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WO1992008726A1 - Gene de rat recepteur de thyrotropine et utilisation dudit gene - Google Patents

Gene de rat recepteur de thyrotropine et utilisation dudit gene Download PDF

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WO1992008726A1
WO1992008726A1 PCT/US1990/006533 US9006533W WO9208726A1 WO 1992008726 A1 WO1992008726 A1 WO 1992008726A1 US 9006533 W US9006533 W US 9006533W WO 9208726 A1 WO9208726 A1 WO 9208726A1
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receptor
antibody
rat
substantially pure
thyrotropin receptor
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PCT/US1990/006533
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English (en)
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Takashi Akamizu
Shoichiro Ikuyama
Motoyasu Saji
Shinji Kosugi
Toshiaki Ban
Leonard D. Kohn
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The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services
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Application filed by The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services filed Critical The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services
Priority to CA 2095013 priority Critical patent/CA2095013A1/fr
Priority to PCT/US1990/006533 priority patent/WO1992008726A1/fr
Priority to EP90917617A priority patent/EP0557273A1/fr
Publication of WO1992008726A1 publication Critical patent/WO1992008726A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a substantially pure nucleotide sequence that encodes the thyrotropin receptor; medicaments and therapeutic compositions comprising said sequence or the receptor expressed therefrom; methods of detecting ligands or molecules that bind to said sequence or to said receptor; and therapeutic methods of detecting ligands or molecules that bind to said sequence or to said receptor.
  • Thyrotropin, or thyroid stimulating hormone (TSH) is a pituitary hormone that regulates the development and activity of the thyroid gland.
  • T 3 also known as triiodothyronine
  • T 4 also known as thyroxine
  • TSH is bound at the surface of hormone-responsive cells, for example the epithelial follicular cells, by a specific integral membrane receptor.
  • Activated receptors stimulate and regulate adenylate cyclase through G proteins, such as G S and G I , and the cAMP signal regulates the expression of a variety of downstream genes and effector functions, such as the breakdown of colloid to mobilize stored T 3 and T 4 as well as the active synthesis of T 3 and T 4.
  • Clinical correlates of abnormal binding of TSH to its specific receptor may be manifest in a variety of syndromes.
  • hypothyroidism or myxedema can result from a TSH receptor that is unable to bind TSH, or if binding with TSH occurs, the abnormal receptor cannot send an appropriate message to influence adenylate cyclase activity.
  • expression of the receptor may be down-regulated, thereby producing a hypothyroid state, such as during oncogene transformation.
  • hyperthyroidism A common form of hyperthyroidism is Graves Disease wherein antibodies that react with the TSH receptor mimic TSH and activate the receptor thereby resulting in a tonic up-regulation of thyroid function.
  • TSH-binding thyroid proteins or protein complexes with estimated molecular weights of about 500, 300 and 150 kd whereas studies using denaturing conditions, such as with sodium dodecyl sulfate gel electrophoresis, have identified 50-70, 30-45 and 15-25 kd components. The latter studies resulted in a postulated TSH receptor structure composed of 2 or 3 subunits.
  • Akamizu et al. identified two proteins that interact with thyrotropin, revealed by virtue of their TSH-dependent binding to TSH-Sepharose. The two proteins, of 43 kd and 70 kd molecular weight, were found to be ⁇ -actin and a member of the hsp70 family, respectively. Biochem Biophys Res Comm 170:351-358 (1990).
  • Thyrotropin receptor genes have been cloned in two species, dog and human. Parmentier et al. cloned the dog gene using degenerate oligonucleotides, corresponding to conserved regions in the transmembrane segment of known receptors that interact with G proteins, in the polymerase chain reaction. That procedure first yielded a receptor-related clone (probably not TSH receptor) which itself was used to probe a thyroid cDNA library. That screen yielded a putative TSH receptor clone. Science 246:1620-1622 (1989).
  • a more favorable starting point for elucidating the structure and function of the thyrotropin receptor would be to study the receptor of a utile animal model.
  • the FRTL-5 rat thyroid cell line has become a widely used in vitro model of a normal, functioning endocrine cell.
  • the growth and function of FRTL-5 cells depend on thyrotropin.
  • the cells can be used to measure and study the action of antibodies in patients with autoimmune thyroid disease.
