©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Functional Expression of a Thyrotropin Receptor cDNA from Rat Fat Cells (*)

Toyoshi Endo , Kazuyasu Ohta , Kazutaka Haraguchi , Toshimasa Onaya (§)

From the (1) Third Department of Internal Medicine, University of Yamanashi Medical School, Tamaho, Yamanashi 409-38, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Thyrotropin receptor (TSH-R) has been thought to be thyroid-specific, but, by Northern blot analysis, we found that rat adipose tissue expressed TSH-R mRNAs in amounts approaching those in the thyroid. To investigate the function of TSH-R from adipose tissue, we screened a rat fat cell gt11 cDNA library for TSH-R sequences using a P-labeled rat thyroid TSH-R cDNA as a probe. Among 10 plaques, we obtained four positive clones. Sequencing of these cDNAs has revealed that two of them (F and F) contained both initiation and termination codons. Comparison of F with the thyroid TSH-R cDNA sequence revealed that F was almost identical to the thyroid TSH-R, except that nucleotides 1041 and 1277 were changed from A to G and from C to T, respectively. In contrast, we found that F contained 21 novel nucleotides between nucleotides 467 and 468 of the thyroid TSH-R cDNA, encoding an additional 7 amino acids. However, when we prepared mRNA from adipose tissue and transcribed it into cDNA, we failed to amplify the F type of TSH-R cDNA by polymerase chain reaction, suggesting that F mRNAs are rare in the tissue. We then ligated F cDNAs into pSG5 and transfected them with pSV-neo into Chinese hamster ovary (CHO)-K1 cells. TSH stimulated cAMP formation in CHO-F cells in a manner similar to that in CHO cells transfected with thyroid TSH-R cDNA. In contrast, no increase of cAMP was observed in CHO-F cells. IgG from patients with Graves' disease ( n = 4) showed thyroid-stimulating antibody activity only in CHO-F cells (1288-4582%). In addition, CHO-F cells and CHO cells transfected with thyroid TSH-R showed similar I-TSH binding activity. These results indicate that the fat cell expresses high levels of a TSH-R whose function is indistinguishable from that in the thyroid and suggest that the TSH-R autoantibody plays an important role in the pathogenesis of the extrathyroidal manifestations of Graves' disease.


INTRODUCTION

Thyrotropin receptor (TSH-R)() , the main autoimmune antigen in Graves' disease (1) , is a key molecule in the regulation of thyroid functions including hormone secretion and cell growth (2) . The cloning of a TSH-R encoding cDNA (3) has revealed that TSH-R is highly homologous to the luteinizing hormone (LH)/chorionic gonadotropin (CG) receptor (4) . Both receptors have a large extracellular domain and have been classified as a new subtype of the G protein-coupled receptor family (5) .

Both TSH-R and LH/CG receptor mRNA are expressed in the thyroid. Frazier et al.(6) have successfully cloned a LH/CG receptor cDNA from a human thyroid gt11 cDNA library and determined its nucleotide sequence. However, they found that the LH/CG receptor mRNA expressed in thyroid was an incompletely spliced form and suggested that tissue-specific splicing may be an important step in the regulation of the glycoprotein hormone receptor family.

On the other hand, the presence of TSH-R in non-thyroid tissues has been controversial. TSH-R has long been considered thyroid-specific (7) . However, high affinity TSH binding activity has been reported in guinea pig adipocytes (8) and human lymphocytes (9) . Recently, we (10) and other groups (11, 12, 13) obtained TSH-R cDNA fragments from rat or human retro-orbital tissue, adipose tissue, and fibroblasts using polymerase chain reaction (PCR). Partial sequencing of cDNA fragments revealed that the cDNA was that of the TSH-R. However, PCR methods are so sensitive that a target cDNA can be amplified from a very small amount of mRNA. In addition, as in the case of the LH/CG receptor mRNA in the thyroid, it was possible that TSH-R mRNA from non-thyroid origins was an incompletely spliced form.

TSH-R expression and function in non-thyroid tissues may be particularly important in the pathogenesis of extrathyroidal manifestations of Graves' disease such as ophthalmopathy and dermopathy (12) . In the present study, in order to resolve the issue, we have isolated full-length TSH-R cDNAs from a rat fat cell gt11 cDNA library.


