EP0557273A1 - Rat thyrotropin receptor gene and uses thereof - Google Patents

Abstract

The thyrotropin receptor rat gene was cloned and expressed. The nucleotide sequence and the substantially pure product expressed from said sequence, or parts of said product, are useful for detecting molecules and ligands capable of binding to said sequence or said product.

Description

RAT THYROTROPIN RECEPTOR GENE AND USES THEREOF The invention described and claimed herein was supported in part by the Department of Health and Human Services, National Institutes of Health. FIELD OF INVENTION 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. BACKGROUND OF THE INVENTION Thyrotropin, or thyroid stimulating hormone (TSH), is a pituitary hormone that regulates the development and activity of the thyroid gland. The thyroid secretes two principle iodine-containing hormones, T₃ (also known as triiodothyronine) and T₄ (also known as thyroxine), which, among other roles, regulate basal metabolism. Secretion of T₃ and T₄ is in turn regulated by TSH.

As with other glycoprotein hormones, 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 T₃ and T₄ as well as the active synthesis of T₃ and T_4.

Clinical correlates of abnormal binding of TSH to its specific receptor may be manifest in a variety of syndromes. For example, 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. Alternatively, expression of the receptor may be down-regulated, thereby producing a hypothyroid state, such as during oncogene transformation. Another example is 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.

The in situ structure of the thyrotropin receptor remains unclear because of a multiplicity of proteins which appear to interact with TSH. Studies using nondenaturing conditions have identified 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 ɤ-actin and a member of the hsp70 family, respectively. Biochem Biophys Res Comm 170:351-358 (1990).

Pure sources of the receptor are unavailable, primarily because of the extraordinarily small number of receptors on thyroid cells. Attempts to use TSH receptor antibodies to purify the receptor have been unsuccessful.

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).

Nagayama et al. were unsuccessful in cloning a rat gene using oligonucleotide probes corresponding to the rat LH/hCG (LH is luteinizing hormone and hCG is human chorionic gonadotrophin, other glycoprotein hormones) receptor sequence. Those authors then used the same strategy of Parmentier et al. employing transmembrane domain-related oligonucleotides to obtain clones of the human gene. Transfectants showed increased levels of cAMP upon exposure to TSH but not in reponse to hCG, ACTH or insulin exposure. Biochem Biophys Res Comm 165:1184-1190 (1989). Libert et al. used a clone containing the complete coding sequence of the dog TSH receptor gene to screen a human cDNA bank. They obtained a full length clone and expression thereof in transfected COS cells. Biochem Biophys Res Comm 165:1250-1255 (1989).

Misrahi et al. also cloned the human gene. Those authors screened a cDNA library with a full length porcine LH/hCG receptor cDNA because of a structural similarity between LH and TSH. Biochem Biophys Res Comm 166:394-403 (1990).

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. Thus, 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. SUMMARY OF THE INVENTION A first object of the instant invention is to provide substantially pure nucleotide sequences encoding a rat thyrotropin receptor gene, and portions thereof.

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.

These and other objects have been achieved by the successful cloning and expression of a rat thyrotropin receptor gene. BRIEF DESCRIPTION OF THE DRAWING Figure 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.

Figure 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.

Below the schematic drawing and to the left are a series of bars drawn to scale with the schematic drawing depicting the extent of the deletion in the extracellular domain of the mutants. The mutants maintained a normal transmembrane domain. Numbers above the bars represent the first and last amino acids deleted. Below the schematic drawing and to the right is a table of data relating to the deletion mutants, that are identified in the first column. TSH binding and cAMP response were determined as described herein. DETAILED DESCRIPTION OF THE INVENTION In order to clone the rat gene, a cDNA library was constructed using the FRTL-5 rat thyroid cell line as the source of RNA. After clones were obtained, the recombinant receptor was expressed and critical regions thereof were identified.

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 CO₂ 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.

Templates of cDNA were synthesized using 5 μg 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.