  • defining the structure of the rat TSH receptor and its function in the growth and properties of FRTL-5 cells is critically important to a multiplicity of research and clinical programs.
  • a second object of the instant invention is to provide the substantially pure polypeptide product produced therefrom, and portions thereof.
  • a third object of the instant invention is to provide medicaments and therapeutic compositions comprising said nucleotide sequences, or portions thereof, or said product, or portions thereof, produced therefrom.
  • a fourth object of the instant invention is to provide assays employing said sequences, or portions thereof, for detecting nucleic acids hybridizable thereto.
  • a fifth object of the instant invention is to provide assays employing said product, or portions thereof, for detecting ligands bindable thereto.
  • a sixth object of the instant invention is to provide uses of said sequences or of said products in the treatment and management of disorders that arise from dysfunction of the receptor.
  • a seventh object of the instant invention is to provide a means of making antibodies to the thyrotropin receptor nucleotide sequence and product produced therefrom.
  • FIG. 1 depicts the nucleotide and amino acid sequence of a rat thyrotropin receptor and flanking noncoding sequences. Potential glycosylation sites are underlined and are referred to in consecutive order with the amino terminal-most site denoted as I. A potential phosphorylation site is denoted with the underscored dashed line. TM1-TM7 denote hydrophobic regions of the transmembrane domain. Wavy lines indicate approximate endpoints of the probe used for the initial screen of the cDNA bank.
  • FIG. 2 presents at the top, a schematic drawing of the receptor.
  • MET represents the signal peptide region.
  • the highlighted region between amino acid residues 300 and 400 represents the peptide not found in the LH/hCG recptor.
  • Roman numerals represent putative glycosylation sites.
  • the FRTL-5 cell line (which is available publicly from the ATCC under accession number CRL 8305 and was deposited in relation to Ambesi-Impiombato, U.S. Pat. No. 4,608,341 and Kohn et al., U.S. Pat. No. 4,609,622) was derived from thyroid of normal Fischer rats.
  • the cells were maintained in culture as described in U.S. Pat. No. 4,609,622. Briefly, the cells were cultured in Coon's modified Ham's F-12 medium supplemented with calf serum, TSH, insulin and various other optional hormones. The cells were incubated in a CO 2 environment at physiologic temperatures.
  • the epithelioid cells grew attached to the cultureware surface, had a doubling time of 1-2 days and were passed biweekly with a split ratio of between 5 to 15.
  • cDNA was synthesized using 5 ⁇ g of rat testis poly(A) RNA (Clontech), murine reverse transcriptase (Pharmacia) and Pharmacia's protocol.
  • a 286 base pair (bp) cDNA fragment comprising transmembrane domains was amplified with Thermus aquaticus DNA polymerase and 25 pmol of each of two 30-mer oligonucleotide primers complementary to sites flanking the region to be amplified and hybridizable to alternative strands.
  • Mullis et al. U.S. Pat. Nos. 4,683,195 and 4,800,159; Mullis, U.S. Pat. No. 4,683,202.
  • Oligomer A has the sequence (5'-GGGCTCTACCTGCTGCTCATTGCCTCCGTG-3 ') and oligomer B has the sequence (5'-CCCACAAGGGGCATCGTGGCGATCAGCG-3 ').
  • Each cycle comprised 1 minute at 94°C for denaturation, 2 minutes at 55°C for hybridization and 3 minutes at 72°C for extension.
  • the amplified fragment was purified from 3% low melting agarose.
  • FRTL-5 cDNA library was constructed in ⁇ gt11 (Clontech), with mRNA obtained using standard procedures from cells maintained for 7 days in the absence of TSH. Plaques were screened using the LH/hCG receptor transmembrane domain-derived cDNA fragment described above, which was labelled by random priming. The initial screen was under low stringency conditions (55°C). Positive plaques were isolated, grown and rescreened with the same cDNA fragment probe. The inserts of plaque-purified clones were subcloned into the EcoRI site of either pGEM-4Z or 7Z (Promega) using standard methodologies. DNA sequencing was by the dideoxy chain termination method.
  • the full length rat thyrotropin receptor sequence encodes a protein of 764 amino acids with an estimated molecular weight of about 87,000, as shown in Figure 1.
