T H Y R O I D - T R H - T S H
Characterization of Thyrotropin Receptor
Antibody-Induced Signaling Cascades
Syed A. Morshed, Rauf Latif, and Terry F. Davies
Thyroid Research Unit, Mount Sinai School of Medicine, James J. Peters Veterans Affairs Medical Center, New York,
New York 10468
The TSH receptor (TSHR) is constitutively active and is further enhanced by TSH ligand binding or
by stimulating TSHR antibodies (TSHR-Abs) as seen in Graves’ disease. TSH is known to activate the
thyroid epithelial cell via both G␣s-cAMP/protein kinase A/ERK and G␣q-Akt/protein kinase C
coupled signaling networks. The recent development of monoclonal antibodies to the TSHR has
enabled us to investigate the hypothesis that different TSHR-Abs may have unique signaling imprints that differ from TSH ligand itself. We have, therefore, performed sequential studies, using
rat thyrocytes (FRTL-5, passages 5–20) as targets, to examine the signaling pathways activated by
a series of monoclonal TSHR-Abs in comparison with TSH itself. Activation of key signaling molecules was estimated by specific immunoblots and/or enzyme immunoassays. Continuing constitutive TSHR activity in thyroid cells, deprived of TSH and serum for 48 h, was demonstrated by
pathway-specific chemical inhibition. Under our experimental conditions, TSH ligand and TSHRstimulating antibodies activated both G␣s and G␣q effectors. Importantly, some TSHR-blocking
and TSHR-neutral antibodies were also able to generate signals, influencing primarily the G␣q
effectors and induced cell proliferation. Most strikingly, antibodies that used the G␣q cascades
used c-Raf-ERK-p90RSK as a unique signaling cascade not activated by TSH. Our study demonstrated that individual TSHR-Abs had unique molecular signatures which resulted in sequential
preferences. Because downstream thyroid cell signaling by the TSHR is both ligand dependent and
independent, this may explain why TSHR-Abs are able to have variable influences on thyroid cell
biology. (Endocrinology 150: 519 –529, 2009)
T
he TSH receptor (TSHR) is a member of the seven-transmembrane receptor subfamily and mainly activates the classical G protein-coupled receptor (GPCR) effectors, G␣s and
G␣q, and their complex signaling systems (1, 2). Because multiple positive and negative feedback mechanisms are common to
postreceptor signaling pathways, most are not considered as linear pipelines but as systems or cascades. They need to be viewed
as intricate signaling networks containing multiple modules of
protein-protein complexes that assemble at various intracellular
compartments to process, integrate, and transmit information
ultimately specifying a particular biological response. The TSHR
has constitutive signaling activity and is further activated by TSH
ligand binding or by unique stimulating autoantibodies to the
TSHR (TSHR-Abs) seen in patients with Graves’ disease. Therefore, inappropriate activation and/or inactivation of signaling
cascades triggered by these different ligands may contribute to
thyroid pathology in a way quite distinct from TSH.
The G␣s activities are largely mediated by an increase in adenylate cyclase (AC) activity, which generates intracellular
cAMP leading to the direct activation of protein kinase A (PKA)cAMP response element-binding protein (CREB) or either the
PKA-dependent Ras family of GTP binding proteins (Rap)1-b-
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2009 by The Endocrine Society
doi: 10.1210/en.2008-0878 Received June 11, 2008. Accepted August 12, 2008.
First Published Online August 21, 2008
Abbreviations: AC, Adenylate cyclase; BCS, bovine calf serum; CHO, Chinese hamster
ovary; CREB, cAMP response element-binding protein; DAG, diacylglycerol; EIA, enzyme
immunoassay; Elk, Ets-like transcription factor; EPAC, exchange protein activated by cAMP;
GPCR, G protein-coupled receptor; 5H, 6H devoid of TSH; 6H, mixture of six hormones; IP3,
inositol 3,4,5-trisphosphate; MEK, MAPK kinase; MTT, 3-(4, 5- dimethylthiazol-2-yl)-2,5diphenyl 2H-tetrazolium bromide; NF␬B, nuclear factor ␬B; p90RSK, ribosomal protein S6
kinase, 90kD protein; PDK1, 3-phosphoinositide-dependent protein kinase-1; PI, phosphatidylinositol; PIP2, phosphatidylinositol bisphosphate; PIP3, phosphatidylinositol
(3,4,5)-trisphosphate; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C;
Rap, Ras family of GTP binding proteins; RTK, receptor tyrosine kinase; TSHR, TSH receptor;
TSHR-Abs, TSHR antibodies.
Endocrinology, January 2009, 150(1):519 –529
endo.endojournals.org
519
520
Morshed et al.
TSHR Signal Transduction
Endocrinology, January 2009, 150(1):519 –529
Growth
Factors
TSH or TSHR antibody
RTK
γ
β Gαq
γ
β Gαs
TSHR
AC
PKC
cAMP
NFκB
PKA
EPACI
DAG PLC
c-Raf
MEK1/2
PI3K
PDK1
Akt
SHC
GRB2
SOS
RAS
c-Raf
MEK1/2
RapI
Erk1/2
MEK1/2
mTOR
p90RSK
Elk1
CREB
FIG. 1. Simplified diagrammatic illustration of the major signaling pathways
evaluated in the current studies. Note the five pathways from left to right: cAMP/
PKA/ERK or PKA/CREB, PI3/Akt/mTOR, PKC/NF␬B, PKC/c-raf/ERK/p90RSK, and
Ras/c-Raf/ERK. In practice, many of these pathways interact, and the diagram is
for illustrative purposes only.
Raf-ERK-Ets-like transcription factor (Elk1) cascade or the PKAindependent exchange protein activated by cAMP (EPAC1)Rap1b-ribosomal protein S6 kinase, 90 kD protein (Raf)-ERKElk1 signaling pathway to regulate thyroid function (Fig. 1) (3,
4). The ERK has been the subject of intense study and it is important to note that ERK is a downstream component of a well
conserved signaling module that is more commonly activated by
the Raf serine/threonine kinases, particularly c-Raf (Fig. 1) (5, 6).