An FRTL-5 cDNA library was constructed in λgt11 (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. Akamizu et al., Biochem Biophys Res Comm 169:947-952 (1990). There is a long extracellular domain comprising at least five N-linked glycosylation sites and a transmembrane region with seven hydrophobic domains.

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.

In Northern blots of FRTL-5 mRNA, two species of message were noted. Cells exposed to TSH exhibited decreased levels of both species of message and the amount of message was dependent on TSH concentration. A similar down-regulation of the two mRNA species was noted when cells were treated with forskolin, cholera toxin or 8-bromo-cAMP, but no change was noted when the cells were treated with a phorbol ester. Down-regulation also occurred when cells were exposed to thyroid-stimulating antibodies, which also increased cAMP levels. Exposure to antibodies that inhibit TSH binding to the receptor increased TSH receptor mRNA levels. Insulin, calf serum and insulin-like growth factor I up- regulated TSH receptor expression. These observations may account for the success of the cloning described herein because RNA was obtained from cells maintained in medium containing insulin and calf serum but no TSH.

The clone and the receptor protein produced therefrom find utility in a variety of circumstances. For example, the recombinant receptor can be used in assays for detecting ligands capable of binding the receptor. Suitable ligands include thyrotropin and anti-receptor antibodies.

Thus, 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. 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.

After an incubation period to assure attachment, the support is washed liberally. Optionally, non-specific sites on the support are blocked with a non-croεs-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. 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. For example, a readily known labelled second ligand, such as 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. Alternatively, 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.

Although solid support assays are preferred because of the facility in performing the methods, other assays can be configured without undue experimentation. Thus liquid phase assays are practicable, as well as competition assays and those where the recombinant receptor is labelled. There are many variations in configuring an assay using the recombinant receptor and the skilled artisan can design an assay of choice within the spirit of the invention. 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.

Because of the low density of receptor on thyroid cells, it has not been possible to obtain specific high titer anti-receptor antibodies. The substantially pure receptor and parts thereof can be used to obtain specific antibody. As descibed below, 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. Accordingly, 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. As described below, 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. Thus 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. If normal tissue transplantation barriers are surmounted, for example using a syngeneic, or at least histocompatible thyroid cell line from another individual or a cell line that does not express major, and possibly minor histocompatibility antigens, it is possible that replacement transfected cells need not come from the patient in need of treatment.

Certain thyroid dysfunctions are treatable with 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.

Pharmaceutical formulations 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. In addition to the active ingredient, 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. For example, 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. Alternatively, treatment may involve temporary removal of tissue and exposure thereof to the claimed compositions before introduction back into the patient. Thus during hemapheresis, plasmapheresis, transfusion or dialysis, for example, 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.

Delivery of the receptor sequence is practiced by art-recognized means such as electroporation, precipitation, microinjection, liposome fusion, microparticle bombardment and the like. Generally 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.

The skilled artisan can determine the most efficacious and therapeutic means for effecting treatment practicing the instant invention. Reference can also be made to any of numerous authorities and references including, for example, "Goodman & Gilman's The Pharmaceutical Basis of Therapeutics" (6th ed., Goodman et al., eds., MacMillan Publ. Co., NY, 1980). The invention will be described in further detail by way of the following non-limiting Examples. EXAMPLE 1 From 8 x 10⁵ plaques screened at low stringency with the rat LH/hCG receptor-related probe described above, 20 FRTL-5 rat thyroid cell clones were obtained. Eighteen, with insert sizes of 1.4-4.2 kilobases (kb), contained transmembrane domain sequences exhibiting about 70% amino acid sequence identity with the comparable region of the rat LH/hCG receptor. Compared to the LH/hCG receptor, the two largest clones, 4.2 and 2.4 kb in length (4A2 and 16B1, respectively) had an incomplete 5' end.

To obtain a full length clone, 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.

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. There is a stretch of amino acids present in the TSH receptor that is not found in the LH/hCG receptor.