  • the sequence was deposited in the GenBank data base on 15 September 1990 and has accession number M34842. It should be noted that in the coding sequence, as noted in Figure 1 wherein the adenine of the ATG codon for the first methionine of the extracellular domain is considered nucleotide 1, the codon for the seventh leucine residue at nucleotides 55-57 is CTG.
  • the GenBank sequence lists that codon as CTC, also a leucine codon.
  • the predicted protein has a 21-23 residue hydrophobic region at its N-terminus which is a signal peptide.
  • TSH-sensitive adenylate cyclase response After transfection into non-thyroid-derived cells, said cells expressed a TSH-sensitive adenylate cyclase response and the ability to bind labelled TSH.
  • the activities were specific for TSH, LH did not stimulate an adenylate cyclase response nor was LH bound by the transfectants.
  • the clone and the receptor protein produced therefrom find utility in a variety of circumstances.
  • the recombinant receptor can be used in assays for detecting ligands capable of binding the receptor.
  • Suitable ligands include thyrotropin and anti-receptor antibodies.
  • recombinant receptor can be attached to a solid phase support, such as the wells of a microtiter plate, plastic beads, dip sticks, membranes and the like. Many such supports have a natural affinity for proteins so attachment of the receptor thereto is accomplished by merely exposing the support to an aqueous solution comprising the receptor.
  • aqueous solution comprising the receptor.
  • Physiologic saline, tissue culture medium, buffers and the like are suitable fluid vehicles for preparing the aqueous solution. Attachment of the receptor to the support can be enhanced if the fluid phase is a buffered solution with a pH of about 9. If mere exposure is inadequate for attachment, art-recognized attachment agents can be used. Suitable agents include glutaraldehyde, poly-L-lysine and dessication.
  • the support is washed liberally.
  • non-specific sites on the support are blocked with a non-cro ⁇ s-reactive carrier protein, such as albumin or gelatin, or with a protein laden mixture, such as serum or a non-fat dried milk solution.
  • the blocking solutions are preparable in the fluid vehicles disclosed above, often as 0.1-30% solutions.
  • the receptor attached blocked support is exposed to a test sample, often a body fluid sample, such as a blood or serum sample, to determine the presence of ligand, and amounts thereof.
  • a test sample often a body fluid sample, such as a blood or serum sample.
  • the support is incubated with the test sample for a period of time to assure ligand-receptor binding. Then the support is washed liberally and detection of bound ligand is conducted.
  • the means of detection can take a variety of forms.
  • a readily known labelled second ligand such as TSH
  • TSH can be exposed to the solid support following exposure of said support to a test sample suspected of carrying a first ligand bindable to the receptor, or part thereof.
  • the amount of label bound thereto is determined, such as by liquid scintillation counting in the case of radiolabelling or by spectrophotometry in the case of enzyme labelling, and measures inhibition of binding of said known second ligand with the receptor, by a first ligand, such as an anti-receptor antibody, in said test sample.
  • the amount of bound label is related inversely to the amount of first ligand in the test sample.
  • a second labelled ligand bindable to the receptor bound first ligand on the support is exposed to the support and the amount of label bound thereto is determined.
  • Suitable second ligands include an appropriate antibody, such as an antibody to the receptor or to TSH.
  • the amount of bound label is related directly to the amount of ligand in the test sample.
  • solid support assays are preferred because of the facility in performing the methods, other assays can be configured without undue experimentation.
  • liquid phase assays are practicable, as well as competition assays and those where the recombinant receptor is labelled.
  • Suitable guidance can be obtained, for example, in U.S. Pat. Nos. 4,486,530 and 4,520,113.
  • Truncated versions of the gene product are also useful. For example, it is the extracellular domain that interacts with TSH and with anti-receptor antibodies. Thus that polypeptide domain alone can be used in place of the intact receptor in the uses disclosed herein. Truncated proteins can be obtained for example, by chemical synthesis of the domain or part thereof, chemical treatment of the intact protein to liberate a domain or part thereof from the remainder of the receptor protein and by altering the nucleotide coding sequence, such as by site-directed mutagenesis or by subcloning an appropriate restriction fragment, so that only the extracellular domain or part thereof is expressed.
  • the substantially pure receptor and parts thereof can be used to obtain specific antibody.
  • a rabbit polyclonal antiserum was raised to a 16 residue polypeptide fragment of the extracellular domain of the receptor. Accordingly, monoclonal antibodies to the receptor can be made by obtaining immune cells suitable for hybridization with known myeloma fusion partners and practicing the fusion, cloning and selection that typifies the making of mAbs.
