogenous RAR is limiting in concentration.
  • a hybrid receptor which contains the RAR ⁇ DNA binding domain and the estrogen receptor hormone binding domain (Fig. 7a). It has been shown above that individual receptor domains can be exchanged to create receptors with novel specificities.
  • the hybrid receptor RAR-E 2 was cotransfected (Fig. 7a) with the TRE-CAT genes into F9 cells, induction of CAT activity in the presence of the hormone estradiol (E 2 ) was observed (Fig. 7b).
  • Other hybrid receptors containing the ER DNA binding domain and the RAR ⁇ hormone binding domain were unable to stimulate transcription from TRE-CAT genes. These data therefore define the DNA binding region of RAR ⁇ as necessary for activating transcription from TREs in F9 cells.
  • RA and T 3 likely modulate gene transcription via their nuclear receptors from a common family of responsive elements.
  • T3 hormone
  • RA vitamin
  • both receptors were cotransfected into the same cells together with a TRE-CAT gene. High CAT activity is observed in the presence of both ligands (T 3 + RA). Approximately 70% of the maximal activity is observed when only T3 is added. Surprisingly, no activity (or very low activity) is observed when only RA is added.
  • F9 cells is observed between RAR ⁇ : TR ⁇ ratios of 1:1 to 4:1.
  • TR repressor mechanism still need to be elucidated; in particular, whether the receptors compete independently for the DNA binding site or whether an additional level of regulatory control by mutual protein- protein interaction exists. If the competition model applies, one would predict that TR ⁇ and TR0 both bind to TREs in the absence of ligand. This would be in agreement with reported findings that several steroid hormone receptors can bind specific DNA sequences in the absence of hormone. See, for example, Willmann, T., et al., Nature, 324, 688-691 (1986), incorporated herein by reference. Our observed repression is highly specific for RAR since the hybrid receptors activating an ERE-CAT gene are not significantly inhibited (Fig. 10c).
  • NUCLEOTIDE SEQUENCE OF RAR ⁇ cDNA AND AMINO ACID
  • the amino acid sequences of the DNA- and hormone-binding domains are compared with the same domains of other human proteins of this family: RAR, retinoic acid receptor Petkovich, et al., supra; and Giguere et al., supra; hTR and hTR ⁇ , thyroid hormone receptor, Benbrook et al., supra; and Weinberger et al., Nature 324, 642-649 (1986); hER, estrogen receptor Green et al., Nature 320, 134-139 (1986) and Green et al., Science 231, 1150-1154 (1986); hGR, glucocorticoid receptor Weinberger et al., Nature 318, 610-612 (1985); hMR, mineralocorticoid receptor Arriza et al., Science 237, 268-275 (1987); hPR, progesterone receptor Misrahi et al., Comm.
  • RNAs from clone HBV-l were used to translate 35 S-labeled RAR protein (lane XR); as controls, human estrogen receptor (lane ER), and human thyroid hormone rece ⁇ tor (lane T-1) were also synthesized using the same in vitro system, as shown in Fig. 2.
  • FIG. 2 Analysis of the protein from the RAR-ER hybrid gene is shown in Fig. 2.
  • An in vitro transcription-translation system was used to analyze the protein encoded by the RAR-ER hybrid gene (lane XER) shown in Figure 3.
  • Fig. 3 presents a schematic representation given to illustrate the construction of an RAR ⁇ -ER chimaeric gene.
  • the brackets indicate the portions of the RAR ⁇ (hXR) and ER DNA-binding domains used in the construct. These exchange regions contain the potential zinc-fingers of the DNA-binding domain.
  • the ⁇ 1-RAR clone of Example I was cut with HindIII and Apal, the Apal ends were blunt-ended with mung-bean nuclease.
  • the plasmid was then religated which resulted in a clone (RAR-S) in which the XhoI and HincII sites of the cloning box were deleted.
  • RAR-9 hXR was digested with XhoI and HincII which cut at unique sites. The staggered end of the XhoI sites was filled in.
  • the 5' end of the ER fragment encodes 13 amino acids more than were present on the excised RAR fragment. Constructs were analyzed by DNA sequencing. The clone with the described features is designated ⁇ 1-RAER (XER). The major protein obtained from this clone by in vitro transcription-translation is indistinguishable in size from the largest RAR ⁇ wild-type translation product (Fig. 4).
  • the RAER gene was cut out with BamHI and KpnI (both sides in the cloning box) and ligated into the BglII Ind KpnI sites of the PECE eukaryotic expression vector, Ellis et al., Cell 45:721-732 (1986), incorporated herein by reference, which yielded clone PECE-RAER.
  • BamHI and KpnI both sides in the cloning box
  • HeLa cells were co-transfected with the PECE-RAER expression vector (RAER) and the oestrogen-responsive CAT gene (ERE-CAT) and grown in the presence of either 5 x 10 -9 M oestradiol (Est) or 5 x 10 -7 M dexamethasone (Dex) or the indicated molar concentrations of various retinoids (Ret): 1,retinoic acid; 2,retinol; 3,retinal; 4,retinylacetate (see Fig. 4).
  • RAER PECE-RAER expression vector
  • ERE-CAT oestrogen-responsive CAT gene
  • HeLa cells (1.5 x 10 6 per dish) were transfected using calcium phosphate, Wigler et al., Cell 11, 223-232 (1977) which is incorporated herein by reference, with 5 ⁇ g of the expression vectors PECE-RAER or pckR2ER and 20 ⁇ g of the ERE-CAT vector or the pSV2CAT vector. After 5 hours, cells were shocked with 10% glycerol for 2 min. Eighteen hours after the start of transfection, steroid hormones or retinoids were added to the medium and cells were incubated for a further 24 hours before harvesting. CAT activity in cell extracts was determined as described by Gormon et al., Biol 2, 1044-1051 (1982), herein incorporated by reference.
