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Document Type and Number:
WIPO Patent Application WO/1994/029447
Kind Code:
The present invention provides a human TRH receptor gene (seq ID No.1), and polynucleotide probes specifically binding to this gene or naturally occurring variants thereof, as well as polypeptide probes specifically binding to the receptor polypeptide or naturally occurring variants thereof. Expression of the gene in a host is also useful for providing means for use in the evaluation of TRH-agonists and -antagonists.

Application Number:
Publication Date:
December 22, 1994
Filing Date:
June 09, 1994
Export Citation:
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International Classes:
G01N33/566; C07K14/72; C12N5/10; C12N15/09; C12N15/12; C12P21/08; C12Q1/68; (IPC1-7): C12N15/12; C12N5/10; C12P21/08; G01N33/53
Other References:
NEUROLOGY, vol.36, 1986 pages 641 - 644 S. MANAKER ET AL.; 'Autoradiographic localization of thyrotropin releasing hormone receptors in human brain'
PROC. NATL. ACAD. SCI. USA, vol.87, 1990 pages 9514 - 9518 R.E. STRAUB ET AL.; 'Expression cloning of a cDNA encoding the mouse pituitary thyrotropin-releasing hormone receptor' cited in the application
J. MOL. ENDOCRINOL., vol.10, 1993 pages 199 - 206 R.E. SELLAR ET AL.; 'Functional expression and molecular characterization of the thyrotrophin-releasing hormone receptor from the rat anterior pituitary gland' cited in the application
BIOCHEM. BIOPHYS. RES. COMMUN., vol.195, 1993 pages 179 - 185 V. MATRE ET AL.; 'Molecular cloning of a functional human thyrotropin-releasing hormone receptor'
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1. A gene encoding human TRH receptor.
2. A gene encoding human TRH receptor having the amino acid sequence of SEQ ID No.l.
3. An isolated DNA sequence encoding human TRH receptor.
4. A recombinent DNA sequence encoding human TRH receptor.
5. An isolated or recombinent DNA sequence for use in expression in a prokaryotic or eukaryotic host cell of a polypeptide product having an amino acid sequence corresponding to a sufficient extent to that of a naturally occurring TRH receptor to provide at least one biological function of said naturally occuring TRH receptor, which DNA sequence is selected from: a) sequence complementary thereto; and b) a DNA sequence which can hybridize to a DNA sequence defined in (a) or to a fragment thereof.
6. A prokaryotic or eukaryotic host cell transformed or transfected with a gene or DNA sequence according to any one of the precending claims.
7. A recombinant vector containing a DNA sequence or gene according to any one of claims 1 to 5.
8. A prokaryotic or eukaryotic host cell transformed or transfected with a recombinant vector according to claim 7.
9. A polynucleotide probe comprising a labelled DNA or RNA sequence capable of specifcally hybridizing to a gene according to claim 1 or claim 2 or a naturally occurring variant thereof.
10. A method of detecting a gene according to claim 1 or claim 2 which method comprises hybridizing a probe according to claim 9 with said gene, and dectecting bound labelled probe.
11. An antibody probe comprising a labelled antibody raised against an amino acid sequence capable of specifically binding to TRH receptor expressed by a gene according to claim 1 or claim 2 or a naturally occuring variant thereof.
12. An antibody probe according to claim 11 which is a monoclonal antibody.
13. A method of detecting TRH receptor expressed by a gene according to claim 1 or claim 2 or a naturally occurring variant thereof, which method comprises allowing a probe according to claim 11 or claim 12 to bind with said receptor, and detecting bound labelled probe.
14. A method of diagnosis for a condition associated with TRH receptor abnormality which comprises hybridizing a polynucleotide probe according to claim 9 with the 8q23 chromosomal region, and detecting bound labelled probe.
15. A method of diagnosis for a condition associated with TRH receptor abnormality which comprises microscopic examination of the 8q23 chromosomal region for gross abnormality.