  • the cloned sequence is useful for detecting nucleic acids hybridizable thereto.
  • a nucleic acid hybridization assay such as filter hybridization (Southern blot), in situ hybridization, dot/slot blot or a solution hybridization assay, with the clone as probe can be used to determine presence of complementary genomic sequences and message, for example.
  • Those procedures are useful, for example, in detecting hypothyroidism resulting from a TSH receptor defect.
  • the nucleic acid assays may comprise an amplification step such as taught in Mullis or Mullis et al. (supra) or in Kramer et al. (U.S. Pat. No. 4,786,600).
  • the cloned sequence is useful for correcting defects at the level of the gene, transcription, translation or processing by, for example gene replacement therapy.
  • non-thyroid cells that normally do not express the thyrotropin receptor were transfected with the full length expressible sequence.
  • the transfectants expressed a functional TSH receptor, bound TSH at the cell surface and exhibited a TSH-dependent activation of cAMP synthesis.
  • cells from a patient that does not express the TSH receptor or expresses a defective receptor can be removed, transfected in vitro, and stable transfectants that express a functional receptor can be introduced back into the patient.
  • compositions comprising said nucleotide sequence or preferably said gene product, or portions thereof, encoded thereby.
  • the compositions comprise a therapeutically effective amount of the nucleotide sequence or gene product thereof and a pharmaceutically acceptable carrier.
  • the composition can be administered in any of a variety of art-recognized modes including orally and parenterally, preferably intramuscularly or intravenously. Appropriate dosages are determinable by, for example, dose-response experiments in laboratory animals or in clinical trials and taking into account body weight of the patient, absorption rate, half life, disease severity and the like. The number of doses, daily dosage and course of treatment may vary from individual to individual.
  • compositions can be of solid form including tablets, capsules, pills, bulk or unit dose powders and granules but preferably are of liquid form including solutions, fluid emulsions, fluid suspensions, semisolids and the like.
  • the formulation would comprise suitable art-recognized diluents, carriers, fillers, binders, emulsifiers, surfactants, water-soluble vehicles, buffers, solubilizers and preservatives.
  • Methods of treatment include those known in the art for administering biologically active agents. Such methods include in vivo and ex vivo modalities.
  • a receptor-containing solution can be delivered intraveously, by a pump means attached to a reservoir containing bulk quantities of said solution, by passive diffusion from an implant, such as a Silastic implant and the like.
  • treatment may involve temporary removal of tissue and exposure thereof to the claimed compositions before introduction back into the patient.
  • the extracorporeal fluid is passed over solid phase bound receptor to entrap ligands bindable to the receptor. The fluid is then returned to the patient.
  • Target cells are obtained, such as from the patient in need of treatment or a cell line, the expressible sequence is inserted into said cells and stable transformants are selected. Said stable transformants expressing said receptor are introduced into the patient in need of treatment by direct infusion into the tissue or by parenteral means.
  • EXAMPLE 1 From 8 x 10 5 plaques screened at low stringency with the rat LH/hCG receptor-related probe described above, 20 FRTL-5 rat thyroid cell clones were obtained.
  • a 177 bp probe of the 5' end of 16B1 was synthesized using 10 ng of pGEM-7Z (Promega) carrying the 16B1 insert and oligo primer C with the sequence (5'-CGCTATACAACAATGGATTTACTTCTT-3 ') and primer D with the sequence (5'-GAAGAGCAGTAACGCTGGTGGAAGACA-3'). That probe was used to rescreen the bank and revealed a 2.8 kb cDNA clone (T8AFB) with characteristics compatible with its encoding the full-length TSH receptor and only a small portion of the 3' noncoding sequences.
  • T8AFB 2.8 kb cDNA clone
  • the nucleotide sequence of T8AFB, 2834 bp long contains an open reading frame encoding a protein, M r 86,528, with 764 amino acids.
  • the first in-frame ATG is followed by codons specifying a hydrophobic sequence defined as a signal peptide in the LH/hCG receptor.
  • Akamizu et al., supra. There is a long hydrophilic region with five potential N-linked glycosylation sites followed by a region with seven hydrophobic, membrane-spanning domains and a cytoplasmic region containing a potential protein kinase C phosphorylation site.