MATERIALS AND METHODS

Northern Blot Analysis and PCR

Total RNAs from rat thyroid gland, epididymal adipose tissue, and liver were prepared by guanidine thiocyanate extraction and CsCl centrifugation (14) . 10 µg of RNA/lane were electrophoresed in agarose and transferred to cellulose acetate membrane (Zeta probe, Bio-Rad). A rat thyroid TSH-R cDNA (2.8 kilobase), kindly donated by Dr. L. D. Kohn (NIH, Bethesda, MD), was labeled with [-P]dCTP using a random primer labeling kit (Takara Shuzo Co., Tokyo, Japan). Blots were prehybridized overnight at 42 °C in 50% formamide, 10 Denhardt's solution (0.2% Ficoll, 0.2% polyvinylpyrrolidone, and 0.2% bovine serum albumin), 5 SSPE (20 SSPE is 3 M NaCl, 0.2 M sodium phosphate, and 20 mM EDTA, pH 7.4), 0.1% SDS, 0.1 mg/ml heat-denatured salmon sperm DNA, and 1.0 µg/ml poly(A). Hybridization was performed at 42 °C for 12 h with radiolabeled probe in fresh hybridization solution, as described above. Filters were washed three times at room temperature in 2 SSC (20 SSC is 3 M NaCl and 0.3 M sodium acetate, pH 7.0) containing 0.1% SDS. Stringent washing was performed three times for 30 min each time at 55 °C in 0.1 SSC containing 0.1% SDS. The filters were exposed for 2 h to an imaging plate and analyzed with a Bas 2000 image analyzer (Fuji Film Co., Tokyo, Japan).

For PCR analysis, RNA was transcribed into cDNA by avian reverse transcriptase (Takara) and then used as a template. Primers used here to amplify TSH-R cDNA fragments were as follows (nucleotide positions are according to Akamizu et al.(15) ): Oligo A, 5`-TTCTTTATACTAGAAATCACA (residues 457-477; sense strand); Oligo B, 5`-TTGTCATCGTTTCTCTTGCAGA (sequence of the unidentified insertion); Oligo C, 5`-GTTCTTCGCGATCAGCTCTTT (residues 748-768; antisense strand); Oligo D, 5`-TCCATAGATGCCACTCTGCAG (residues 250-270; sense strand); and Oligo E, 5`-AGGTAAACAGCATCCAGCTT (residues 601-620; antisense strand). PCR reactions contained 10 ng of template cDNA, 1.5 units of AmpliTaq DNA polymerase (Perkin-Elmer), and 125 ng of each primer in buffer containing 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl, 50 mM KCl, and 0.1% (w/v) gelatin in a 50-µl volume. PCR was performed under mineral oil for 30 cycles using a TP cycler 100 (TOYOBO Engineering, Tokyo, Japan) as follows: 1.0 min at 92 °C, 2 min at 53 °C, and 2 min at 72 °C with 30 s of ramping time. When needed, the PCR products were ligated into pCRII vector (Invitrogen, San Diego, CA) and then their nucleotide sequence was determined.

Cloning of the TSH-R from Rat Fat Cells

A rat fat cell gt11 cDNA library (RL1035b) constructed from 4-week-old Sprague-Dawley testicular fat cells that had been separated from other types of cells was obtained from Clontech Laboratories, Inc. (Palo Alto, CA). The library was screened with P-labeled rat thyroid TSH-R cDNA. After three rounds of screening, inserts of the positive clones were cut with EcoRI and ligated into pSG5 (Stratagene, La Jolla, CA). The nucleotide sequence of single-stranded DNA from pSG5 was determined by deoxynucleotide methods (16) using 20-mer synthetic oligonucleotides corresponding to rat thyroid TSH-R cDNA or pSG5 as primers.

Expression of Rat Fat Cell TSH-R

Rat fat cell TSH-R cDNA ligated into pSG5 in the correct orientation was transfected into CHO-K1 cells (1 µg/10 cells) using Lipofectin reagent (Life Technologies, Inc.). Cotransfection with pSV-neo (17) was performed with resistance to G418 used as a selectable marker for transfection. The mixed transfected CHO-K1 cells were further cloned by limited dilution, and their responsiveness to TSH and expression of TSH-R mRNA were determined.