The homology between the entire coding regions defined by the rat TSH and LH/hCG receptors is relatively low, 64% and 48% for nucleotides and amino acids, respectively. The homology in the transmembrane region is slightly greater, 60% and 70%, respectively. The overall amino acid homology with the human and dog TSH receptors is 86% and 89%, respectively. 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 μg in 10 μl of water) for 10 minutes in an ice-water bath before being pulsed with 330 V and 25 μF. The cells then were plated in dishes at 1.5-4 × 10⁶ 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.

Highly purified bovine TSH (NIDDK-bTSH-I-1, 30 units/mg) and LH (USDA-bLH-B5, 2.1 units/mg) were obtained from the hormone distribution program of the National Institute of Diabetes and Digestive and Kidney Diseases. 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 × 10⁶ cpm of ¹²⁵I-labeled TSH (120 μCi/μg) and unlabeled TSH or LH. Specific binding was calculated by subtracting values obtained in the presence of 0.1 μM unlabeled TSH.

Levels of cAMP 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).

When T8AFB in the correct orientation was transfected into COS-7 cells, the expressed protein caused the cells to become sensitive to TSH in cAMP assays. The relative increase in total cAMP induced by 0.1 nM TSH in the transfected cells was 5-fold above basal (>20 pmol/mg of protein). By comparison, LH did not increase significantly levels of cAMP above basal level when tested at a 10-fold higher (1 nM) concentration. COS-7 cells transfected identically with constructs containing the cDNA insert in the opposite orientation did not have a TSH-induced increase of adenylate cyclase activity.

The development of a TSH-sensitive adenylate cyclase response in the transfected COS-7 cells was accompanied by the appearance of specific binding of TSH. Binding of ¹²⁵I-labeled TSH to COS-7 cells transfected with the insert in the correct orientation, but not in the opposite orientation, exhibited a curvilinear isotherm similar to that of FRTL-5 thyroid cells (Tramontano & Ingbar, supra) and was inhibited 50%, 75% and >90% by 0.3, 3 and 30 nM unlabeled TSH, respectively, but was not inhibited by 10 nM LH. 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. (Mol Endo 3:1681-1692 (1989)). The probes used were the purified inserts from clone T8AFB, 16B1 or 4A2 and as a control, a β-actin cDNA (kindly provided by B. Paterson, National Cancer Institute). The final wash of the filters was in 1 × SSPE (0.15-0.18 M NaCl/10 mM phosphate, pH 7.4/0.5-1.0 mM EDTA) containing 0.1% SDS at 65°C. Quantitation of RNA amounts was 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.

Northern analyses of poly(A)⁺ RNA from FRTL-5 cells identified two mRNA species, 5.6 and 3.3 kb in size. The same two mRNA's were detected barely in poly(A)⁺ RNA of rat ovary and were not detected in rat testis, brain, liver, lung or spleen (RNA samples obtained from Clontech). A probe derived from the midportion of the extracellular domain of the T8AFB clone hybridized with both species of transcripts. A 0.7 kb cDNA probe derived from the 3' nontranslated portion of clone 4A2 hybridized with only the 5.6 kb transcript. That suggests that the 5.6 kb mRNA transcript is larger primarily because it contains a longer 3^' noncoding region.

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. Measured at the same time and under the same conditions, TSH binding to cells decreased about 60% whether TSH, cholera toxin, forskolin, or 8-bromo-cAMP was the agent. The addition of insulin, insulin-like growth factor-1 or calf serum to FRTL-5 cells that had been maintained for 7 days with no TSH or insulin and little (about 0.2%) or no calf serum, up-regulated the TSH receptor gene and was required for down-regulation by TSH or compounds known to affect the expression of the adenylate cyclase complex.

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 μg/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. 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.