  • RNA (20 ⁇ g per lane) extracted from different rat tissues.
  • Lane 1 brain; lane 2, pituitary; lane 3, atrium; lane 4, lung; lane 5, diaphragm; lane 6, kidney; lane 7, liver; lane 8, spleen; lane 9, colon; lane 10, uterus; lane 11, ovary; lane 12, testis; lane 13, prostate; lane 14, seminal vesicle; lane 15, adrenal; lane 16, eye (see Fig. 5). Markers on the side are in kb.
  • RNA was isolated from tissues extracted from adult BK1: (SD) rats.
  • Northern blots were prepared from 1% formaldehyde-agarose gels containing 20 ⁇ g RNA per lane, prehybridized at 42oC overnight in 50% formamide, 5x SSPE (1X SSPE is 0.18 M NaCl, 10 mM NaPO 4 , pH 7.7 1 mM EDTA), 5x Denhardt's reagent, 0.1% SDS, and 250 ⁇ g/ml -1 sheared salmon sperm DNA.
  • Hybridizations were at 42oC in 50% formamide, 5x SSPE, 1x Denhardt's reagent, 0.1% SDS, 100 ⁇ g/ml -1 sheared salmon sperm DNA, and 20 ng/m l-1 of probe 1 for all lanes except 10, 12, 13, and 14 for which 2 x 10 6 cpm/ml of probe 2 was used.
  • Probe I was labeled by nick-translating the ⁇ 1-RAR whole plasmid (10 8 cpm/ ⁇ g -1 ) and probe 2 consisted of an EcoRI-SphI fragment of the 5' end of RAR labeled with Klenow using random oligonucleotides as primers (10 9 cpm/ ⁇ g -1 ).
  • F9 cells were cotransfected with 5 ⁇ g of the expression vectors PECE-RAR ⁇ or PECE-TR ⁇ and 20 ⁇ g of the reporter plasmids G17-2 CAT or MHC-CAT. As a control, cells were also transfected with the reporter plasmids only.
  • the G17-2 CAT construct contains three copies of a synthetic TRE derived from the rat GH gene. Glass, C. K., et al., supra, incorporated herein by reference.
  • the MHC construct contains nucleotides -163 to -81 upstream of the CAP site of the rat ⁇ MHC gene, Izumo, S., et al., supra.
  • Fold activation is expressed as the ratio of relative CAT activity after hormone induction to relative CAT activity without hormone induction.
  • the mean of at least three experiments is given in Figure 6(a).
  • Ligand dependant activation of RAR ⁇ and the endogenous F9 cell RAR is shown in Figure 6(b).
  • F9 cells were cotransfected with 5 ⁇ g of PECE-RAR ⁇ and 20 ⁇ g of G17-2 CAT ( ⁇ - ⁇ ) or with 20 ⁇ g of the expression vector alone ( ⁇ - ⁇ ). Indicated amounts of hormones were added 24 hours after transfection.
  • F9 cells were cultured as monolayer on gelatin-coated dishes in alpha-MEM (GIBCO, Santa Clara, CA), supplemented by 10% fetal calf serum (FCS), glutamine and nucleosides as described in Grover, A., et al., J. Cell Biol. 96, 1690-1996 (1983) which is incorporated herein by reference.
  • FCS fetal calf serum
  • FCS fetal calf serum
  • nucleosides as described in Grover, A., et al., J. Cell Biol. 96, 1690-1996 (1983) which is incorporated herein by reference.
  • FCS fetal calf serum
  • Two to five hours before transfection cells were fed the above medium containing charcoal-treated FCS. Twenty-four hours after transfection, cells were fed with charcoal treated medium together with the appropriate amount of hormone (final hormone concentrations 5 x 10 -8 M). Forty-eight hours after transfection, cells were harvested, lysed and as
  • B1-RAR clone was digested with ApaI and HincII to remove a 1 kB fragment with the hormone-binding domain and the COOH-terminus of the RAR ⁇ .
  • a 1.15 kb NaeI - ApaI fragment of the human ER clone B1-ER, Benbrook, D. and Pfahl, M., Science, 238, 788-791 (1987) was ligated to the RAR ⁇ fragment which encodes the aminoterminal and DNA binding domains.
  • DNA sequencing confirmed the in-frame connection between the RAR DNA binding domain and the ER hinge region.
  • a 1.8 kb BamHI-SalI fragment from B1-RAR-E2 was cloned into the BgII & SalI sites of PECE.
  • F9 cells were cotransfected with 5 ⁇ g of PECE-RAR-E 2 and 20 ⁇ g of G17-2 CAT or MHC-CAT.
  • Estradiol (E 2 ) was added 24 hours after transfection (final concentration 5 x 10 -7 M).
  • CAT activity was determined as described in Example VI. In the presence of E 2 , MHC-CAT is induced twofold by RAR-E 2 and G17-2 CAT is induced 20 fold (fold induction is calculated from 3 independent experiments) (See Figure 7b).
  • CV-1 cells were cotransfected with 5 ⁇ g of PECE-RAR ⁇ , PECE-TR ⁇ or PECE-TR/5 as indicated and 20 ⁇ g of G17-2 CAT.
  • Final concentrations of hormone were 6 x 10 -7 (RA) and 10 -7 (T 3 ) (See, Figure 8). Fold activation was determined as the mean of 3 separate experiments.
  • RAER-II contains amino acids 1-287 of the human ER (which includes the DNA binding domain up to the hinge region) and amino acids 169 - 448 of RAR (which includes a portion of the hinge region and the hormone binding domain).
  • Figure 10(c) showns RAER-I and RAER-II activate transcription from an ERE-TK-CAT reporter gene in the presence of TR ⁇ .
  • CV-1 cells were cotransfected with 2.5 ⁇ g of PECE-RAER-I or PECE-RAER-II and 2.5 ⁇ g PECE-TR ⁇ , together with 20 ⁇ g of ERE-TK-CAT reporter gene as described above.
  • the ERE-TK-CAT contains an estrogen responsive element (ERE), Klein-Hitpass, L., et al., Cell 46, 1053-1061 (1986), incorporated herein by reference. Transfected cells were treated with 6 x 10 -7 M RA, 10 -7 M T 3 , or RA + T 3 as indicated.
  • ERP estrogen responsive element