This Raf-MAPK kinase (MEK)-ERK pathway is also a key
downstream effector of the Ras small GTPase, which requires
receptor tyrosine kinase (RTK) activation by various growth
factors (7). It is, therefore, interesting that in TSHR signaling,
ERK1/2 can also be activated by either the PKA-Rap1 or EPAC1Rap1 pathways as depicted in Fig. 1 (8, 9). Both Elk-1 and ribosomal protein S6 kinase, 90 kD protein (p90RSK) as well as
immediate-early genes are important downstream transcription
factors in these ERK1/2 activation pathways (10, 11).
An alternate TSHR effector pathway, via G␣q, mediates the
activation of the phospholipase C (PLC)-␤ and the G␤␥ subunit
(12). Among multiple isoforms, PLC␤1 and -3 are stimulated by
G␣q, whereas PLC␤2 is stimulated by the G␤␥ subunit complex.
Once activated, PLC hydrolyzes phosphatidylinositol (PI) bisphosphate (PIP2) to inositol 3,4,5-trisphosphate (IP3) and diacylglycerol
(DAG) (13, 14). Akt is activated as a result of PI3-kinase activity,
because Akt requires the formation of the PI-3,4,5-trisphosphate
molecule to be translocated to the cell membrane (15) and in the
presence of PI-3,4,5-trisphosphate, Akt is then phosphorylated by
another kinase called 3-phosphoinositide-dependent protein kinase-1 (PDK1), and is thereby activated (16). The Akt signaling
pathway is required most notably for cellular proliferation and survival (17, 18). PI3 and DAG are also involved in the activation of
Ca2⫹ and protein kinase C (PKC) isoenzymes, respectively, whereas
PDK1 kinase is involved in the activation of atypical PKC isoenzymes (16, 19). Hence, PDK1 plays a central role in many signal
transduction pathways involving Akt, PKC isoenzymes, p70 S6 kinase, and RSK (18, 20, 21).
Activation of transcription factors of the nuclear factor ␬B
(NF␬B) in FRTL-5 cells by TSH or stimulating TSHR antibodies
has been reported previously (22, 23). NF␬B-activating agents can
induce the phosphorylation of I␬B’s, targeting them for rapid degradation through a ubiquitin-proteasome pathway, releasing NF␬B
to enter the nucleus, where it regulates gene expression (24, 25).
In addition to the TSH- or TSHR antibody-induced downstream TSHR signaling responses, each arm (G␣) of the effectors
is under the influence of a wide variety of growth factors (26, 27)
(Fig. 1). These factors work via MAPK cascades, which are key
signaling systems involved in the regulation of normal cell proliferation, survival, and differentiation (2, 28).
The recent development of various types of monoclonal antibodies to the TSHR has enabled us to investigate their potential
signaling events in a model system providing information quite distinct from previous reports using polyclonal sera from patients with
TSHR-Abs. Monoclonal TSHR-Abs, which either stimulate as
TSH agonists or block TSH action, have begun to be characterized
previously, but their signaling cascades have not been evaluated in
depth (29 –34). In addition, TSHR-Abs of neutral type have been
defined as binding to the receptor without any influence on cAMP
generation (35) and what role they play in the signal transduction
remains completely unknown. We hypothesized that different
TSHR-Abs might have unique signaling imprints at the TSHR altering cellular functions in distinct patterns not seen with TSH itself.
Using rat thyrocytes (FRTL-5) as targets, we have, therefore, performed sequential studies to examine selected signaling events and
their downstream effectors initiated by a series of monoclonal antibodies to the TSHR. These studies were not envisaged as determining new signal transduction pathways but rather as exercises in
illuminating differences between TSHR-Abs and TSH ligand. Our
observations demonstrated that stimulating TSHR-Abs used signaling
pathwayssimilartoTSHactivationmodulesdependingonthestrength
of the signals generated. Some blocking and neutral TSHR-Abs, although previously considered as nonsignaling, also showed unique
molecular signatures affecting c-Raf-ERK-p90RSK signal transduction pathways, indicating that these antibodies have the potential to act
as weak agonists. Because thyroid cell proliferation is one of the downstream effector mechanisms of signaling events, our studies also
showed that both G␣s and G␣q signaling networks contributed to this
process. Forward acceleration or interruption of signaling networks by
TSHR-Abs may, therefore, explain some of the variable thyroid pathology observed in patients with Graves’ disease.
Materials and Methods
Cell cultures
FRTL-5 rat thyroid cells were used as the model system (28, 36). TSH
signal transduction is known to differ in the FRTL-5 rat thyroid cell line
Endocrinology, January 2009, 150(1):519 –529
endo.endojournals.org
when compared with normal cells (37). In particular, TSH signaling may
be PKA independent in the FRTL-5 cell (28). Nevertheless, in the absence
of a more normal thyroid cell line, the FRTL-5 served our purpose well.
Cells were diploid and between their fifth and 20th passage after receipt.
The doubling time of these cells was 36 ⫾ 6 h when cultured in the
presence of bovine TSH, and proliferation was absent without TSH.
Cells were grown as previously described (28) on 60-mm culture dishes
in modified Ham’s F12 medium (Cellgro; Mediatech Inc., Herndon, VA)
supplemented with 5% bovine calf serum (BCS, Serum Supreme; Cambrex BioScience Walkersville Inc., Walkersville, MD) and a mixture of
six hormones (designated 6H) (all from Sigma Chemical Co., St. Louis,
MO): bovine TSH (1 U/liter), insulin (10 mg/liter), hydrocortisone (0.4
mg/liter), human transferrin (5 mg/liter), glycyl-L-histidyl-L-lysine acetate (10 ␮g/liter), and somatostatin (10 ␮g/liter). Before any stimulation
experiments, cells were grown to 80% confluence in a humidified 5%
CO2 atmosphere and then made quiescent by switching to medium containing 5H (6H devoid of TSH) and 5% BCS for 3 d and synchronized
by starvation in BCS-free basal medium (modified Ham’s F12) containing 0.3% BSA for 2 d. Quiescent and synchronized cells were then stimulated for different time periods using TSH or TSHR antibodies as described below. A different cell line of Chinese hamster ovary (CHO) cells
expressing recombinant wild-type human TSHR (JPO9 cells, kind gift of
Dr. G. Vassart, Brussels, Belgium) were used to measure phosphoprotein
activities and cAMP levels. Cells were maintained in Ham’s F12 medium
supplemented with 10% fetal bovine serum (Mediatech), 100 U/ml penicillin, and 100 U/ml streptomycin (Invitrogen Corp., Grand Island, NY)
as previously described (29).