Background of invention Field of invention This invention relates to the discovery of cDNA sequences encoding the human thyrotrophin-releasing hormone (TRH) receptor, and the application of this discovery to inter alia 1) the development and use of nucleotide sequences derived from the receptor for the measurement (qualitative and quantitative) and regulation of gene expression and 2) the development and use of TRH peptide agonist and antagonist analogues for clinical and therapeutic use in the management of human pituitary and thyroid dysfunction, cancers of the pituitary and thyroid and TRH related disorders of brain function . Description of the prior art

The initial step in TRH action centres on the binding of TRH to a specific membrane bound receptor. Multiple steps are then involved in triggering the synthesis and secretion of thyrotrophin (TSH) from thyrotrophs and prolactin (PRL) from lactotrophs. Both cell types are located in the anterior pituitary. The binding of this hormone to its receptor has species-specific characteristics related to the peptide sequence and three-dimensional conformation of both ligand and the receptor. A number of TRH agonist and antagonist analogues eg. [N 3iιn -methyl]TRH and [β-pyrazol-1- yl-Ala 2 ]TRH (Szirtes et al., 1984 J. Med. Che . 27, p741- 745) have been produced since the structure of TRH was established in 1969. Synthetic TRH is used clinically in a specific provocative test of hormonal function, known as

the TRH-Test. This test is used to asses the status of TSH and prolactin secretion by the pituitary gland. TRH agonists have also been used in clinical trials. Thus, for example, the analogue RX77368 has been used in controlled trials of patients with motor neurone disease, (Guiloff R.J. et al., 1986. J. Neurology, Neurosurgery and Psychiatry 49, 969-973), but with only limited success. TRH analogues are also believed to have potential therapeutic uses in Alzheimer's disease, (Metcalf G., 1982 Brain Res. Rev. 4, 389-408) and stroke, (Holaday J.W. et al., 1981 Science 213, 216-218), narcotic depression, psychiatric disorders, narcolepsy, hyperalgesia and colonic paralysis. In addition TRH analogues are expected to be beneficial in reducing symptoms associated with neurological disorders such as motor neurone disease, spinocerebellar degeneration, (Sobue et al., 1980 Lancet i, 418-419) and brain and spinal injuries. TRH has been shown to be more effective than Naloxone an opiate receptor antagonist in improving cardiovascular function and survival in experimentally induced spinal injury in cats, (Faden A.I. et al., 1981 New Eng. J. Med. 305, 1063-1067). Other properties of TRH analogues may be useful in treating narcotic and anaesthetic overdose and opiate dependence, (Metcalf G., 1982 Brain Res. Rev. 4, 389-408). Accordingly there is a need for potent synthetic TRH analogues for such purposes also. Treatments with TRH itself or with known analogues are however inconvenient to administer, of limited efficacy, and/or difficult to control and adapt to individual requirements. Knowledge of the comparative

characteristics of TRH receptors in the brain and pituitary would be helpful both in attempting to use TRH or its analogs in behavioural pharmacotherapy without endocrine side effects and in attempting to understand the evolution of TRH's possible roles in the CNS.

Species differences in the kinetics of the TRH receptor are known to exist, based on the binding of TRH analogues (Taylor St Burt 1982, J Neurochemistry 38,1649-1656) Furthermore sub-types exist of most receptors within a single species. This is in line with what is known for other transmembrane receptors where subtypes and alternatively spliced versions have been demonstrated.

Recent attempts have been made to elucidate the structure of the TRH receptor. (Straub et al., 1990. PNAS 87 9514- 9518) have cloned the cDNA coding for the mouse TRH-R gene and have shown it to code for a 393 amino acid protein. We have recently cloned the rat TRH-R cDNA, (Sellar et al 1993) and found it to be 96% identical to the mouse gene in the coding region, but having 19 extra amino acids at the carboxy terminal (COOH) tail, resulting in a 412 amino acid protein. The peptide sequences deduced from their DNA constructs conform in broad structural terms to the family of seven-transmembrane G-protein coupled receptors. The