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Abstract

Cette invention concerne un récepteur d'acide rétinoïque epsilon (RARepsilon) pratiquement purifié, ayant la séquence d'aminoacides représentée par la figure 1, ainsi que l'acide nucléique isolé codant le RARepsilon. Elle concerne également des polypeptides comprenant, et des acides nucléiques codant le domaine de liaison d'ADN et de liaison d'hormones du récepteur, ainsi que des procédés de détection de RARepsilon et des acides nucléiques codant le RARepsilon et ses domaines.
PCT/US1989/002636 1988-06-16 1989-06-15 Recepteur d'acide retinoique WO1989012687A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0545554A2 (fr) * 1991-11-04 1993-06-09 Institut National De La Sante Et De La Recherche Medicale (Inserm) Anticorps spécifiques pour le récepteur d'acide rétinoique (RAR)-gamma ou une isoforme spécifique de RAR-gamma
EP0552302A1 (fr) * 1990-10-10 1993-07-28 La Jolla Cancer Research Foundation Procede d'inhibition de transcription utilisant des recepteurs nucleaires
EP0575528A1 (fr) * 1991-03-18 1993-12-29 The Salk Institute For Biological Studies Compositions a elements de reaction et dosages les incorporant
WO1996029400A1 (fr) * 1995-03-23 1996-09-26 Institut National De La Recherche Agronomique (Inra) Procede de regulation de l'expression d'un gene dans un baculovirus, par un site de fixation d'un recepteur de l'acide retinoique, et vecteur pour la mise en ×uvre dudit procede
EP0769023A1 (fr) * 1994-06-24 1997-04-23 Human Genome Sciences, Inc. Recepteur epsilon d'acide retinoique
US5922596A (en) * 1990-12-10 1999-07-13 Bristol-Myers Squibb Co. Promoter of the retinoic acid receptor gene for directing gene expression
EP0960941A1 (fr) * 1990-04-10 1999-12-01 CANJI, Inc. Thérapie génique des proliférations cellulaires diseases