Culture conditions for stimulations
To obtain a good timeframe for synchronizing signaling events to
basal levels of signaling molecules, we starved cells for different time
points. We started with 1 h starvation and then went for 24 and 48 h and
finally for 72 h. After 24 –72 h starvation, FRTL-5 cells exhibited fewer Akt
responses but produced more responses on multiple signaling events, particularly PKC and PKA, and so 48 h starvation was chosen. All of the above
conditions had no effect on the c-Raf/ERK/p90RSK pathway or NF␬B activities. In two separate experiments at two different passages, both FRTL-5
and JPO9 cells demonstrated similar reproducible data (not illustrated).
TSHR antibodies
The monoclonal TSHR antibodies examined are listed in Table 1.
They included three stimulating (M22, MS1, and RSR-12), two blocking
(Tab-8 and RSR-B2), and two neutral (7G10, Tab-16) monoclonal antibodies. M22 and the RSR monoclonals were kindly supplied by Dr. B.
Rees Smith (RSR Ltd., Cardiff, Wales, UK). The remaining monoclonal
antibodies have been described previously (30, 31, 33, 34). Each of these
521
antibodies was used to stimulate FRTL-5 cells to study signal transduction mechanisms.
Treatment and cell lysates
Before stimulation, medium was discarded from the 60-mm culture
dishes, and the cells were washed three times with a fresh medium containing Hanks’ balanced salt solution (HBSS; Life Technologies, Inc.
Laboratories, Grand Island, NY). After the indicated stimulations in
fresh modified Ham’s F12 basal medium, cells were washed twice with
ice-cold PBS (pH 7.2) without calcium and magnesium (Mediatech),
scraped into cold Phosphosafe lysis buffer (EMD Biosciences, San Diego,
CA) containing proteinase inhibitor cocktail (Complete, Mini; Roche
Applied Science, Indianapolis, IN) and phenylmethylsulfonyl fluoride
(Sigma) and sonicated for 10 –15 sec. After measuring protein concentrations by the Bradford or BCA assay kits (Bio-Rad Laboratories, Hercules,
CA), samples were boiled for 5 min in NuPAGE LDS sample buffer with
reducing agent (Invitrogen, Carlsbad, CA). The samples were then centrifuged at 13,000 rpm for 5 min at room temperature, and the supernatants
were removed and stored in aliquots at ⫺80 C. Because culture conditions
may influence signaling events, the same condition was applied to all treatments and was strictly maintained in each experiment. All stimulation experiments were performed at least twice at two different time points.
Detection of signaling molecules by immunoblot
Sixty micrograms of total proteins were resolved by 4 –15% gradient
SDS-PAGE mini-gel (Bio-Rad) and transferred onto polyvinylidene difluoride membranes (Bio-Rad). Antibodies that recognize signaling molecule-specific phosphoproteins or non-phosphoproteins were immunodetected according to the instructions provided with the phosphospecific
antibody kits (Cell Signaling Technology, Beverly, MA). Antibody substrate reaction was performed by SuperSignal chemiluminescence kit
(Pierce, Rockford, IL) and was visualized on x-ray film using variable
exposures (38). The same blots were used repeatedly after stripping off
and reprobing with different antibodies. Relative quantification of signal
intensities on x-ray film was analyzed using scanning densitometry (AlphaImager 2200; Alpha Innotech, San Leandro, CA).
Two pathway-specific antibody sampler kits containing ERK (pcRaf, Ser338; pMEK1/2, Ser217/221; pERK1/2, Thr202/Tyr204; pElk-1,
Ser383; pp90RSK, Ser380) and PKC (pan pPKC␤⌱⌱ Ser660, pPKC␣/␤⌱⌱
Thr638/641, pPKC␦ Thr505, pPKC␦ Ser643, pPKD/PKC␮ Ser744/748,
pPKD/PKC␮ Ser916, ␪ Thr538, pPKC␨/␭ Thr410/403) (Cell Signaling)
were used. pAkt (Ser473), pPKC-␨/␭ (⌻hr410/403), pPKA-C (Thr197),
pCREB (Ser133), non-phospho-PKAc␣, pERK1/2 (Thr202/Tyr204),
pp90RSK (Ser380), and pNF␬B (p65, Ser536) were also from Cell Signaling. Nonphosphorylated mouse monoclonal ␤-actin and unrelated
control monoclonal (IgG2, ␬-chain) antibodies were from Sigma and BD
Biosciences (Franklin Lakes, NJ), respectively.
TABLE 1. Characteristics of antibodies used to study signaling mechanisms
Type
Stimulators
MS1
RSR-12
M22
Blockers
Tab-8
RSR-B2
Neutrals
Tab-16
7G10
Species
Isotype
␭/␬-Chain
NC35a
Hamster
Mouse
Human
IgG2
IgG2
IgG1
␭
␬
␭
Yes
Yes
Yes
Hamster
Mouse
IgG2
IgG2
␭
␬
Yes
Yes
1
1.7
Hamster
Hamster
IgG2
IgG2
␬
␭
No
No
1
1
Fold stimulation
⬃10
⬃10
⬃25
% Blocking
Epitope
Ref.
80
ND
ND
Conformational
Conformational
Conformational
29
32
33
80
80
Conformational
Conformational
30
34
0
0
322–341
337–356
30
30
Fold stimulation was determined by intracellular cAMP assay using CHO cells expressing human TSHR, and blocking was expressed as the percent reduction of cAMP
produced by test antibodies in the presence of TSH divided by cAMP produced by medium (29, 34). ND, Not determined.
a
NC35 indicates that the antibody-binding target was CHO-NC35 cells expressing a TSHR variant lacking residues 316 –366 and included amino acid substitutions at
367–369 (58).