412 amino acid sequence of the rat receptor is encoded by a DNA that contains 69 different nucleotides within the comparable coding regions plus 57 extra nucleotides at the COOH terminus (representing 90% overall homology) . In the

comparable coding region (393 amino acids) 21 nucleotide changes result in 17 amino acid sequence changes. The significance of species differences has moreover been emphasized recently (Oksenberg et al., 1992 Nature 360: 161-163) where a single amino acid difference between human and rodent 5-HT 1B receptor proteins was found to result in major pharmacological differences. Thus there is a particular need for providing probes and other assay materials and methods for use in the detection and/or identification, directly or indirectly of abnormalities in human TRH-R as well as means for use in the production of improved TRH-R analogues for use in the treatment of such abnormalities.

It is an object of the present invention to avoid or minimise one or more of the above problems or disadvantages. Summary of Invention We have now isolated and cloned the cDNA encoding a human TRH receptor molecule(s), and identified polynucleotide sequences thereof, and established amino acid sequences of human TRH receptor corresponding thereto. In more detail a DNA sequence has now been discovered for human TRH receptors, which is presented as the basis of this invention. This sequence is presented in interleaved format (see Fig. 1) . The sequence of the human TRH receptor could not have been deduced from the published nucleotide sequence of the TRH receptors of mouse and rat.

Thus, in more detail, the present invention provides: a gene encoding human TRH receptor; and preferably a gene encoding human TRH receptor having the amino acid SEQ ID No:l disclosed herewith. It will be understood that the genes of the present invention may include nucleic acid sequences (upstream and/or downstream of the receptor coding sequence) which are utilized in the expression of the gene such as promoter, operator, and terminator sequences as well as other sequences which do not inhibit its expression. Thus the expression "gene" includes DNA and/or RNA sequences as well as plasmid or viral "genes" containing the receptor gene and expression vectors for the gene. In one aspect therefore the present invention provides new methods and means based upon the newly discovered TRH-R polynucleotide sequence for use in the clinical diagnosis and therapeutic management of those processes and abnormalities thereof that involve the action of TRH and its receptor, including but not exclusive of:

Acromegaly, Primary hypothyroidism in children. Diabetes mellitus. Anorexia nervosa, Schizophrenia, Cirrhosis of liver. Chronic renal failure. Protein-calorie malnutrition. The above conditions show paradoxical GH responses to TRH.

Primary Thyroid Disease including Hyperthyroidism (any cause) , Subclinical hyperthyroidism; Diffuse nontoxic goiter (15%); Treated hyperthyroidism (6-12 months); Opthalmic Graves' disease (30%); Subclinical


Pituitary Disease including Acromegaly, Cushing's disease, Nelson's syndrome, Prolactinomas, "Non-functional" adenomas; TSH-feedback adenoma; 'TSH toxicosis" without adenoma; "TSH toxicosis" with adenoma, and Familial hypopituitarism with enlarged sella in childhood Hypothalamic Disease (any cause) including Idiopathic GH deficiency; Idiopathic hypopituitarism in childhood; Neuropsychiatric Disorders including Anorexia nervosa and Unipolar depression;

Other Endocrine/Metabolic Disorders including Chronic renal failure; Starvation; Elderly sick/euthyroid The above conditions all show alterations in TSH responses to TRH.

Other conditions in which TRH/TRH-R action is thought to be involved

Altzhei er's disease (Albert et al. 1993 Biological

Psychiatry 33:267-271); Epilepsy (Kubek et al, 1993. Ann. Neurology 33:70-76);

PMS (Schmidt et al. 1993. J Clin Endo & Metab. 76:671-674); Ataxia due to spinocerebellar lesions (Engel et al. 1983. Lancet ii:73-75 and Faden 1983. Trends Neurosci 6:375- 377.); Narcolepsy, Hyperalgesia and Colonic paralysis.

