Molecular cloning of a human thyrotropin receptor cDNA fragment. Use of highly degenerate, inosine containing primers derived from aligned amino acid sequences of a homologous family of glycoprotein hormone receptors

Autoantibodies to the thyrotropin (TSH) hormone receptor (TSH-R) are present in the sera of patients with thyroid autoimmune disease which are pathogenetic leading to hyperthyroidism of Graves' disease. Considerable interest has been focused on the cloning of the human TSH-R, which has until very recently, proven exceedingly difficult due to the very low receptor level expression on thyroid cells. We have used polymerase chain reaction and highly degenerate, inosine containing oligonucleotides derived from sequence alignments of the transmembrane regions 2 and 7 of a number of G-binding protein receptors including the lutropin/choriogonadotropin (LH/CG) receptors to amplify various cDNAs from human thyroid cDNA. Sequencing analysis of 27 different clones revealed that they fall into eight different groups. The very recent publication of the complete nucleotide sequence of the human TSH-R revealed that one of the groups (GT1) containing seven clones which had been sequenced belong to the human TSH-receptor. The sequence of all 7 GT1 clones was identical and in complete concordance with transmembrane regions 2 and 7 of the published TSH-R sequence. Our results show that by designing oligonucleotides to common transmembrane regions of G-binding proteins where the primers are biased in their sequence to the LH/CG receptors it is possible to amplify the TSH-R receptor sequence.     

Biochemical characterization and modulation of LH/CG-receptor during human trophoblast differentiation

Due to the key role of the human chorionic gonadotropin hormone (hCG) in placental development, the aim of this study was to characterize the human trophoblastic luteinizing hormone/chorionic gonadotropin receptor (LH/CG-R) and to investigate its expression using the in vitro model of human cytotrophoblast differentiation into syncytiotrophoblast. We confirmed by in situ immunochemistry and in cultured cells, that LH/CG-R is expressed in both villous cytotrophoblasts and syncytiotrophoblasts. However, LH/CG-R expression decreased during trophoblast fusion and differentiation, while the expression of hCG and hPL (specific markers of syncytiotrophoblast formation) increased. A decrease in LH/CG-R mRNA during trophoblast differentiation was observed by means of semi-quantitative RT-PCR with two sets of primers. A corresponding decrease ( approximately 60%) in LH/CG-R protein content was shown by Western-blot and immunoprecipitation experiments. The amount of the mature form of LH/CG-R, detected as a 90-kDa band specifically binding (125)I-hCG, was lower in syncytiotrophoblasts than in cytotrophoblasts. This was confirmed by Scatchard analysis of binding data on cultured cells. Maximum binding at the cell surface decreased from 3,511 to about 929 molecules/seeded cells with a kDa of 0.4-0.5 nM. Moreover, on stimulation by recombinant hCG, the syncytiotrophoblast produced less cyclic AMP than cytotrophoblasts, indicating that LH/CG-R expression is regulated during human villous trophoblast differentiation.     

The testicular receptor for follicle stimulating hormone: structure and functional expression of cloned cDNA

Cloned cDNA encoding the rat Sertoli cell receptor for FSH was isolated from a cognate library and functionally expressed in cultured mammalian cells. The FSH receptor (FSH-R), as predicted from the cDNA, is a single 75K polypeptide with a 348 residue extracellular domain which contains three N-linked glycosylation sites. This domain is connected to a structure containing seven putative transmembrane segments which displays sequence similarity to G protein-coupled receptors. Thus, the FSH-R is identical in its structural design to the LH/CG receptor (LH/CG-R). Furthermore, both receptors display 50% sequence similarity in their large extracellular domains and 80% identity across the seven transmembrane segments. Expression of the cloned cDNA in mammalian cells conferred FSH-dependent cAMP accumulation. The selectivity for FSH is attested by the fact that the related human glycoprotein hormones human CG and human TSH do not stimulate adenylyl cyclase in FSH-R expressing cells even when these hormones are present at high concentrations.     