522
Morshed et al.
TSHR Signal Transduction
Endocrinology, January 2009, 150(1):519 –529
Pathway-specific inhibitors
tyl-1-methylxanthine (Sigma) in the basal medium was used for measuring intracellular cAMP levels.
All inhibitors were purchased from EMD Biosciences (Gibbstown,
NJ). H89, GF109203X, LY294002, and PD98059 were used to inhibit
PKA, PKC, PI3-kinase (Akt), and MEK/ERK activities, respectively.
MTT cell proliferation assay
An MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl 2H-tetrazolium bromide] proliferation assay was carried out according to instructions provided (Sigma). Cell proliferation experiments were performed
in a 96-well plate in triplicate in basal medium without any hormones.
Cells (3 ⫻ 103 per well) were seeded and maintained in 200 ␮l 6H
medium in a humidified incubator. Cells were then made quiescent by
switching to medium containing 5H plus 5% BCS for 3 d and synchronized by starvation in the basal medium containing 0.3% BSA for 2 d.
The colorimetric MTT proliferation assays were performed at d 3 after
treatment. MTT (400 ␮g/ml) was added to each well, incubated for 3 h,
and solubilized with 0.04 M HCl/isopropanol for 30 min. The OD was
determined using a spectrophotometer (DTX 880 Multimode Detector;
Beckman Coulter, Fullerton CA) at a wavelength of 570 nm.
To ensure reproducibility, all stimulation and inhibition experiments
were repeated twice, and the basal level of analytes in all assays used,
when analyzed either densitometrically or colorimetrically, were comparable (coefficient of variation ⫽ 4.6 –11%).
PKA and PKC activity assays
Determination of PKA and PKC activity in whole-cell lysates was
achieved with commercial enzyme immunoassay (EIA, StressXpress;
Stressgen Bioreagents, Ann Arbor, MI) kits according to the manufacturer’s instructions. The assay was based on a solid-phase EIA that used a
specific synthetic peptide as a substrate for kinases and polyclonal antibodies
that recognized the phosphorylated forms of the substrate. These assays
were designed for the analysis of PKA and PKC activities in the solution
phase. Treated cells with specific agents were lysed after the indicated time
points. Five micrograms of total protein extracts were used for the assays.
Results are expressed as relative units of absorbance (450 nm).
cAMP assay
FRTL-5 (80% confluent) and JPO9 (3 ⫻ 104) cells were used for
intracellular cAMP levels measured by EIA (Assay Designs, Inc., Ann
Arbor, MI, or Amersham cAMP Biotrak EIA System, GE Healthcare
Bio-Sciences Corp., Piscataway, NJ) using polyclonal antibody to cAMP
to bind, in a competitive manner, the cAMP in the standards or samples
or a conjugate molecule that had cAMP covalently attached. The intensity of the bound color was inversely proportional to the concentration
of cAMP in either standards or samples. In each treatment, 2 mM 3-isobu-
Fold Increase (cAMP)
A
Statistical analysis
The paired t test was used to evaluate the significance of differences in
means for continuous variables. A P value of ⱕ0.05 was used to determine
statistical significance. Data are presented as the mean ⫾ SD of the mean.
2.5
0.1
mAb: µg/ml
1.0
TSH: mU/ml
10
2
1.5
1
0.5
0
M22
R12
MS1
Stimulating
T16
7G Control Basal
Neutral
C
800
pmol/ml
Blocking
Basal
M22
TSH
B2
600
400
200
0
03
15
30
60 min
pmol/well
7.5
pmol/well
B
T8
B2
4.0 3.2 2.4 1.6 0.8 0
6.0
µg/ml
TSH
10
4.5
3.0
1.0
0.1
1.5
0
B2-Dose Response
0
03
15
30
60 min
FIG. 2. Intracellular cAMP generation in quiescent FRTL-5 cells. Increasing concentrations of TSH (0.1, 1.0, and 10 mU/ml) or TSHR-stimulating monoclonal antibodies (0.1,
1.0, and 10 ␮g/ml) induced cAMP during a 60-min exposure as illustrated by fold increases in picomoles per milliliter (A). At fixed concentrations of TSH (1 mU/ml) and
antibodies (1 ␮g/ml), an hour of stimulation produced significantly increased levels of cAMP (TSH, P ⬍ 0.007; M22, P ⬍ 0.01; R12, P ⬍ 0.01, MS1, P ⬍ 0.001; B2, P ⬍ 0.009;
T8, P ⬍ 0.035) when compared with the medium alone or a control antibody. B, Detailed changes of selected stimulators or blocker over time. C, cAMP
responses in JPO9 cells produced by stimulators or blocker as in B. B2 dose-response is also shown in C. These demonstrate endogenous (basal) cAMP (without
stimulation) and stimulated cAMP [using 1 mU/ml TSH or 1 ␮g/ml monoclonal antibody (mAb)]. The individual antibodies are labeled according to Table 1 (R12, RSR-12; B2, RSR-B2; T8,
Tab-8; 7G, 7G10; T16, Tab-16).
Endocrinology, January 2009, 150(1):519 –529
endo.endojournals.org
Fold Increase (Cell Proliferation)
9
8
0.1
1.0 mAb: µg/ml
TSH: mU/ml
10
7
6
5
4
3
2
1
0
TSH M22 R12 MS1 B2
Stimulating
T8 T16
Blocking
7G Cont Basal
Neutral
FIG. 3. Induction of FRTL-5 cell proliferation. Quiescent and starved cells were
stimulated with increasing concentrations of TSH (0.1, 1.0, and 10 mU/ml) or TSHR
antibodies (0.1, 1.0, and 10 ␮g/ml) in the basal medium. At a fixed concentration,
TSH (1 mU/ml) stimulating and blocking antibodies (1 ␮g/ml) demonstrated
significantly higher cell proliferation (TSH, P ⬍ 0.0004; M22, P ⬍ 0.0005; R12, P ⬍
0.0004; MS1, P ⬍ 0.003; B2, P ⬍ 0.0008; T8; P ⬍ 0.001) than medium alone (basal)
or a control antibody (Cont; 1 ␮g/ml). One of the neutral (T16; 1 ␮g/ml) antibodies
also showed significantly higher proliferation (P ⬍ 0.001), whereas 7G showed
suppressive effects (P ⬍ 0.02) at a higher concentration (10 ␮g/ml). These
experiments were repeated twice in triplicate.