In general the TRH-R cDNA sequences are also useful for the design of oligonucleotide probes capable of specifically hybridising with the genes of the present invention, and for the synthesis of polypeptides which may be used in

immunoassays. Both oligonucleotide probes and the polypeptides may be useful for the diagnosis of TRH-R abnormalities. Polypeptides encoded within the cDNA sequences may also be used to raise antibodies against selected regions of normal or abnormal TRH-R polypeptide corresponding to one or more domains, or portions thereof of the TRH-R polypeptide, which are particularly implicated in ligand building i.e. the first, second, third and fourth extracellular domains of the TRH-R polypeptide (see Figs.l and 2); or in signal transduction i.e. the first, second, third and fourth intra cellular domains, especially the main cytoplasmic loop or third intracellular domain, most preferably in the region of the interface between the third intracellular domain and the sixth transmembrane region. e.g. one of the four extra-cellular domains identified hereinbelow, and for the purification of antibodies directed against such regions. These antibodies may be useful in immunoassays for detecting normal or abnormal TRH-R in individuals. Thus the present invention provides various means for detecting naturally occurring TRH-R polypeptides or genes i.e. both fully functional normal forms and abnormal forms which present one or more deinations whether this be in relation to altered ligand binding, or signal transduction, which may be found to occur within the human population.

Furthermore the invention provides screening means for use in the evaluation of new TRH agonists and antagonists, comprising a cell transformed with a recombinant expression

system comprising an open reading frame (ORF) of DNA derived from a TRH-R genome or TRH-R cDNA, said ORF being operably linked to a control sequence compatible with said cell, as well as such expression systems per se.

Thus the present invention further includes a method of producing TRH receptor which method includes the step of expressing the genes of the present invention in a host, as well as TRH receptor produced by such a method. Various suitable hosts are known in the art though eukaryotic hosts are generally preferred, e.g. Xenopus oocytes and COS-1 cells. Prokaryotic hosts that may be used include E. coli. and B. Subtilis. Fungi e.g. yeast may also be used.

The preferred method of restriction enzyme analysis in this invention depends on Restriction Fragment Length Polymorphisms (RFLPs) . A sample is taken from any suitable tissue such as blood. DNA is extracted from the cells in any conventional way. It is then digested with an appropriate restriction enzyme e.g. one which cuts in CG- rich sequence. The fragments of different length are separated by gel electrophoresis in any conventional way. A restriction fragment pattern is generated. Probing of the fragments will generally be necessary for clearer detection of the pattern and of the fragment(s) of interest, e.g. a fragment which extends from restriction sites "n" to "n + 2" (where "n" denotes any arbitrary number) , seemingly not being restricted at the normal site "n + 1" lying between "n" and "n + 2" due to an abnormality

at the "normal" restriction site. Alternatively, a polymorphism might generate restriction enzyme sites and thereby given rise to a plurality of shorter fragments where the normal DNA provides longer ones. Whether it is appropriate to probe for long or short fragments will therefore depend on the circumstances of the polymorphism. In some instances, the probe will extend outside the region designated.

Although the RFLP method is the currently preferred method of assay, it cannot be ruled out that direct hybridisation of probes to the TRH-R genomic region will be of interest. Thus suitable biopsy or other samples can be subjected to cloning techniques, to isolate a library of genomic DNA. Clones containing the TRH-R gene can be amplified by

Polymerase Chain Reaction (PCR) and probes complementary to the said region used directly on PCR products, which need not be first restricted by enzymes.

It will be appreciated, therefore, that the cDNA of the invention also has uses in assays which are not of the RFLP type. Accordingly, the polynucleotides per se are part of this invention, as 'intermediates' suitable (when labelled) for use as probes. Both double-stranded and single- stranded polynucleotides are included as well as sense and anti-sense forms. Suitable polynucleotide probes will normally be a polynucleotide of from 10 to 50, preferably from 16 to 30 nucleotides in length. Shorter probes are unlikely to be sufficiently specific for the sequence of