Analysis of TSH-R Functions

cAMP response of TSH-R-expressing cells to TSH (bovine TSH, Sigma) or IgG from the patients with Graves' disease was assayed as described previously (18) using a commercially available radioimmunoassay kit (Yamasa Shoyu Co., Tokyo, Japan). TSH binding by the cells was studied essentially by the methods of Chazenbalk et al.(19) as previously reported (20) .

Preparation of Isolated Fat Cells

Isolated fat cells were prepared from epididymal adipose tissue essentially by the methods of Honnor et al.(21) . Epididymal fat pads were obtained from Sprague-Dawley rats killed by decapitation. The major blood vessel was removed with forceps, and the pads were minced with a chopper. Digestion was performed with 1 g of minced pad in 3 ml of 1 mg/ml collagenase (Wako Chemical Co., Tokyo, Japan) for 30 min at 37 °C in Krebs-Ringer solution buffered with 25 mM Hepes at pH 7.4 (KRH) containing 1% defatted bovine serum albumin (BSA) and 400 nM adenosine. Cells were filtered over nylon mesh. After 3 cycles of brief centrifugation at 200 g, aspiration of infranatant, and resuspension in 1% BSA in KRH, the cells were suspended in KRH containing 4% BSA. The fractional occupation of the suspension by fat cells was determined by the microhematocrit centrifugation procedure (22) .

Binding ofI-TSH to Isolated Fat Cell Membrane

Isolated fat cells were homogenized in 10 volumes of 10 mM Tris-HCl, pH 7.5. After centrifugation at 800 g for 10 min at 4 °C, the supernatant was centrifuged at 15,000 g for 15 min. The pellet was washed three times with 50 mM NaCl and 10 mM Tris-HCl, pH 7.5, containing 1 mg/ml BSA (23) and then solubilized with the buffer containing 1% Lubrol. TSH binding to the solubilized membrane was assayed according to the methods of Smith and Hall (24) . As a control, we used membrane from FRTL-5 cells (rat thyroid epithelial cells, American Type Culture Collection, Rockville, MD; no. CRL8305) cultured in the presence of TSH and BRL-3A cells (rat liver cells, American Type Culture Collection; no. CRL1442). Effect of TSH on Lipolysis in Isolated Fat Cells-Glycerol production in isolated fat cells served as the measure of lipolysis and was assayed according to Honnor et al.(21) as follows. A 100-µl aliquot of the adipocyte suspension was added to 700 µl of KRH containing 4% BSA and 1 unit/ml adenosine deaminase plus TSH or isoproterenol as indicated. The incubations were performed in polypropylene tubes for 45 min at 37 °C, terminated by the addition of 200 µl of 50 mM EDTA buffered with Tris at pH 8.0. After homogenization of the cells, the homogenate was centrifuged at 10,000 g for 15 min. The amounts of glycerol in the infranatant were measured with the glycerol F-kit (Boehringer Mannheim), and the results were expressed as the amounts/unit volume of packed fat cells.


RESULTS

TSH-R mRNA in the Rat Adipose Tissue

Recently, we and others have demonstrated the existence of TSH-R mRNA in non-thyroid tissues by PCR (10, 11, 12, 13) , but the amounts and size of the mRNA in these tissues remained unknown. Fig. 1shows the result of Northern blot analysis of TSH-R mRNA in rat thyroid gland and in non-thyroid tissues. As reported by Akamizu et al.(15) , two species of TSH-R mRNA, containing 5.6 and 3.3 kilobase pairs, were observed in the thyroid (Fig. 1, lane 1). We could detect the same size of mRNAs in the epididymal adipose tissue (Fig. 1, lane 2) in amounts that were, unexpectedly, comparable with those in the thyroid. However, no bands were detected in the liver RNA (Fig. 1, lane 3).


Figure 1: Expression of TSH-R mRNA in various rat tissues. Northern blot analysis of RNA (10 µg/lane) isolated from rat thyroid ( lane 1), epididymal adipose tissue ( lane 2), and liver ( lane 3) was performed with P-labeled rat thyroid TSH-R cDNA. kb, kilobase.