Results
Traditional readouts: cAMP generation and cell
proliferation
We first characterized the TSH and TSHR antibody responses
in rat thyroid FRTL-5 cells and CHO cells, under our defined
experimental conditions, by using intracellular cAMP accumulation and cell proliferation as endpoints (Table 1). As expected
from previous studies, both TSH and three stimulating antibodies to the TSHR-induced cAMP generation and enhanced proliferation of thyroid cells (Figs. 2 and 3). Both induced intracellular production of cAMP in a dose- and time-dependent manner
and in high concentrations suppressed the thyroid cell responses
(Fig. 2, A and B). When fixed concentrations of TSH (1 mU/ml)
and TSHR-Abs (1 ␮g/ml) were applied, stimulators and one
blocker exhibited significantly higher levels of cAMP than medium alone (TSH, P ⬍ 0.007; M22, P ⬍ 0.01; RSR-12, P ⬍ 0.01,
MS1, P ⬍ 0.001; RSR-B2, P ⬍ 0.009). TSHR neutral antibodies
showed no effect on cAMP production (P ⫽ not significant).
Using a fixed concentration of TSH (1 mU/ml), the ligand response was first detectable at 3 min in thyroid cells with peak
levels reached after 30 min stimulation (Fig. 2B). With a fixed
concentration of TSHR stimulating antibody (M22, 1 ␮g/ml), a
slower response was seen, but the peak was also reached by 30
min (Fig. 2B).
After 48 h serum deprivation, human TSHR-expressing CHO
cells were also treated with a fixed concentration of TSH (1
mU/ml) or antibodies (1 ␮g/ml) over time, and a similar but more
rapid response was observed by the human stimulating antibody
(M22) (Fig. 2C) compared with the rat TSHR (Fig. 2B). B2 blocking antibody (1 ␮g/ml) also induced cAMP production as seen in the
rat cells. Increasing concentrations of B2 showed a peak at 1 ␮g/ml
followed by a suppressive effect at 10 ␮g/ml. This finding was different from that of our earlier observations where cells were not
523
starved for 2 d (29). Serum starvation decreased the basal cAMP
and thus caused a detectable increase in cAMP production similar
to what we observed in FRTL-5 cells. By contrast, an unrelated
control antibody produced no effect on cAMP induction.
Increasing concentrations of TSH and TSHR-Abs showed
different effects on the rat thyroid cell proliferation assay. The
highest proliferation was observed at 1 mU/ml TSH and 1 ␮g/ml
antibodies. A robust increase in proliferation was produced by
stimulating antibodies. and much less robust stimulation was
seen with some blocking and neutral antibodies (Fig. 3). At a
fixed concentration, TSH (1 mU/ml) stimulating and blocking
antibodies (1 ␮g/ml) activated thyroid cell proliferation (TSH,
P ⬍ 0.0004; M22, P ⬍ 0.0005; RSR-12, P ⬍ 0.0004; MS1, P ⬍
0.0003; RSR-B2, P ⬍ 0.0008; Tab-8; P ⬍ 0.001) when compared
with the basal medium alone or control antibody (Fig. 3). Interestingly, one of the neutral and one of the blocking antibodies
(Tab-8 and Tab-16) that showed no significant increase in cAMP
production consistently induced thyroid cell proliferation (Tab16, P ⬍ 0.002), whereas another (7G10) exhibited suppressive
effects (P ⬍ 0.02) at 10 ␮g/ml. This suggested that different
pathways to proliferation via G␣q existed in this cell model.
However, overall, the results of the cell proliferation studies
mostly paralleled the cAMP responses with down-regulation of
the proliferative responses seen with concentrations greater than
10 mU/ml TSH or 10 ␮g/ml antibody (Fig. 3).
These experiments confirmed that our cell culture conditions
were appropriate for the subsequent signal transduction studies.
Constitutive activity of Akt, PKA/CREB, PKC, NF␬B, and
c-Raf/ERK/p90RSK
To define the activity and stability of selected key signaling
molecules, we next performed time-dependent studies to examine their constitutive activity under our experimental conditions.
Quiescent and starved cells were left untreated in the fresh basal
medium at intervals for up to 60 min and then lysed as described.
Immunoblot analyses of these lysates exhibited decreased expression of c-Raf/ERK/p90RSK, PKC-␨/␭, and Akt in a timedependent fashion, whereas PKAc␣, PKAc (phospho), CREB,
and NF␬B activities remained unchanged (Fig. 4, A and B). The
constitutive activity of ERK and c-Raf decreased faster than
p90RSK or Akt. To further confirm key signaling cascades
unique to FRTL-5 cells and to define their specificities, we examined the effect of molecular inhibitors on the different signaling cascades, being aware that not all such inhibitors are always
totally specific. FRTL-5 cells were treated with a fixed concentration of inhibitor and analyzed at three time points (30 min and
1 and 2 h). Cell lysates were then immunoblotted and analyzed
by antibodies specific for phosphoproteins. All the inhibitors
used in this study produced a reduction in the phosphorylation
of their specific proteins (Fig. 4, C and D). Inhibitors specific for
PI3-kinase uncovered multiple feedback mechanisms in quiescent thyroid cells. The PI3-kinase inhibitor (LY294002; 1 ␮M)
suppressed Akt activity but activated signaling cascades including ERK/p90RSK, PKA/CREB, PKC-␨/␭, and NF␬B, whereas the
PKA inhibitor (H89; 50 ␮M) increased the constitutive activity of
Akt. The PKC inhibitor (GF109203X; 50 ␮M) reduced PKC-␨/␭
activity and also suppressed c-raf/ERK/p90RSK (Fig. 4, C and
Morshed et al.