interest. Longer nucleic acid probes of 100 nucleotides or more are likely to be inconveniently long, but up to 250 or more might be useful in some cases e.g. for chromosomal in- situ buybridisation as further described hereinbelow in the detailed examples. Preferably the probes relate to parts of the polynucleotide sequence corresponding to one or more domains, or portions thereof of the TRH-R polypeptide, which are particularly implicated in ligand building i.e. the first, second, third and fourth extracellular domains of the TRH-R polypeptide (see Figs. 1 and 2); or in signal transduction i.e. the first, second, third and fourth intra cellular domains, especially the main cytoplas ic loop or third intracellular domain, most preferably in the region of the interface between the third intracellular domain and the sixth transmembrane region. The probe will usually be of DNA and labelled in any suitable manner e.g. by labelling with an enzyme, radioisotope, fluorescent, luminescent, or chemiluminescent labels or biotinylation. It could also be of RNA.

The fragments are probed under any appropriate conventional hybridisation conditions, the fragments being conveniently first transferred to a filter. The complexes thus formed are detected by autoradiography or other detection means appropriate to the particular kind of label used.

Abnormalities in the polynucleotide sequence of restriction fragments of the DNA coding for TRHR which are as small as single-point mutations can also be detected by means of

Temperature Gradient Gel Electrophoresis in which a temperature gradient is superimposed, parallel to or transversely of, the electrical field in gel electrophoresis. The method is based on the fact that the temperature of denaturation of double stranded (ds) DNA is altered by changes in polynucleotide sequence. Furthermore, partial denaturation of a DNA duplex causes a change in electrophoretic mobility. Further details, of this technique are described in the literature by Reisner et al, 1989, Birmse et al, 1990, and Wartell et al.

The human cDNA cloned and sequenced is shown in Fig. 1. The nucleic acid and amino acid sequences shown in SEQ ID No. 1 herein are substantially identical to those disclosed in our earlier British Patent Application No. 9311854.5 from which priority has been claimed herein except for two nucleotides which had been misread but have now been corrected herein as follows: A at 728 now reads C resulting in Lys now reading as Thr in the polypeptide, and A at 897 now reads T resulting in Lys reading as Asn.

We have now further found that the chromosomal location of the TRH-R receptor gene is at 8q23 and thus the present invention now also provides further diagnostic means for use in the detection of conditions associated with TRH-R abnormalities, which comprises one or more of hybridisation of a polynucleotide probe of the invention with the 8q23 chromosome region; and examination of the 8q23 chromosome region for gross abnormalities.

Fig. 2 illustrates schematically the transmembrane, intracellular and extracellular domains of the receptor molecule. The transmembrane domains consist of seven stretches of hydrophobic in nature amino acids which span the membrane. The extracellular domain consists of four hydrophilic in nature amino acid stretches which exist exterior to the cells membrane. This region is believed to be important for the recognition of specific ligands. The intracellular domain consists of four hydrophilic stretches of amino acids which are thought to be involved in signal transduction.

It will be appreciated that abnormality in human TRH-R and/or its expression may be "assayed" in a number of ways. Thus the DNA encoding the TRH-R may itself be assayed for the presence or absence of abnormalities or the TRH-R polypeptide may be assayed for such purposes, where this is actually expressed.

The former case generally involves the use of labelled polynucleotide probes to hybridise with DNA within the region coding for human TRHR polypeptide for the purposes of indicating the presence of absence of particular polynucleotide sequences. In the latter case antibody probes are used to form antigen-antibody complexes with regions of the expressed TRH-R polypeptide for the purposes of indicating the presence or absence of particular polypeptide sequences.

It will be understood that once more or less common or typical TRH-R abnormalities have been specifically identified e.g. by initially probing with 'normal' polynucleotide and then sequencing, polynucleotide probes can be synthesized or otherwise produced with sequences corresponding to or complementary to the "abnormal" sequences, to allow screening of tissue samples for specific TRHR gene abnormalities.