  • the TAA stop codon is followed by a polyadenylation signal at nucleotides 2686-2691.
  • EXAMPLE 2 Transfection experiments with COS-7 cells (which is a publicly available non-patented cell line with ATCC accession number CRL 1651), or other non-thyroid cell, used a commercially available electroporation device and the technique recommended by the manufacturer (Bio-Rad).
  • the expression vector was constructed by subcloning the EcoRI T8AFB cDNA insert into the EcoRI site of SV40 promoter-driven pSG5 (Stratagene).
  • the cells (about 10 per ml), which were washed and resuspended in 0.8 ml of sucrose/phosphate buffer, were incubated with the plasmid DNA (80 ⁇ g in 10 ⁇ l of water) for 10 minutes in an ice-water bath before being pulsed with 330 V and 25 ⁇ F. The cells then were plated in dishes at 1.5-4 ⁇ 10 6 cells per dish. Cell viability was ⁇ 50% after electroporation. After a 40-48 hour stabilization culture period, TSH-stimulated cAMP production and TSH binding were measured.
  • TSH was radioiodinated and binding thereof was measured using standard techniques, for example as described in Tramontano & Ingbar (Endo 118:1945-1951 (1986)) with the exception that the incubation and wash buffer was modified Hanks balanced salt solution (wherein NaCl is replaced by 222 mM sucrose) containing 0.5% bovine serum albumin and 20 mM Hepes at pH 7.4.
  • the incubation mixtures contained about 4 ⁇ 10 6 cpm of 125 I-labeled TSH (120 ⁇ Ci/ ⁇ g) and unlabeled TSH or LH. Specific binding was calculated by subtracting values obtained in the presence of 0.1 ⁇ M unlabeled TSH.
  • cAMP levels were assayed using a standard technique, for example as described in Kohn et al. (supra). Briefly, the test substance, for example TSH or thyroid stimulating antibody, was added to the culture medium or to cells washed and maintained in Hank's balanced salt solution or the modified Hank's balanced salt solution described above, along with a cAMP phosphodiesterase inhibitor, such as 3-isobutyl-1-methylxanthine. After a brief incubation of 0.5-3 hours the cells were separated and the amount of cAMP in the medium was determined, and in cell lysates if desired. Presence of cAMP was determined by commercially available radioimmunoassay kits (for example DuPont or New England Nuclear). Cell pellets were in each case solubilized with 1 M NaOH for protein determinations. Protein was measured using a commercially available kit with bovine serum albumin as standard (Bio-Rad).
  • TSH-sensitive adenylate cyclase response in the transfected COS-7 cells was accompanied by the appearance of specific binding of TSH.
  • the K d values for the high-affinity and low-affinity binding sites were estimated at about 1.3 x 10 -10 M and 5.1 x 10 -8 M, respectively. Those values compare favorably with the values that were obtained for FRTL-5, 5.9 x 10 -10 M and 1.7 ⁇ 10 -8 M, respectively, by Tramontano & Ingbar (supra).
  • EXAMPLE 3 Poly(A) + RNA's from FRTL-5 cells were prepared using standard procedures. The RNA's were separated by size and transferred to Nytran membranes ( Schleicher & Schuell ) using standard Northern blot methods, see for example Zarrilli et al. (Mol Endo 3:1498-1508 (1989)) and Isozaki et al.
  • RNA amounts were inferred from densitometric scanning (LKB laser densitometer) of the hybridized bands with the value obtained in the lane containing RNA of cells not exposed to TSH at time zero serving as an arbitrary reference value.
  • EXAMPLE 4 Poly(A) + RNA from FRTL-5 cells maintained in the absence of TSH for 7 days had significantly higher levels of the 5.6 kb and 3.3 kb transcripts than did cells maintained in the presence of TSH, suggesting that TSH down-regulated expression of the gene. Down-regulation was rapid, 3-4-fold within 8 hours of TSH challenge, and was dependent on TSH concentration. A comparable down-regulation was found when cells were exposed to cholera toxin, forskolin or 8-bromo-cAMP, compounds known to affect the adenylate cyclase complex. Down-regulation was not found when cells were exposed to phorbol 12-myristate 13-acetate.
  • TSH binding to cells decreased about 60% whether TSH, cholera toxin, forskolin, or 8-bromo-cAMP was the agent.