TSHR Signal Transduction
A
Endocrinology, January 2009, 150(1):519 –529
B
(mins)
pAkt
pPKC-ζζ/λ
PKAcα
pPKAc
pCREB
pc-Raf
pErk1/2
pp90RSK
pNFκB
β-actin
1.4
1.2
pAKT
pPKCζ/λ
PKAcα
pPKAc
pCREB
pc-Raf
pErk1/2
pp90RSK
pNFκB
β-actin
1
0.8
0.6
0.4
0.2
0
0 3
C
30
D
E C A P No
pAkt
pPKC-ζ/λ
PKAcα
pPKAc
pCREB
pc-Raf
pErk1/2
pp90RSK
pNFκB
β-actin
60
min
PI3 kinase inhibitor
2.5
Control
Inhibitors
2
1.5
1
0.5
0
p
pP Ak
K t
C
PK ζ/λ
A
c
pP α
K
pC Ac
R
E
pc B
-R
pE af
pp rk1
90 /2
R
pN SK
Fk
β- B
ac
tin
3 30 60
Fold Activity
0
Fold Change in Relative Unit
524
FIG. 4. The constitutive activity of signaling molecules in untreated FRTL-5 cells. Starved quiescent cells were left untreated for multiple different time points, and their
signaling activities were examined by immunoblotting with phosphospecific antibodies. Signaling molecule activity decreased in a time-dependent manner over 60 min
as demonstrated by repeated experiments. A, Representative immunoblots containing phospho- or non-phosphoproteins. The lowercase letter p in front of each
molecule indicates phosphoprotein detection. B, Densitometric quantitation of each band on the autoradiographs produced from immunoblots. Quiescent FRTL-5 cells
were treated with a fixed concentration of specific kinase inhibitors as described in Materials and Methods. Both reductions and increases in phosphorylated proteins
were observed by these specific inhibitors. Of note, unlike suppressing Akt, PI3-kinase inhibitor (Ly294002; 1 mM) activated PKC, CREB, and ERK/p90RSK (C and D).
D). To show effects of ERK on other pathways, we also used a
MEK1/2 inhibitor (PD98059; 50 ␮M), the immediate upstream
regulator of ERK. This inhibitor suppressed ERK and its downstream molecule p90RSK. All the inhibitors used with rat thyroid
cells were also used to suppress kinase activities in human TSHRtransfected CHO cells. Unlike the potentially different signaling
cross talk and/or feedback loops, these inhibitory effects were no
different from those seen in the FRTL-5 cells. These multiple
signaling events by different pathway-specific inhibitors indicated that cross talk and/or feedback mechanisms were essential
steps in the fine tuning of the signaling cascades and were not
apparently specific for thyroid cell function.
TSH activates CREB but not the PKA/ERK or c-Raf /ERK/
p90RSK pathway
The ERK pathway was analyzed by phosphospecific sampler
antibodies before and after exposure to TSH (Fig. 5, A and B).
The constitutive expression level of phosphorylated c-Raf, ERK,
and p90RSK was higher than MEK (immediately upstream of
ERK) and Elk1 (downstream of ERK) in the rat thyroid cells.
Because 1 mU/ml TSH and 1 ␮g/ml TSHR-Abs showed comparable effects on cAMP and proliferation indices, we used these
concentrations for subsequent time-response immunoblot experiments. TSH (1 mU/ml), a dose that induced thyroid cell pro-
liferation, did not increase the activity of ERK even after 60 min
exposure, and the constitutive loss described earlier continued
(Fig. 5, A and B). The loss of ERK activity paralleled the p90RSK
activity. Hence, a proliferative concentration of ligand-dependent TSHR stimulation did not enhance the activity of the c-Raf/
ERK/p90RSK pathway, but CREB, a downstream molecule of
the PKA pathway, indicated higher activity over time. Because
the constitutive activity of PKA was high in these quiescent cells,
it was difficult to discern changes by immunoblot or bioassay in
this model (Figs. 5 and 6). Nevertheless, small TSH-induced increases were seen by 30 min, and such small PKA dynamic
changes may have been sufficient to phosphorylate downstream
molecules such as CREB.
TSH activates the Akt and PKC pathways
Both time-dependent and dose-dependent studies demonstrated that TSH also activated Akt and PKC effectors (Figs. 5
and 6). PKC-␨/␭, atypical isoforms, were identified as highly
expressed phosphoproteins in rat thyroid cells (Figs. 5 and 6).
Other highly expressed classical and novel phosphorylated PKC
isoforms such as ␤⌱⌱, ␣/␤⌱⌱, ␦, and ␪ were also detectable (not
illustrated). A robust increase in Akt and PKC-␨/␭ activity was
observed by 30 min TSH stimulation (Fig. 5). Increased PKC
activity was also confirmed by enzyme assay (Fig. 7). NF␬B
Endocrinology, January 2009, 150(1):519 –529
0
525
B
3
30
60 (mins)
pAkt
pPKC-ζζ/λ
PKAcα
pPKAc
pCREB
pc-Raf
pErk1/2
pp90RSK
pNFκB
β-actin
Fold Change in Relative Unit
A
endo.endojournals.org
2
pAKT
pPKCζ/λ
PKAcα
pPKAc
pCREB
pc-Raf
pErk1/2
pp90RSK
pNFκB
β-actin
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0 3
30
60 min
FIG. 5. TSH uses both G␣s and G␣q pathways. In 60 min, TSH repeatedly activated Akt, PKC, and CREB but not PKA and ERK as illustrated in these representative
immunoblots (A). Densitometric quantitation of each band was plotted as fold activation (B), which indicated that the activity of the phosphoproteins had either
reduced or increased.
showed a small increase in activity after exposure to TSH in line
with the increased PKC levels.
These observations indicated that CREB, Akt, and PKC-␨/␭
were important molecules involved in TSH-induced thyroid cell
signaling cascades. The PKA independence of these cascades has
been previously reported in these cells (28).