The mRNA levels of thyrotropin receptor, thyroglobulin and thyroid peroxidase in neoplastic human thyroid tissues

Expression levels of thyrotropin receptor (TSH-R), thyroglobulin (Tg) and thyroid peroxidase (TPO) mRNA in normal and neoplastic human thyroid tissues (6 adenomas and 7 carcinomas) were investigated by Northern-blot and slot-blot analyses. We found that TSH-R mRNA levels were significantly lower in carcinoma tissues than in normal tissues. The levels of Tg mRNA were also significantly lowered in adenoma and carcinoma tissues as compared to normal tissues. In contrast, no significant difference was observed in the expression levels of TPO mRNA between these tissues. Furthermore, TSH-R mRNA levels were well-correlated with Tg mRNA levels in neoplastic tissues. These results suggest that mRNAs of TSH-R and Tg are expressed in relation to their degree of differentiation.     

Thyrotropin receptor non-mediated thyroid stimulating immunoglobulin in Graves' disease.

There exists a consensus that hyperthyroid Graves' disease is caused by thyrotropin receptor (TSH-R) autoantibodies. To test the possibility that the TSH-R is the sole antigen for thyroid stimulating antibodies (TSAb), we compared bioactivities of Graves' IgGs between non-thyroid mammalian cells transfected with human TSH-R cDNA and the reference thyroid bioassay. A Graves' IgG with TSH-binding inhibitor immunoglobulin (TBII) activity (89%) markedly stimulated cAMP formation in both CHO-K1 cells transfected with TSH-R cDNA (340 microU/ml of TSH equivalent) and rat thyroid cells, FRTL-5, (410 microU/ml of TSH equivalent). In contrast, a TBII negative (-1.5%) IgG from another patient with Graves' disease showed a strong thyroid stimulating activity (87 microU/ml of TSH equivalent) when FRTL-5 cells were used for the assay. But no stimulating activity was observed in this IgG when CHO-K1 cells transfected with TSH-R cDNA were used, suggesting a possible existence of TSH-R non-mediated thyroid stimulating immunoglobulin in some cases of Graves' disease.     

A cloned chromogranin A (CgA) cDNA detects a 2.3Kb mRNA in diverse neuroendocrine tissues

CgA is a 72Kd protein of unknown function that is present in many neuroendocrine tissues and co-secreted with their resident hormones. We prepared a cDNA library to the mRNA from CgA-producing human medullary thyroid carcinoma (MTC) cells in the expression vector lambda gt11. The library was screened with a panel of one polyclonal and two monoclonal antibodies to CgA. The specificity of the antibodies for CgA was demonstrated by immunoassay, immunohistology, and immunoprecipitation of the in vitro translation products of mRNA from CgA-producing tissues. A chromogenic second antibody identified five immunoreactive clones. Their cDNA inserts were isolated after EcoRI digestion and agarose gel electrophoresis. These cDNAs were 32P-labelled and used as probes in Northern hybridization studies. An mRNA of approximately 2.3Kb was detected with the cDNA probes in human cell lines from MTC and lung cancers that were shown to produce CgA and in human pheochromocytoma and bovine adrenal medulla tissue. To confirm its identity, one of the putative CgA cDNAs was subcloned into a plasmid and was used to hybridization-arrest the in vitro translation of CgA mRNA. These studies demonstrate the cloning of cDNAs which hybridize with CgA mRNA from diverse neuroendocrine tissues.     

Cloning, sequencing and expression of human TSH receptor

Complementary cDNA clones encoding the TSH (thyroid stimulatory hormone) receptor were isolated from a human thyroid lambda gt10 library using Iow stringency hybridization with LH/hCG (luteinizing hormone-human choriogonadotropic hormone) receptor probes. Sequencing of the clones showed a 764 amino acid open reading frame. The first 21 amino acids probably correspond to a signal peptide, the mature protein thus contains 743 amino acids (calculated molecular weight: 84,501 daltons). Its putative structure consists of a 394 amino acid extracellular domain, a 266 amino acid membrane spanning domain with 7 putative transmembrane segments and a 83 amino acid intracellular domain. A high degree of homology is observed with LH/hCG receptor suggesting the definition of a new subfamily of G-protein coupled receptors. Computer search showed the presence in the putative third intracellular loop of a motif resembling that described in the non receptor type protein tyrosine kinases (c-src, c-yes, c-fgr, etc...). RNA blots showed that the receptor messenger RNA consists of two major species of 4300 and 3900 nucleotides. The cDNA was inserted into an expression vector and after transfection into COS 7 cells it was shown to produce a functional TSH receptor.     