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0321362A1 (fr) * 1987-12-16 1989-06-21 Institut Pasteur Récepteur de l'acide rétinoique et ses derivées, DNA codant pour ces deux substances et l'usage des protéines et des DNA

Patent Citations (1)

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EP0321362A1 (fr) * 1987-12-16 1989-06-21 Institut Pasteur Récepteur de l'acide rétinoique et ses derivées, DNA codant pour ces deux substances et l'usage des protéines et des DNA

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Title
NATURE, Volume 330, issued 17 December 1987, DE THE, "A novel steroid thyroid hormone receptor-related gene inappropriately expressed in human hepatocellular carcinoma". See pages 667-670 particularly figures 1-4. *
NATURE, Volume 330, issued 17 December 1987, GIGUERE, "Identification of a receptor for the morphogen retinoic acid". See pages 624-629, particularly figures 1-6. *
NATURE, Volume 330, issued December 1987, PETROVICH, "A human retinoic acid receptor which belongs to the family of nuclear receptors". See pages 440-450 particularly figure 4. *
NATURE, Volume 332, issued 28 April 1988, BRAND, "Identification of a second human retinoic acid receptor". See pages 850-853. *
NATURE, Volume 333, issued 16 June 1988, BENBROOK, "A new retinoic acid receptor identified from a hepatocellular carcinoma". See pages 669-672 particularly figures 1-5. *
NATURE, Volume 336, issued 17 November 1988, UMESONO, "Retinoic acid and thyroid hormone induce gene expression through a common responsive element". See pages 262-265 particularly figures 2-4. *
SCIENCE, Volume 240, issued 13 May 1988, EVANS, "The steroid and thyroid hormone receptor superfamily". See pages 889-895 particularly figures 2-5. *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0960941A1 (fr) * 1990-04-10 1999-12-01 CANJI, Inc. Thérapie génique des proliférations cellulaires diseases
EP0552302A1 (fr) * 1990-10-10 1993-07-28 La Jolla Cancer Research Foundation Procede d'inhibition de transcription utilisant des recepteurs nucleaires
EP0552302A4 (en) * 1990-10-10 1993-11-10 La Jolla Cancer Research Foundation Method of inhibiting transcription utilizing nuclear receptors
US5922596A (en) * 1990-12-10 1999-07-13 Bristol-Myers Squibb Co. Promoter of the retinoic acid receptor gene for directing gene expression
EP0575528A1 (fr) * 1991-03-18 1993-12-29 The Salk Institute For Biological Studies Compositions a elements de reaction et dosages les incorporant
EP0575528A4 (en) * 1991-03-18 1994-09-14 Salk Inst For Biological Studi Response element compositions and assays employing same
EP0545554A2 (fr) * 1991-11-04 1993-06-09 Institut National De La Sante Et De La Recherche Medicale (Inserm) Anticorps spécifiques pour le récepteur d'acide rétinoique (RAR)-gamma ou une isoforme spécifique de RAR-gamma
EP0545554A3 (en) * 1991-11-04 1994-05-25 Inst Nat Sante Rech Med Antibodies specific for rar-gamma or a specific isoform of rar-gamma
EP0769023A4 (fr) * 1994-06-24 1999-05-26 Human Genome Sciences Inc Recepteur epsilon d'acide retinoique
EP0769023A1 (fr) * 1994-06-24 1997-04-23 Human Genome Sciences, Inc. Recepteur epsilon d'acide retinoique
FR2732035A1 (fr) * 1995-03-23 1996-09-27 Agronomique Inst Nat Rech Procede de regulation de l'expression d'un gene dans un baculovirus, par un site de fixation d'un recepteur de l'acide retinoique, et vecteur pour la mise en oeuvre du dit procede
US5939285A (en) * 1995-03-23 1999-08-17 Institut National De La Recherche Scientifique Agronomique (Inra) Method for regulating the expression of a gene in a baculovirus using a retinoic acid receptor binding site and vector therefor
WO1996029400A1 (fr) * 1995-03-23 1996-09-26 Institut National De La Recherche Agronomique (Inra) Procede de regulation de l'expression d'un gene dans un baculovirus, par un site de fixation d'un recepteur de l'acide retinoique, et vecteur pour la mise en ×uvre dudit procede

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