Cloning of Rat Fat Cell TSH-R cDNA

Four positive clones (termed here TSH-R F, F, F, and F) were isolated by screening a rat fat cell gt11 cDNA library (10 plaques). Nucleotide sequencing revealed that F and F were 2.3-kilobase pair cDNAs, both of which lacked the translation initiation codon and started from nucleotide 1407 of the thyroid TSH-R cDNA (Fig. 2 A). In contrast, the inserts of both F and F were about 3.0 kilobase pairs; the former corresponded to nucleotides -54-2903 and the latter to nucleotides -54-2946 of the published thyroid receptor cDNA (15) .


Figure 2: Cloning and sequencing of TSH-R cDNA from rat fat cell. A, schematic representation of TSH-R cDNA clones from a rat fat cell library. We isolated four positive clones for TSH-R (designated F, F, F, and F) from a rat fat cell gt11 cDNA library. The direction and extent of the individual clones are represented by thin bars. The thick bar represents a rat TSH-R cDNA from the thyroid, and nucleotide numbers are according to Akamizu et al. (15). B, the differences between thyroid and fat cell TSH-R cDNAs. Amino acids encoded are designated by single letters.



To further compare F and F with thyroid receptor cDNA, we determined the complete nucleotide sequence of F and F cDNA. Comparison of F with thyroid TSH-R cDNA sequence revealed that the F cDNA was almost identical to that from thyroid except that nucleotide 1041 was G instead of A and nucleotide 1277 was T instead of C. The first substitution resulted in no amino acid change, but the other substitution changed the 426th amino acid, proline, to leucine (Fig. 2 B). We found the same changes at nucleotides 1041 and 1277 in F cDNA. In addition, however, F contained a 21-nucleotide insertion between nucleotides 467 and 468 of the thyroid receptor cDNA. Thus, the sequence encodes seven additional amino acids (cysteine, histidine, arginine, phenylalanine, serine, cysteine, and arginine) between leucine at position 156 and glutamic acid at position 157 of the thyroid receptor (Fig. 2 B).

Expression of F and F TSH-R mRNA in the Thyroid and the Epididymal Adipose Tissue

In order to study the distribution of F and F mRNA in thyroid and adipose tissue, we performed reverse transcription-PCR using the oligonucleotide corresponding to the unidentified inserted portion of F cDNA (Oligo B) or to the wild type sequence (Oligo A). When Oligo A and Oligo C (270 bp downstream from the insertion) were used as primers (Fig. 3 A), we successfully obtained the predicted size of major bands (311 bp) from both the thyroid and the adipose tissue (Fig. 3 B), whereas no products were amplified from both tissues when Oligo B and Oligo C were employed as primers.


Figure 3: Detection of F and F TSH-R mRNAs by PCR. A, schematic representation of the oligonucleotides ( A-E) used as primers for amplifying F and F TSH-R cDNAs from the thyroid or the adipose tissue. The dotted segment represents the 21 inserted nucleotides found in F TSH-R cDNA. B, results of reverse transcription-PCR. The mRNAs from thyroid ( lanes 1) and epididymal adipose tissue ( lanes 2) were reverse transcribed into cDNAs and used as a template. The gel shows the PCR products when oligos A and C ( a), oligos B and C ( b), or oligos D and E ( c) were used as primers. Molecular size markers were as follows: 2000, 1500, 1000, 700, 500, 400, 300, 200 and 100 bp.



Next, using single-stranded cDNAs from the thyroid or the adipose tissue as a template, we also carried out reverse transcription-PCR with Oligo D and Oligo E. A 371-bp cDNA was amplified from both tissues. Then the cDNA from the adipose tissue was subcloned into pCRII vector. Nucleotide sequencing of 10 clones has revealed that all clones lacked the insert portion recognized in F cDNA (data not shown), suggesting that a very small amount of the F type of mRNA was expressed in the epididymal adipose tissue.

