From the
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
Thyrotropin receptor (TSH-R)
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
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
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
As shown in Fig. 5 A,
In the present study, we have cloned two TSH-R cDNAs (F
In
contrast, the nucleotide sequence of the F
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
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.
(
)
, 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) .
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.
gt11 cDNA library.
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).
, 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 of
Isolated fat cells were homogenized in 10
volumes of 10 mM Tris-HCl, pH 7.5. After centrifugation at 800
I-TSH to Isolated
Fat Cell Membrane
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.
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
In order to study the
distribution of F and F
TSH-R mRNA in the Thyroid
and the Epididymal Adipose Tissue
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
F and F
cDNAs into CHO-K1 Cells
and Their Responsiveness to TSH or Thyroid-stimulating
Antibody
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.
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.
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.
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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.