When IgG preparations from patients with Graves disease were tested, those antibodies, which increased cAMP levels as does TSH, also down-regulated TSH receptor mRNA levels to that comparable to what is found in cells exposed to TSH. IgG preparations from patients with primary hypothyroidism, which have inhibitory activity, increased TSH receptor mRNA levels about 2-fold over baseline. Reactivity of both types of antibodies with FRTL-5 cells was associated with the presence of the TSH receptor on the cell as both types of antibody reacted with FRTL-5 cells but not with FRT rat thyroid cells. (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. For example, 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.

To derive mutants of potential glycosylation sites, the asparagine residue (AAT) 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. After removal of the parental strand with 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 EcoRI inserts in correct orientation of positive clones were used to produce 9 deletion mutants after reconstruction and subcloning. Mutant Ml lacked amino acids 37-121 (where the first residue is the initiating methionine); M2 lacked amino acids 110-307; M2A lacked amino acids 173-231; M2B lacked amino acids 233-265; M2C lacked amino acids 268-303; M3 lacked 308-410; M3A lacked 338-399; M3B lacked 339-367 and M3C lacked 374-400 (see Figure 2).

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.

Deletion mutants spanning portions of the extracellular domain that included potential glycosylation sites (M1, M2, M2A, M2B and M2C) and lacking the hydrophobic signal peptide region (amino acids 5-23) did not exhibit TSH binding nor did TSH elevate cAMP levels in transfected COS-7 cells. To further define the actual sites responsible for the loss of function in M1, M2, M2A, M2B and M2C, mutants with individual carbohydrate deletions (I through V) were created. Carbohydrate units II, III and V did not influence TSH binding or receptor function. In contrast, carbohydrate units I and IV limited TSH binding but did not influence the TSH-induced or stimulating antibodyinduced increase in cAMP. 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. Furthermore, the antibodies bound to FRTL-5 cells, which express TSH receptor, but not to FRT cells, which do not express TSH receptor.

For the ELISA, 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 μg/ml, and adding 100 μl 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. An appropriate amount of 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). In the case of radiolabelling, a gamma or liquid scintillation counter is used to determine the amount of well bound label. In the case of 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.

For example, it is clear that changes can be made to the nucleotide or amino acid sequence without affecting the capability thereof to hybridize to homologous sequences or to serve as a functional receptor. Thus 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.

The described embodiments are thus to be considered illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than the foregoing description. All changes that come within the meaning and range of equivalency are to be embraced within the scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A substantially pure nucleic acid fragment hybridizable to a rat thyrotropin receptor gene or functional equivalent thereof.

2. The fragment of claim 1 that is hybridizable to said gene.

3. The fragment of claim 1 that comprises a polynucleotide hybridizable with sequences of said gene that encode the extracellular domain of the receptor expressed therefrom.

4. A substantially pure nucleic acid fragment comprising sequences encoding a rat thyrotropin receptor or functional equivalent thereof.

5. The fragment of claim 4 that encodes said receptor.

6. The fragment of claim 4 that comprises sequences encoding the extracellular domain of said receptor.

7. The substantially pure nucleic acid sequence, or functional equivalent thereof, of Figure 1.

8. The substantially pure nucleic acid sequence of Figure 1.

9. The substantially pure amino acid sequence, or functional equivalent thereof, of Figure 1.

10. The substantially pure amino acid sequence of Figure 1.

11. The substantially pure amino acid sequence of Figure 1 comprising the extracellular domain.

12. A substantially pure rat thyrotropin receptor or functional equivalent thereof.

13. A substantially pure rat thyrotropin receptor.

14. The extracellular domain of a rat thyrotropin receptor or functional equivalent thereof.

15. The extracellular domain of a rat thyrotropin receptor.

16. A medicament comprising:

(a) a therapeutically effective amount

of at least one member selected from the group consisting of a rat thyrotropin receptor, the extracellular domain of said receptor and functional equivalents of either or both of said receptor and said domain; and

(b) a pharmaceutically acceptable

carrier.

17. A method of detecting nucleic acids hybridizable to a rat thyrotropin receptor gene comprising the steps of:

(a) exposing at least one member

selected from the group consisting of a substantially pure polynucleotide comprising a rat thyrotropin receptor gene and a functional equivalent thereof to a test sample comprising molecules bindable to said receptor gene; and

(b) determining the presence of bound

molecules.