  • Patients with autoimmune thyroid disease have circulating antibodies that increase cAMP levels or that inhibit TSH binding.
  • Representative antibodies were obtained from diagnosed patients using Protein G (Genex) and the manufacturer's recommended procedure or a standard procedure for immunoglobulin purification by affinity chromatography. Stimulating antibodies were identified by their ability to induce cAMP using the assay described above with FRTL-5 cells and with the immunoglobulin at a concentration of 1 mg/ml.
  • Antibodies that inhibit TSH binding were identified using a solid phase assay. Briefly, microtiter plates optionally were precoated with 0.1 ml of a 20 ⁇ g/ml poly-L-lysine (M r 70,000 from Sigma) solution prepared in water for one hour at room temperature. The solution was replaced with 0.1 ml of thyroid membranes, obtained by standard procedures, diluted appropriately in 20 mM Tris-acetate, pH 7.0. Controls consisted of wells containing 0.5% bovine serum albumin (BSA) in place of membranes. After 4 hours or more of incubation at 4°C, the wells were washed with buffer comprising 0.5% BSA in 20 mM Tris-acetate, pH 6.7 for 30 minutes at room temperature.
  • BSA bovine serum albumin
  • the buffer was replaced with a test sample diluted appropriately in the same buffer and the plates were allowed to incubate. The wells then were exposed to labelled TSH, incubated, washed and bound label determined. The inhibition activity is related directly to the decrease in labelled TSH bound when compared to the decrease observed with IgG from a normal individual.
  • FRT is a continuously growing line that, like FRTL-5, is derived from Fischer rats and has an apparently normal adenylate cyclase complex sensitive to cholera toxin and forskolin (Ambesi-Impiombato & Coon, Int Rev Cytol Supp 10:163-171 (1979), Ambesi-Impiombato, supra and Kohn et al., supra). FRT cells did not express the two species of TSH receptor mRNA's in Northern blots.) EXAMPLE 5 The technique of site-directed mutagenesis enabled the identification of critical sites on the extracellular domain including sites that are important for TSH binding, that impart TSH binding specificity on the receptor, species specificity and antibody binding sites.
  • two sites on the extracellular domain are important for TSH receptor function and stimulating antibody action, but not for high affinity TSH binding; a third site is important immunologically but is not important functionally and is not important for either inhibiting or stimulating antibody interaction; and a fourth site adjacent to the third contributes more to receptor function than to TSH binding.
  • Oligonucleotide mediated site-directed mutagenesis was performed using the T7-GEN In Vitro Mutagenesis kit of U.S. Biochemical Corp. Two phosphorylated oligos which imparted new restriction sites unique to the full length clone or vector were annealed with a single strand preparation of the EcoRI T8AFB construct inserted into M13mpl8.
  • the asparagine residue was converted to glutamine (CAG) using a 27-mer complementary to the target sequence and having the CAG codon located centrally.
  • a second strand comprising methylated cytosine was generated with T7 polymerase and T4 ligase.
  • MspI or Sau3AI
  • Hhal and exonuclease III competent cells were transfected with the in vitro synthesized, mutated single stranded DNA. Restriction mapping and dideoxy sequencing validated mutations in the resulting clones.
  • the M3B transfectant was able to bind TSH and showed induced cAMP synthesis upon reaction with TSH or thyroid stimulating antibody.
  • M3A and M3C transfectants showed no TSH binding or TSH-induced increase in cAMP response.
  • the M3 mutant showed a low level of TSH binding capability.
  • An interpretation of the data is the deletion which preserves TSH binding and function, amino acids 339-367, defines a region that is not critical for receptor function, nor for stimulating or inhibiting antibody binding. Adjacent regions, amino acids 308-339 and 367-399 define regions that contribute to TSH binding and receptor function since the M3 mutant did show some binding capability and lacked that peptide. The region defined by amino acids 308-339 appears to be more important to receptor function than to TSH binding.
  • EXAMPLE 6 Amino acids 339-367 are part of a region found on the TSH receptor and not on the LH/hCG receptor.
  • the TSH receptor-specific region contains a stretch of hydrophilic amino acid residues. It is known that certain amino acids and combinations thereof are likely to be immunonogenic and the probability of immunogenicity of the stretch of hydrophilic amino acids in the TSH receptor is high.