In the case of assays of the TRHR polypeptide itself, suitable stretches of amino acids based on the cDNA sequence information provided by the present information, may be synthesised on a peptide synthesiser. These peptides would generally have a length of from 10 to 50, preferably 15 to 30, amino acids but could be even shorter or longer. Polyclonal antibodies to these peptides may be produced by conventional approaches such as the immunisation of host animals (rabbit, goat etc.) with said peptides and recovery of the desired antibody material therefrom. Monoclonal antibodies could also be raised using conventional monoclonal antibody production procedures.

It will also be understood that "assay" may be either qualitative or quantitative (e.g. where detection of under or over-expression of the receptor is required) .

Detailed Description

Isolation and Sequencing of Human TRH-R Gene Preparation of Human pituitary cDNA 5' stretch library Human pituitary 5' stretch libraries were obtained from Clontech Laoratories, Inc., 4030 Fabian Way, Palo Alto, CA 94303-4607, USA. 5' stretch libraries are prepared from mRNA which has been completely denatured by methyl mercuric hydroxide and have a higher representation of 5' sequences than do regular cDNA libraries. The library was constructed from poly A+ mRNA prepared from normal human, whole pituitary gland, taken 1.5 to 2.5 hours post-mortem from a pool of 9 Caucasians (5 females and 4 males) , aged 15, 34, 35, 43 53, 64, 70, 80 & 83. Cause of death certified as trauma. The cDNA was cloned into the Eco Rl site of the lambda gtlO vector.

Screening of the human pituitary cDNA 5' stretch library An aliquot containing human pituitary cDNA 5' stretch libraries (described above) was subjected to selective amplification by using TRH-R specific olignucleotide primers, produced on an Applied Biosystems 391 PCR-Mate DNA Synthesiser, which were 24 nucleotides in length and selected on the basis of conserved regions of the mouse TRH receptor sequences (Straub et al, 1990) and rat TRH receptor sequences (Sellar et al, 1990). The template was amplified using Taq polymerase (Promega Ltd, Southampton) in Hybaid Intelligent Heating Block and separated on a 1% preparative agarose gel. The PCR reaction product was sequenced, thereby providing provisional information covering approximately 300 nucleotides of the human

receptor. A 26-mer oligonucleotide was synthesised on the basis of this human sequence and used as a probe to screen the human pituitary cDNA 5' stretch library. The probe was labelled with 3 2P by random hexamer priming. One positive clone was isolated from approximately lxlO 6 plaques. A secondary screen with the same human 26-mer oligonucleotide TRH-R cDNA probe isolated several positive clones. Purification of DNA from plate lysate stocks of six. putative TRH-R clones were performed according to a protocol described by Maniatis et al (1985) . These clones were subsequently sequenced to establish the structure of the human TRH receptor. DNA Sequencing DNA sequencing was carried out using an Applied Biosystems 373A automated DNA sequencer and Taq dye-deoxy terminator and primer sequencing protocols. Oligonucleotide primers for sequencing were produced on an Applied Biosystems 391 PCR-Mate DNA synthesiser. Analysis was carried out on Apple Macintosh computers and analysed using the sequence processor program GeneJockey (Biosoft, Cambridge, U.K.). Sequencing of this clone indicated the largest open reading frame as being 1086 bp. The ATG initiation codon was identified by comparison with both the rat and mouse TRH-Rs with which the human receptor shares high sequence homology. The nucleotide and deduced amino acid sequences for the human receptor are shown in Fig. 1. The human TRH- R was shown to encode a 398 amino acid protein (Fig. 2) , compared to 393 for the mouse TRH-R (Straub et al. 1992); and 412 (Zhao et al., 1992; de la Pefia et al., 1992); 411

(Sellar et al. , 1993) and 387 (de la Pena et al. , 1993) for the rat TRH-R. Our human clone contained only 130 bp of 3' untranslated region (UTR) and approximately 1 kb of 5'UTR (not sequenced) . The poly (A) tail was not present. Comparison of the human/mouse/rat TRH-R amino acid sequences indicated high homology between all isoforms, except for the COOH terminal tail where the major variation occured (Fig. 3) . This region has already been shown to be functionally important, and has been implicated in receptor desensitisation of the -adrenergic receptor (Hausdorff et al., 1989). More recently, particular domains in the COOH terminal tail of the mouse TRH-R have been identified as affecting ligand/receptor internalisation (Nussenzveig et al., 1993) Expression of Human TRH-R gene