Characterization of a third human thyroid hormone receptor coexpressed with other thyroid hormone receptors in several tissues

A cDNA that encodes a third type of human thyroid hormone receptor (hTR alpha 1) has been isolated from a skeletal muscle library. The cDNA encodes a 410 amino acid protein, Mr = 46,820. When expressed and translated in vitro, hTR alpha 1 binds T3 with an association constant (ka) of 1.8 x 10(9) M-1. Comparison of the DNA sequence of hTR alpha 1 and a previously identified alpha type thyroid hormone receptor (hTR alpha 2) suggests that they could be transcribed from the same gene, and that alternative RNA splicing results in the synthesis of either hTR alpha 1 or hTR alpha 2. Two mRNA (3.2 kilobases and 6 kilobases) of hTR alpha 1 have been detected in several tissues. At least three types of thyroid hormone receptors (hTR alpha 1, alpha 2, beta), which possess similar affinities for hormone ligands, can be expressed in the same tissue.     

Hormonal regulation of rat androgen-binding protein (ABP) messenger ribonucleic acid and homology of human testosterone-estradiol-binding globulin and ABP complementary deoxyribonucleic acids

Complementary DNA clones coding for rat androgen-binding protein (rABP) were isolated from a rat testis cDNA library constructed in the bacteriophage lambda gt11. The library was screened immunochemically, using two different antibodies against rABP. The identity of the isolated clones was confirmed by epitope selection and DNA sequence analysis. The mRNA encoding rABP could be detected in the testes of 20- and 46-day-old-rats, but not in the 10-day-old rats by hybridization with 32P-labeled rABP cDNA in a Northern blot of poly(A)+-RNA fractioned by agarose gel electrophoresis. No hybridization signal was seen with poly(A)+-RNA isolated from kidney and liver. The rABP mRNA appeared as a single species with a size of 1.65 kilobase, sufficient to encode a protein of 42,000 daltons. The concentration of rABP mRNA in the testes of 37-day-old hypophysectomized rats increased after treatment with testosterone and FSH, given alone or in combination. Sequence and hybridization analysis of cDNAs for rABP, human testosterone-estradiol-binding globulin, and human ABP demonstrates that the cDNAs for human testosterone-estradiol binding globulin and human ABP have greater sequence similarity with each other than either has with rABP.     

Development of serum-free bioreactor production of recombinant human thyroid stimulating hormone receptor

For the detection of autoantibodies to thyroid stimulating hormone receptors (TSH-R) in Graves' disease based on a novel coated tube assay system, human TSH-R is needed in large amounts. Whereas expression of TSH-R in bacteria, yeast, or insect cells results in nonfunctional, denaturated receptor, mammalian cells such as COS, CHO, and HeLa are able to express functional TSH-R, but only in very low amounts. Furthermore, for all of these cultivations expensive standard media containing 10% fetal calf serum are needed to obtain functional receptor. Here we report on the development of a serum-free production-scale process based on a stable transformed and highly productive human leukemia cell line K562 (1). Starting with K562-TSH-R cells growing in medium containing 10% fetal calf serum the cell line was adapted to serum-free medium. The adaptation medium was optimized in regards to amino acid and protein concentrations, since the use of unadjusted medium caused cell death after 2 days. The adapted cells were stable and could be cultivated without antibiotics for more than 50 cell doublings without losing their productivity. The obtained receptor showed improved TSH binding. The process development was based on cultivations in a 2-L bench-scale bioreactor. Cultivations in batch mode and chemostat mode and perfusion cultivation with the usage of an internal microfiltration device and a spin-filter device were compared. After process optimization a continuous process using spin-filter was set up and run in a 20 L-pilot-scale bioreactor. The presented results were the prerequisite for the production of the novel assay for the diagnosis of autoantibodies to TSH-R in Graves' disease.     

Assignment of chromosomal locus and evidence for alternatively spliced mRNAs of a human sperm membrane protein (hSMP-1).