  • a sixteen residue peptide (Tyr-Tyr-Val- Phe-Phe-Glu-Glu-Gln-Glu-Asp-Glu-Ile-Ile-Gly-Phe-Cys; commercially synthesized under contract) from this region was tested on FTRL- 5 cells and COS-7 cells transfected with the full length TSH receptor cDNA. It was found that the 16-mer had no effect on TSH binding, TSH-induced cAMP synthesis, stimulating antibody- induced cAMP synthesis or the binding of inhibiting antibodies.
  • the 16-mer readily produced antibodies within three weeks of injection into rabbits using an immunization schedule recommended by Hazelton Laboratory.
  • the resulting antisera were reactive in an ELISA, as described below, using the 16-mer as antigen as described below.
  • protein antigen was bound to the wells of a microtiter plate by dilution of antigen in bicarbonate buffer, pH 9.6, containing about 0.1% BSA (Calbiochem) at a concentration of about 5-20 ⁇ g/ml, and adding 100 ⁇ l of the solution to the wells.
  • the plate was incubated at 37°C for about 2 hours.
  • the plates were washed with PBS-T (phosphate-buffered saline containing 0.05% Tween-20).
  • the test sample for example antipeptide antibody, patient serum or patient IgG preparation, was diluted appropriately in 1% BSA in PBS-T and next added to the wells. The plate was incubated for about 90 minutes at room temperature and then washed as described above.
  • a detection molecule diluted with 1% BSA in PBS-T was added to the wells, the plate was incubated for about 90 minutes, washed and the amount of peptide-reactive material in the test sample was determined.
  • Suitable detection molecules include an avidin-biotin system or an appropriate antibody radioactively labelled or enzyme conjugated (available, for example, from Zymed or Amersham).
  • radiolabelling a gamma or liquid scintillation counter is used to determine the amount of well bound label.
  • enzyme labelling a suitable substrate is added to the well and appropriate detection is effected, for example by luminometry or spectrophotometry.
  • the peptide was used in the ELISA to determine the presence of reactive antibodies in IgG preparations from a variety of patients including those with Graves' Disease. Preparations from 29/34 patients with Graves' Disease reacted positive in the assay whereas samples obtained from 22 patients with non-thyroid diseases (including rheumatoid arthritis, systemic lupus erythematosus and non-autoimmune thyroid disease, such as adenoma) and from 15 normal individuals were non-reactive with the peptide. Publications and references referred to and recited herein are expressly incorporated by reference. While preferred embodiments of the instant invention have been described, it will be apparent to those skilled in the art that many changes and modifications can be made to the products and processes without departing from the spirit of the invention.
  • a functional equivalent of a nucleic acid fragment can be defined in terms of capability of hybridization or in terms of capability of expressing a polypeptide product therefrom that comprises residues of a rat thyrotropin receptor.
  • a functional equivalent of a polypeptide can be defined in terms of carrying a function normally associated with the intact protein, such as a peptide that defines an antibody binding site, a peptide that comprises the extracellular domain or a peptide that comprises a carbohydrate binding site on said extracellular domain.

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Abstract

Le gène de rat récepteur de thyrotropine a été cloné et exprimé. La séquence de nucléotides et le produit sensiblement pur exprimé à partir de ladite séquence, ou des parties dudit produit, sont utiles pour détecter des molécules et des ligands pouvant se lier à ladite séquence ou audit produit.