18. A method of detecting ligands bindable to a rat thyrotropin receptor comprising the steps of:

(a) exposing at least one member

selected from the group consisting of substantially pure rat thyrotropin receptor

and a functional equivalent thereof to a test sample comprising a ligand bindable to said receptor; and

(b) determining the presence of bound

ligand.

19. The method of claim 18 which further comprises the step of binding either said substantially pure thyrotropin receptor or functional equivalent thereof, or both, to a solid support prior to exposing step (a).

20. The method of claim 18 wherein said ligand is thyrotropin.

21. The method of claim 18 wherein said ligand is an antiglycoprotein hormone receptor antibody.

22. The method of claim 21 wherein said glycoprotein hormone is thyrotropin.

23. The method of claim 18 wherein said functional equivalent of substantially pure rat thyrotropin receptor comprises the extracellular domain.

24. The method of claim 23 wherein said extracellular domain comprises peptides, wherein said peptides are epitopes for antibodies.

25. The method of claim 24 wherein said peptide comprises Tyr-Tyr-Val-Phe-Phe-Glu-Glu-Gln-Glu-Asp-Glu-Ile-Ile-Gly-Phe-Cys.

26. A therapeutic method comprising administering to a patient in need of treatment a therapeutically effective amount of a composition comprising at least one member selected from the group consisting of substantially pure rat thyrotropin receptor, extracellular domain thereof and functional equivalents of either or both said receptor and said domain.

27. A therapeutic method comprising treating a patient in need of therapy with a therapeutically effective amount of at least one member selected from the group consisting of substantially pure rat thyrotropin receptor, extracellular domain thereof and functional equivalents of either or both said receptor and said domain.

28. The method of claim 27 wherein said treating step occurs ex vivo.

29. A method of detecting thyrotropin receptor or functional equivalents thereof comprising the steps of:

(a) exposing an antibody, or binding

site thereof, that binds specifically to the rat thyrotropin receptor or functional equivalents thereof to a test sample comprising said receptor or functional equivalents thereof; and

(b) determining the presence of bound

receptor or functional equivalents thereof.

30. The method of claim 30 wherein said antibody is a monoclonal antibody.

31. A method of making an antibody that binds specifically to thyrotropin receptor comprising immunizing a mammal with substantially pure rat thyrotropin receptor or functional equivalents thereof.

32. The method of claim 31 which further comprises fusing antibody secreting cells of said immunized mammal with a myeloma cell.

33. An antibody that binds specifically to thyrotropin receptor.

34. The antibody of claim 33 wherein said receptor is rat thyrotropin receptor.

35. The antibody of claims 33 or 34 wherein said antibody is a polyclonal antibody.

36. The antibody of claims 33 or 34 wherein said antibody is a monoclonal antibody.

37. A medicament comprising:

(a) a therapeutically effective amount

of an anti-thyrotropin receptor antibody or binding site thereof; and

(b) a pharmaceutically acceptable

carrier.

38. A therapeutic method comprising treating a patient in need of therapy with a therapuetically effective amount of a composition comprising an anti-thyrotropin receptor antibody or binding site thereof.

39. The method of claim 38 wherein said treating step occurs ex vivo.

40. A medicament comprising:

(a) a therapeutically effective amount

of at least one member selected from the group consisting of a rat thyrotropin receptor gene and functional equivalents thereof; and (b) a pharmaceutically acceptable

carrier.

41. A therapeutic method of treating a patient in need of therapy comprising the steps of:

(a) obtaining cells compatible with

said patient;

(b) introducing into said cells an

expressible rat thyrotropin receptor gene, or functional equivalents thereof, to produce transfected cells;

(c) obtaining stable transfected cells

expressing said gene or functional equivalents thereof; and

(d) introducing said stable

transfected cells into said patient.