Transfection of F and F cDNAs into CHO-K1 Cells and Their Responsiveness to TSH or Thyroid-stimulating Antibody

F or F cDNA that were ligated into the EcoRI site of pSG5 in the correct orientation were transfected into CHO-K1 cells (CHO-F or CHO-F cells). After confirming expression of their mRNAs by Northern analysis, we studied their responsiveness to TSH and compared it with that of CHO-K1 cells expressing rat thyroid TSH-R. TSH stimulated cAMP formation in CHO-F cells with a profile similar to that in CHO-K1 cells expressing rat thyroid TSH-R (Fig. 4 A). In contrast, no increase of cAMP levels was observed in CHO-F cells incubated with TSH. Fig. 4 B shows the I-TSH binding activity of these cells. CHO-F cells and CHO-K1 cells expressing rat thyroid TSH-R possessed similar TSH binding activity ( K= 0.25 10 M), but CHO-F cells lacked this activity. Finally, we studied the reactivity of IgGs from patients with Graves' disease to CHO-F or CHO-F cells. All Graves' IgGs tested showed thyroid-stimulating antibody activity (1288-4582%) only in CHO-F cells (Fig. 4 C).


Figure 4: Receptor functions of the fat cell TSH-R. A, cAMP responsiveness to TSH. TSH was added to CHO-F cells (), CHO-F cells (), and CHO-thyroid TSH-R cells () at indicated concentrations, and the increase of cellular cAMP in these cells was measured (18). The data are the means of duplicate assays. The value obtained at 1.0 milliunit/ml TSH was taken as 100%. B, I-TSH binding to CHO-F cells (), CHO-F cells (), and CHO-thyroid TSH-R cells (). The values are the means of triplicate assays. C, thyroid-stimulating antibody ( TSAb) activity of IgG from patients with Graves' disease measured using CHO-F cells ( shaded bars) and CHO-F cells ( open bars). Thyroid-stimulating antibody activity was calculated (18) as follows: the percentage of thyroid-stimulating antibody activity = (cAMP increase in the presence of test IgG/cAMP increase in the presence of normal control IgG) 100.



TSH Binding to Isolated Fat Cell Membrane and Lipolytic Activity of TSH

To confirm the existence of TSH-R in rat fat cells, we have further studied the binding activity of TSH to the membrane prepared from isolated fat cells and the effect of TSH on lipolysis in these cells.

As shown in Fig. 5 A, I-TSH specifically binds to the fat cell membrane, as well as to FRTL-5 cell, but not to the membrane prepared from BRL-3A cells (rat liver cells). Small doses of unlabeled TSH suppress this binding, and fat cell membrane shows similar TSH binding characteristics to FRTL-5 cell membrane. The binding activity from unit protein of fat cell membrane is about 48% of that from FRTL-5 cell membrane.


Figure 5: TSH binding and the effect of TSH on lipolysis in isolated fat cells. A, binding of I-TSH to the membrane from isolated rat fat cells (). Results are expressed as activities/unit of fat cell volume. Equivalent amounts of membranes from FRTL-5 cells (cultured rat thyroid epithelial cells) () and BRL-3A cell (rat liver cells) () were used as a control (1 unit of fat cell volume = 1.5 mg of membrane protein). The values are the means of duplicate assays. B, effect of TSH on lypolysis in isolated fat cells. Isolated fat cells from epididymal fat pads were incubated with TSH as indicated for 45 min, and the amounts of glycerol were determined. The level of glycerol in the cells stimulated by 10 µM isoproterenol is shown (). The values are the means of triplicate assays.



Fig. 5B shows the lipolytic activity of TSH. TSH stimulated glycerol production in isolated fat cells dose dependently. The amount of glycerol produced by 30 milliunits/ml TSH is 75% of that produced by 10 µM isoproterenol.