Subcloning of the human TRH-R cNDA. The coding region from -18 bp to 1238 bp of the receptor was amplified by polymerase chain reaction (PCR) . The following oligonucleotide primers were designed based on the sequence analysis of the human lambda gtlO clone:- 5'Ti (- 18>) 5'dAGCTTCAATCCACTGAAGATGG3'; and 3'Tp (<1238) 5 , dTTCTCAATTTCTTTGTCATCC3' . The amplified 1.2 kb PCR product was purified through CHROMA SPIN TE-100 spin columns (Clontech) and subcloned into the EcoRl site of the pcDNA-1 vector (Invitrogen) in both orientations. The reverse orientation was used as a control during expression studies. The pcDNA-1 subclone was then sequenced again to ensure that no base changes had occurred as a result of mis-priming during the PCR amplification. Any base

changes that resulted in amino acid changes could affect expression of the TRH-R protein in a human cell line. No such changes were discovered.

Transfection of COS-1 cells. Monolayer cultures of COS-1 cells (5xl0 5 cells/100mm dish) were transiently transfected overnight with the human TRH-R/pcDNA-1 subclone (3μg) using Lipofectin reagent (50μg, Gibco BRL, Paisley, UK) in serum-free Dulbecco's modified Eagles medium (DMEM) . Following transfection, calcium imaging studies were carried out as previously described (Anderson et al., 1992) on sterilized glass cover slips (22mm x 0.175mm, A.R. Horwell, West Hampstead, London, UK) . Functional studies and ligand binding assays were carried out 72 hours later. Receptor Binding. Transfected COS-1 cells were washed twice with phosphate buffered saline and harvested by scraping. The cells were then suspended in assay buffer (20mM TRIS.HC1, 2mM MgCl 2 , pH7.4). Membranes were prepared following homogenisation and centrifugation (20,000xg, 30 min. , 4 C) . The membrane pellet was resuspended in assay buffer. Ligand binding assays were carried out with 3 H- labelled [3-Me-His 2 ]TRH in assay buffer at a final volume of 0.5ml. Following incubation on ice for 60 min., the membranes were filtered through Whatman GF-C filters and washed 3 times with assay buffer. The membranes with bound labelled TRH were then incubated with unlabelled TRH (10" 6 M) which progressively exchanged with bound labelled TRH displacing the latter intfo the medium. Monitoring of the displaced labelled TRH and analysis of the vairation of the discplacement rate with time indicated the existence of a

single high affinity binding site with a Kd of 6.2nM.

Calcium Image Analysis. COS-1 cells, transiently transfected with the human TRH-R, were loaded with the fluorescent calcium dye fura-2 AM (4μM) . Intracellular calcium ([Ca 2 *]^ was subsequently measured in single cells using dual wavelength fluorescence microscopy combined with dynamic video imaging as previously described (Anderson et al., 1992). TRH (10" 6 M) also produced a rapid elevation of [Ca 2 *]^ in single COS-l cells expressing the human TRH-R. [Ca 2 *] 5 rose from a resting level of 27nM to a peak of 177nM with levels returning to control values approximately 1.6 min. after addition of TRH (Fig.6). In this experiment 2 cells out of a field of 10 single cells responded to TRH, owing to the random nature of the transfection process. The ability of TRH to cause both a rise in total IP production and [Ca 2 *]^ confirms that the human TRH-receptor behaves as a functional G-protein coupled receptor. Inoεitol phosphate (IP) production . Following transfection (24h) cells were trypsinized, transferred to 12-well plates and labelled with myo-[ 3 H]inositol (2μCi/ml for 48h) in inositol free DMEM medium. IP was then extracted and separated as previously described (Anderson et al., 1993). To test for receptor functionality, the human TRH-R was subcloned (in both orientations) into a eukaryotic expression vector driven by the CMV promotor. Binding studies on membranes prepared from COS-l cells transiently transfected with the human TRH-R subclone showed the existence of a single high affinity binding site with a K d of 6.2 nM (Fig. 4). COS-l cells expressing the