TSHR-stimulating antibodies activate TSH signaling
pathways
TSHR-stimulating antibodies showed a signal pattern generally similar to TSH-mediated changes (Fig. 6). Each of the three
stimulating antibodies activated Akt and PKC-␨/␭ at variable
concentrations and time points. For example, both M22 and
MS1 antibodies induced an increase in Akt and PKC-␨/␭ at a dose
of 0.1 ␮g/ml after 1 h stimulation, whereas R-12, which was
much less active, increased Akt and PKC-␨/␭ activities at a maximum dose of 10 ␮g/ml. None of the stimulating antibodies activated the c-Raf/ERK/p90RSK pathway. Rather, they produced
a dose-dependent suppressive effect on this pathway as seen with
TSH (Fig. 6). However, M22 showed a marked increase in PKA
activity and repeatedly exhibited an increase in p90RSK presumably via a different pathway. However, as far as activation of
CREB was concerned, whereas M-22 and MS-1 induced CREB
in the same way as seen with TSH, TSHR-Ab R-12 had no such
effect.
TSHR-blocking antibodies induce signaling cascades
indicative of weak agonist activity
Designated TSHR-blocking antibodies showed a diverse effect on multiple signaling cascades in keeping with their known
weak agonist activity. RSR-B2, which induced low-level cAMP
generation and cell proliferation (Figs. 2 and 3), increased Akt,
CREB, c-Raf, and ERK (Fig. 8A), whereas Tab-8 activated PKA
and PKC (Fig. 8A), in line with their proliferative effects seen
earlier (Fig. 3).
TSHR-neutral antibodies induce different signaling
cascades
The two designated neutral antibodies were not neutral in
their signaling activity but produced different signaling events
dose dependently. One of them (Tab-16) showed modest activation of many signaling molecules such as Akt, PKC, PKA,
CREB, ERK, and p90RSK, whereas the other (7G10) tended to
consistently reduce constitutive signaling molecular activities as
seen with PKC, c-Raf, ERK, and p90RSK (Fig. 8B). Control
antibody did not induce any effect on these effectors (Fig. 8C).
FIG. 6. TSHR-stimulating antibodies activated TSH signaling pathways. Increasing concentrations (0.1, 1.0, and 10 ␮g/ml) of TSHR-stimulating antibodies MS1, R12,
and M22 over a 60-min incubation showed similar changes in signaling molecules as seen with TSH ligand in these representative immunoblots. M22 produced dosedependent significant increases in Akt, PKA, and CREB activities compared with TSH and the other antibodies. These dose-response studies were repeated twice.
526
Morshed et al.
TSHR Signal Transduction
Endocrinology, January 2009, 150(1):519 –529
Fold Increase
(Relative Activity)
5
4.5
PKA
4
PKC
3.5
3
2.5
2
1.5
1
0.5
0
TSH
M22
R12
MS1
Stimulating
B2
T8
Blocking
T16
7G
Cont
Basal
Neutral
FIG. 7. PKA and PKC activity assessed by EIA. In our model system, TSH (1 mU/ml)
did not activate PKA enzyme activity, whereas the M22 TSHR-stimulating antibody (1
␮g/ml) did demonstrate a highly significant increase (P ⬍ 0.0001). R12 (P ⬍ 0.013)
and MS1 (P ⬍ 0.03) stimulating antibodies showed significantly reduced activities.
Both neutral antibodies also exhibited similar effects (T16 and 7G, P ⬍ 0.02).
Although blocking antibodies did not produce any significant effects. In contrast,
both TSH (P ⬍ 0.0005) and TSHR-stimulating antibodies increased PKC (M22, P ⬍
0.001; R12, P ⬍ 0.003) activity significantly. One of blocking (B2, P ⬍ 0.0001) and
both neutral (T16, P ⬍ 0.0001; 7G, P ⬍ 0.0002) antibodies also demonstrated a
significant increase in PKC activity when compared with the medium alone (basal) or
a control monoclonal antibody (cont). The experiments were repeated three times
and confirmed the illustrated findings.
Discussion
The overall aim of our studies was to examine the signal transduction pathways initiated by antibodies to the TSHR in comparison with each other and with TSH itself. We began with the
hypothesis that stimulating TSHR-Abs would act just like TSH
and that blocking and neutral antibodies would produce no signal transduction activities. In fact, we found monoclonal examples of each of these varieties of TSHR-Abs that were able to
initiate complex signaling cascades. As expected, both cAMP
generation and cell proliferation assays identified stimulating
antibodies as true TSH agonists. However, one apparent TSHblocking antibody induced the generation of cAMP both in
FRTL-5 and CHO cells expressing human TSHR and induced
cell proliferation in FRTL-5 cells. This finding was in agreement
with earlier observations where blocking antibodies were proposed as weak agonists (39, 40). In addition, we found evidence
that a totally neutral TSHR-Ab was also capable of initiating
A
0
T8
0.1 1 10
B2
0 0.1 1 10
B
signal transduction cascades. These data strongly suggested that
a variety of changes in molecular shape of the TSHR induced by
antibody binding would result in the initiation of signaling.
To determine key signaling molecules in quiescent thyroid
cells, we first characterized the constitutive activity of different
signaling molecules in a time-dependent fashion. We identified
several key modules that demonstrated reduced activities when
rat thyroid cells were deprived of TSH and serum. We then illustrated continuing system cross talk, i.e. feedback inhibition or
activation of one signaling module by other signaling modules,
using pathway-specific inhibitors (41). For example, a PI3-kinase inhibitor reduced its downstream effector, Akt, but activated the ERK-p90RSK pathway along with PKA/CREB and
PKC. These findings indicated that ERK-p90RSK could be controlled by either PKA or PKC or by both. PKA-dependent and
-independent ERK activation and growth factor RTK-induced
ERK activation have already been described in thyroid cells (7,
28). Because PI3-kinase inhibition activated PKC/ERK/p90RSK
and the PKC inhibitor did the opposite, we reasonably assumed
that PKC also controlled the ERK-p90RSK pathway.