DISCUSSION

In the present study, we have cloned two TSH-R cDNAs (F and F) that contained the full coding sequence from a fat cell gt11 cDNA library. Comparison of the nucleotide sequence of F with that of the thyroid TSH-R cDNA has demonstrated that F contained an extra 21 bp, which encode seven additional amino acids, between residues 156 and 157 of the thyroid receptor. Although the rat TSH-R gene has not been isolated, the position corresponds to the junction of the fifth and sixth exon of the human TSH-R gene (25) . Indeed, the nucleotide sequence of 10 bp from the 3` end of the insert, 5`-TCTCTTGCAG, was identical to the intronic sequence at the fifth intron/sixth exon boundary of the human TSH-R gene, suggesting that F is an alternatively spliced form of the TSH-R. However, reverse transcription-PCR analysis of the TSH-R mRNA from adipose tissue, as well as subsequent nucleotide sequencing of 10 clones (Fig. 3), indicated that little F mRNA is present in epididymal adipose tissue measured here; but the form may be more highly expressed in other fat tissues. When the F type of TSH-R cDNA was transfected into CHO-K1 cells, they lacked responsiveness to TSH and TSH binding activity (Fig. 4). We do not know, at present, whether this was due to the conformational change of the receptor produced by inserted amino acids or due to a failure of its expression at cell surface. So, the role of the form of TSH-R remains unknown and further study will be needed to clarify it.

In contrast, the nucleotide sequence of the F type of TSH-R cDNA was almost identical to that of the thyroid clone. Responsiveness of cAMP to TSH and TSH binding activity of the F type of receptor was indistinguishable from that of the thyroid. These data along with the results of Northern analysis of adipose tissue strongly suggest that about the same level of TSH-R is expressed and functions in the fat cells as in the thyroid.

Graves' disease is characterized not only by thyrotoxicosis but also by other extrathyroidal manifestations such as ophthalmopathy and dermopathy. Evidence that IgGs from the patients with Graves' disease stimulated cAMP formation in non-thyroid cells expressing recombinant TSH-R (3) supports a direct role of thyroid-stimulating antibody in generating thyrotoxicosis in the disease.

However, the pathogenesis of the extrathyroidal manifestations remains obscure and has been debated actively. Kohn and Winand (26) previously proposed the existence of a TSH-like molecule, designated an exophthalmos producing factor, in patients' sera. They showed that this molecule and TSH specifically bound to and increased cAMP levels in guinea pig harderian gland and human fat cells as well as in thyroid membranes, and they also demonstrated that its activity could be modified or substituted by autoantibodies from exophthalmic patients (27, 28) . Based on these data, they postulated that it and/or a TSH-R autoantibody was important in the development of the extrathyroidal manifestations of Graves' disease. On the other hand, several groups presumed the existence of a common antigen such as an eye muscle 64-kDa protein shared between the thyroid and other tissues (29, 30) .

Recently, using TSH-R peptide antibody, we have succeeded in detecting TSH-R immunoreactivity in rat retro-orbital and adipose tissues (10) . Therefore the data presented here may demonstrate a comprehensive role of TSH-R antibody in the pathogenesis of the thyroidal and extrathyroidal manifestations of Graves' disease.

In the present study, we demonstrated the existence of specific binding sites for TSH in membranes prepared from isolated fat cells (Fig. 5 A), and these observations were compatible with previous reports from other laboratories (23, 31, 32, 33, 34, 35) . Using isolated fat cells, we also showed that TSH possesses lipolytic activity (Fig. 5 B). The data are also consistent with previous findings reported by Birnbaumer and Rodbell (36) , Marcus et al.(37) , and Farmer et al.(38) . However, although the reasons remain unclear, a notable difference was the finding that the effective dose of TSH on lipolysis was 1 or 2 orders of magnitude higher than required for suppressing I-TSH binding to fat cell membrane. One of the possibilities is the co-existence of free fatty acid in the medium induced by TSH (39) , which had been reported to inhibit the adenylate cyclase activity (40) , but further elucidation will be needed to clarify it. However, in addition to suggesting a mechanism for the extrathyroidal complications of Graves' disease, our results support the previous findings that TSH has an effect on fat metabolism.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-552-73-1111; Fax: 81-552-73-7108.

The abbreviations used are: TSH-R, thyrotropin receptor; TSH, thyrotropin; CHO, Chinese hamster ovary; LH, luteinizing hormone; CG, chorionic gonadotropin; PCR, polymerase chain reaction; KRH, Krebs-Ringer solution buffered with 25 mM Hepes at pH 7.4; BSA, bovine serum albumin; bp, base pair(s).


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