TRH-R in the correct orientation were exposed to TRH (10 " *M) . Total IP production increased approximately twofold. In either untransfected cells, or cells containing the receptor in the incorrect orientation (data not shown) , TRH was without effect. Results

Figure 1 shows the gene encoding the human TRH receptor. The polynucleotide sequences specifically elucidated thus far are indicated along with the deduced amino-acid sequence;

Figure 2 shows schematically the 7 transmembrane domains of the human TRH receptor and the 4 extracellular and 4 intracellular domains; and Figure 3 highlights amino acid differences at the COOH terminal tails of the TRH receptor in mouse, rat and human. Preparation and use of Polynucleotide Probes Chromosome preparation

Using standard cytogenetic methods, metaphase chromosome spreads were obtained from two human males of normal karyotype. Chromosome spreads in marked slide areas were banded, photographed and destained prior to hybridization as previously described (Garson et al., 1987; Boyd et al., 1989) . Preparation of Labelled Polynucleotide Probes Two human TRH-R cDNA probes were prepared and used. The first clone consists of a 2.3 kb insert in XgtlO vector and includes the entire 1.2 kb coding region of the gene, 132 bp of 3' untranslated region and approximately 1 kb of 5' untranslated region. The second clone consists of the TRH

receptor coding region, 18 bp of 5' untranslated region and 43 bp of 3' untranslated region inserted into the TA cloning vector pCRII (Duthie et al., 1993). The probes were labelled by nick translation with biotin-11-dUTP (Sigma) .

In-situ hybridization

The labelled probes were used at a concentration of 10-30 ng/ul for fluorescence in-situ hybridization according to the procedure described by Pinkel et al. (1986) . Modifications detailed by Carter et al. (1992) were employed but the pre-hybridization step was omitted for this single copy sequence probe. Hybridization was detected by incubation with avidin-fluroescein isothiocyanate (avidin-FITC, Vector Laboratories) and the signal amplified twice by two further incubations with biotinylated anti-avidin (Vector Laboratories) followed by avidin-FITC. Chromosomes were counterstained by mounting the slides in antifade AFl (Citifluor) containing 0.8 ug/ml 4,6-diaminidino-2-phenylindole (DAPI) and 0.4 ug/ml propidium iodide and examined using a Zeiss Axioplan fluorescence microscope. FITC and propidium iodide were excited at 490 nm (Zeiss filter combination 9) . Hybridization signals appear as yellow-green spots against the red propidium iodine counterstain. Previously photographed, DAPI-stained cells were relocated using Zeiss filter combination 1. Signals visualised on the post- hybridization metaphases using filter set 9 were marked on the photographs of pre-hybridization banded metaphases. The disribution of hybridization in chromosome spreads was

analysed using the chi-square test. Results

Forty-one metaphases were scored following hybridization to the lambda-incorporated sequence and the position of 73 signals recorded. Of these, a highly significant (p<0.005) 15 signals (20.5%) were located on chromosome 8 with 13 (17.8%) comprising a signal peak at 8q23. Forty-four metaphases were scored following hybridization to the plasmid-incorporated probe and the position of 186 hybridization signals recorded of these, a highly significant (p<0.005) 35 signals (18.8%) were located on chromosome 8 with 26 (14%) comprising a signal peak at 8q23. References Albert , M. , Jenike, M. , Nixon, R. , Nobel, K. (1993) Biological Pyschiatry 33, 267-271.

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Pelmear AH, Leversha MA, Glancy MT, Wood SL, Cook K, Dyson HM, Ferguson-Smith ME, Willatt LR (1992) Reverse chromosome painting: method for the rapid analysis of aberrant chromosomes in clinical cytogenetics. J Med Genet 29:229-

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