The gene (HSD-1) coding a human sperm membrane protein (hSMP-1) was isolated from a human testis cDNA expression library using antibodies found in the serum of an infertile woman. HSD-1 was localized to a single locus on chromosome 9 and assigned to band 9p12-p13 by fluorescent in situ hybridization (FISH) mapping and DAPI (4,6-diamidino-2-phenylindole) banding, using rat/human somatic cell hybrids and metaphase chromosomes of human lymphocytes. In rescreening a testis lambdagt10 cDNA expression library, the full-length cDNA (HSD-1) and several truncated cDNAs with heterologous regions were isolated from positive clones. The heterology consisted of deletion, insertion and alteration of the 5'-end. These heterologous truncated fragments may be produced by alternative splicing of mRNAs. Two recombinant prokaryotic expression vectors were constructed with one of the heterologous fragment (clone #26) with and without the alternative 5'-end. Escherichia coli transfected with the construct containing the alternative 5'-end failed to produce the recombinant product, whereas those transfected with the vector lacking the 5'-end produced hSMP-1. DNASIS analysis of the structure of #26 mRNA suggests that the 5'-end has a stable secondary configuration that may maintain the mRNA in an inactivated state, whereby hindering its translation and preventing the expression of the gene.     

Molecular cloning of hepatic mRNAs in Rana catesbeiana responsive to thyroid hormone during induced and spontaneous metamorphosis

Amphibian metamorphosis affords a useful experimental system in which to study thyroid hormone regulation of gene expression during postembryonic vertebrate development. In order to isolate gene-specific cDNA probes which correspond to thyroid hormone-responsive mRNAs, we employed differential colony hybridization of a cDNA library constructed from poly(A)+ RNA of thyroxine-treated premetamorphic tadpole liver. From an initial screening of about 6000 transformants, 32 "potentially positive" colonies were obtained. The recombinant cDNA-plasmids from 13 of these colonies plus two "potentially negative" colonies were purified for further study. Southern blot analysis of the plasmid DNA was employed to determine whether different cDNAs encoded for the same mRNA. The effect of thyroid hormone on the relative levels of specific mRNA species was examined by Northern analysis of liver RNA from premetamorphic tadpoles, thyroxine-treated tadpoles, and adult bullfrogs. Three independent cDNA clones were obtained which encoded thyroid hormone-enhanced mRNAs. We also obtained two independent cDNA clones encoding thyroid hormone-inhibited mRNAs and three independent clones encoding thyroid hormone-unresponsive mRNAs. The levels of two thyroid hormone-enhanced mRNAs and one thyroid hormone-inhibited mRNA were essentially the same in the thyroid hormone-treated tadpole liver and adult liver, suggesting that thyroid hormone induces stable changes in liver gene expression during spontaneous metamorphosis. Using selected cDNAs, RNA dot blot analysis of liver mRNA from tadpoles at different stages of metamorphosis showed that the level of one thyroid hormone-enhanced mRNA increased during late prometamorphosis and metamorphic climax. Similarly, a mRNA which was strongly inhibited by thyroid hormone treatment was observed to decline during prometamorphosis and reach undetectable levels during metamorphic climax. One mRNA was detected which was reproducibly inhibited by thyroid hormone treatment but which remained essentially unchanged during spontaneous metamorphosis. These results provide the first direct evidence for the coordinate and selective pretranslational regulation by thyroid hormone of several liver genes during the developmental process of metamorphosis.     

The chromosomal gene structure and two mRNAs for human granulocyte colony-stimulating factor.

Two different cDNAs for human granulocyte colony-stimulating factor (G-CSF) were isolated from a cDNA library constructed with mRNA prepared from human squamous carcinoma cells, which produce G-CSF constitutively. The nucleotide sequence analysis of both cDNAs indicated that two polypeptides coded by these cDNAs are different at one position where three amino acids are deleted/inserted. When the two cDNAs were introduced into monkey COS cells under the SV40 early promoter, both of them produced proteins having authentic G-CSF activity and some difference in the specific activity was suggested. A human gene library was then screened with the G-CSF cDNA and the DNA fragment containing the G-CSF chromosomal gene was characterized by the nucleotide sequence analysis. The human G-CSF gene is interrupted by four introns and a comparison of the structures of the two G-CSF cDNAs with that of the chromosomal gene indicated that the two mRNAs are generated by alternative use of two 5' splice donor sequences in the second intron of the G-CSF gene. When the G-CSF chromosomal gene was expressed in monkey COS cells by using the SV40 enhancer two mRNAs were detected by S1 mapping analysis.