PCT/US1990/006533 1990-11-15 1990-11-15 Gene de rat recepteur de thyrotropine et utilisation dudit gene WO1992008726A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA 2095013 CA2095013A1 (fr) 1990-11-15 1990-11-15 Gene recepteur de la thyrotropine murine et ses usages
PCT/US1990/006533 WO1992008726A1 (fr) 1990-11-15 1990-11-15 Gene de rat recepteur de thyrotropine et utilisation dudit gene
EP90917617A EP0557273A1 (fr) 1990-11-15 1990-11-15 Gene de rat recepteur de thyrotropine et utilisation dudit gene

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0506890A4 (en) * 1989-12-20 1993-06-30 Nichols Institute Diagnostics Recombinant thyrotropin receptor
GB2376016A (en) * 2001-05-30 2002-12-04 Nadir R Farid Thyrotropin receptor (TSHR) domain cleavage catalysed by the metalloprotease ADAM 10
EP0959896A4 (fr) * 1996-06-05 2005-03-02 Basil Rapoport Compositions a base de recepteur de la thyrotropine humaine

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US4472508A (en) * 1982-12-30 1984-09-18 The Beth Israel Hospital Association Test for detecting and measuring the graves' disease-specific immunoglobulins
US4609622A (en) * 1983-05-31 1986-09-02 Interthyr Research Foundation Inc. Clinical determination and/or quantification of thyrotropin and a variety of thyroid stimulatory or inhibitory factors performed in vitro with an improved thyroid cell line, FRTL-5
US4973578A (en) * 1988-03-17 1990-11-27 Mayo Foundation For Medical Education And Research Synthetic peptides derived from the alpha-subunit of human lycoprotein hormones

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US4609622A (en) * 1983-05-31 1986-09-02 Interthyr Research Foundation Inc. Clinical determination and/or quantification of thyrotropin and a variety of thyroid stimulatory or inhibitory factors performed in vitro with an improved thyroid cell line, FRTL-5
US4973578A (en) * 1988-03-17 1990-11-27 Mayo Foundation For Medical Education And Research Synthetic peptides derived from the alpha-subunit of human lycoprotein hormones

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Biochemical and Biophysical Research Communications, Vol. 166, No. 1, issued 15 January 1990, M. MISRAHI et al, "Cloning, Sequencing and Expression of Human TSH Receptor", pages 394-403. *
Biochemical and Biophysical Research Communications, Volume 165, No. 3, issued 29 December 1989, F. LIBERT et al, "Cloning, Sequencing and Expression of the Human Thyrotropin (TSH) Receptor: Evidence for Binding of Autoantibodies", pages 1250-1255. *
Biochemical and Biophysical Research Communications, Volume 165, No. 3, issued 29 December 1989, Y. NAGAYAMA et al, "Molecular Cloning, Sequence and Functional Expression of the cDNA for the Human Thyrotropin Receptor", pages 1184-1190, see entire document. *
Endocrinology, Volume 122, No. 6, issued 1988, B.L. HILL et al., "Monoclonal Antibodies to the Thyrotropin Receptor Raised by an Autoanti-idiotypic Protocol and Their Relationship to Monoclonal Antibodies from Graves's Patients," pages 2840-2850, see entire document. *
Proceedings National Academy of Sciences, Volume 79, issued November 1982, W.A. VALENTE et al, "Monoclonal Antibodies to the Thyrotropin Receptor: Stimulating and Blocking Antibodies Derived from the Lymphocytes of Patients with Grave's Disease", pages 6680-6684, see entire document, especially Abstract. *
S.L. BERGER et al., "Methods in Enzymology" Vol. 152, published 1987 by Academic Press (CA), see pages 572-575. *
Science, Volume 245, issued 4 August 1989, McFARLAND et al., "Lutropin Choriogonadotropin Receptor: An Unusal Member of the G Protein-coupled Receptor Family." pages 494-499, see entire document, esp. page 494, 2nd paragraph. *
Science, Volume 246, issued 22 December 1989, M. PARMENTIER et al., "Molecular Cloning of the Thyrotropin Receptor", pages 1620-1622, see entire document. *
Science, Volume 254, issued 04 August 1989, H. LOOSFELT et al., "Cloning and Sequening of Porcine LH-HCG Receptor cDNA: Variants Lacking Transmembrane Domain", pages 525-528, see entire document esp. page 525 1st paragraph, page 528 last paragraph. *
Sciences, Volume 244, issued 05 May 1989, F. LIBERT et al, "Selective Amplification and Cloning of Four New Members of the G Protein-Coupled Receptor Family, pages 569-575. *
See also references of EP0557273A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0506890A4 (en) * 1989-12-20 1993-06-30 Nichols Institute Diagnostics Recombinant thyrotropin receptor
EP0959896A4 (fr) * 1996-06-05 2005-03-02 Basil Rapoport Compositions a base de recepteur de la thyrotropine humaine
GB2376016A (en) * 2001-05-30 2002-12-04 Nadir R Farid Thyrotropin receptor (TSHR) domain cleavage catalysed by the metalloprotease ADAM 10

Also Published As

Publication number Publication date
CA2095013A1 (fr) 1992-05-16
EP0557273A4 (fr) 1994-01-12
EP0557273A1 (fr) 1993-09-01

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