We saw no effect of TSH on ERK activation. In FRTL-5 cells,
ERK activation by cAMP is independent of PKA and linked to
Rap1 activation through EPAC (3, 4, 9, 28). However, in other
models, TSH or forskolin activated Rap1 but failed to activate
ERK (9, 42). Because different results have been obtained in
different cell model systems (1, 43), the role of ERK in the proliferation of normal thyrocytes still remains unclear. Because
cAMP (via G␣s) and PI3-PDK1-PKC (via G␣q), and Ras (via
RTK) are all able to regulate ERK phosphorylation, we assume
that the ERK pathway in FRTL-5 cells is controlled by at least
three individual modular networks, which have both forward
activation and feedback attenuation and are under the influence
of multiple cross talk. This may explain why our study did not
produce steady-state ERK activation with TSH.
Our study confirmed earlier observations that TSH activated
Akt (44) and that this activation occurred via PI3-kinase. Suh et
al. (44) identified ribosomal S6 kinase (S6K1) as the main effector of PI3-kinase but not Akt in FRTL-5 cells and suggested that
this occurred via cAMP/PKA activation. Although we did not
study the regulatory mechanisms of PI3/Akt activation, there
7G
0 0.1 1
T16
10
0 0.1 1 10
C
Control
0 0.1 1 10
(µg/ml)
pAkt
pPKC-ζ/λ
PKAcα
pPKAc
pCREB
pc-Raf
pErk1/2
pp90RSK
pNFκB
β-actin
FIG. 8. Increasing concentrations (0.1, 1.0, and 10 ␮g/ml) of TSHR blocking (A) and neutral (B) antibodies over a 60-min incubation showed variable changes in
signaling molecules as shown in these representative immunoblots. The data (representing one set of experiments repeated three times) showed different signaling
cascades induced by blocking and neutral antibodies. For example, the B2 blocking antibody increased several signaling molecules, the most important of which
appeared to be the c-Raf/ERK pathway, whereas the 7G neutral TSHR antibody suppressed signaling molecules such as c-Raf/ERK. T16, also a neutral antibody, showed
increasing activities of many signaling molecules including Akt, PKC, CREB, and c-Raf/ERK/p90RSK. By contrast, an isotype control monoclonal antibody (IgG2) did not
produce such dose-dependent activities.
Endocrinology, January 2009, 150(1):519 –529
was no doubt that under our conditions Akt was activated by
TSH. Indeed, the TSH effect on Akt phosphorylation paralleled
that of the proliferation index indicating that this pathway may
be an important signaling module contributing to cell proliferation. Interestingly, the mitogenic effect of TSH on Akt also
correlated well with cAMP levels similar to that observed by
others (37). It is most likely that some activation of Akt may arise
from cAMP through EPAC in FRTL-5 cells as suggested by Mei
et al. (45). Although our study was unable to show a notable
TSH-induced increase in PKA activity, CREB, a key downstream
molecule of PKA, was found activated by TSH, showing that
CREB is one of the important signaling effectors in FRTL-5 thyrocytes (46). Clearly, these studies indicated that a complex network was involved in TSH-induced Akt and CREB activation in
a cell-specific manner.
It is well known that TSH stimulates PLC and increases intracellular free Ca2⫹ concentrations and DAG (16, 19). Kim et
al. (47) confirmed these findings and found that TSH activated
PKC via PC-PLC-DAG, which required Rho activation but not
PKA. In the present study, TSH activated PKC as evidenced by
both PKC enzyme and immunoblot assays. Using a phosphospecific PKC antibody, we identified PKC-␨/␭ as highly expressed in
these cells on activation by TSH. Dominance of PKC-␨ and
PKC-␦ has been described in PCCL3 and PCE1Araf thyrocytes
(48). Because TSH activated cAMP, CREB, Akt, and PKC and
also induced FRTL-5 proliferation, these findings demonstrated
the multiple mitogenic factors involved in thyroid cell activation
and proliferation.
The naturally conformed TSHR is most effectively presented
as an autoantigen to the immune system, causing the production
of stimulating TSHR-Abs. Accumulating evidence indicates that
stimulating TSHR-Abs, either from Graves’ patients or animal
models of the disease, recognize conformational binding sites on
the ␣-subunit involving the leucine-rich repeats. Recently, Sanders and co-workers (49) defined the atomic structure of human
stimulating TSHR antibody (M22) used in our studies, and they
found that the amino acids in the TSHR ectodomain that determined the bioactivity of the antibody did not appear to be important for TSH binding and stimulation. Most of the amino
acids important for M22 interactions were, however, also found
to be important for the activity of six other stimulating monoclonals. They concluded that amino acid differences in the binding epitope were probably responsible for differences in the relative biological activities of the antibodies. Our study of three
stimulating monoclonals, which showed some variation in their
patterns of signal transduction, was also consistent with this
conclusion. These data, therefore, suggested that different stimulating antibodies interacted with the same region of the TSHR,
but there were likely to be subtle differences in the actual amino
acids that made contact with the different stimulators.
Although our stimulating antibodies used primarily TSH signaling pathways, there were differences in signaling activities
and signal strengths. Most importantly, the M22 antibody,
which had the highest affinity for the TSHR, demonstrated significantly increased cAMP/PKA/CREB activity as well as Akt
and PKC phosphorylation, which paralleled a high proliferation
index. Because TSH activated CREB to only a modest degree,
endo.endojournals.org
527
there were subtle differences in PKA/CREB activity induced by
stimulating antibodies, suggesting that antibody binding to
amino acids not contacted by TSH on the TSHR may be responsible for these effects. Furthermore, crystal structure studies of
the binding site topography of stimulating M22 antibody indicated that electrostatic potentials were also different from that of
TSH binding and may induce different effects (50). In particular,
signaling differences between TSH and M22 may be impacted by
amino acid arginine at position 255 in the TSHR ectodomain,
which was identified as an important site for interaction with
M22 but not with TSH. This may be responsible for p90RSK
activation, suggesting that M22 may also have activated additional signaling modules for downstream ERK regulation.
There are also antibodies that reduce TSH action at the TSHR
or decrease its constitutive activity without a potent signaling
capacity (51, 52). Igs from Graves’ and autoimmune (Hashimoto’s) thyroiditis patients have also recently been shown to compete with a blocking monoclonal antibody to the N terminus of
the TSHR ␤-subunit (amino acids 382-415) (53). Hence, blocking antibodies are most likely heterogeneous, and their epitopes
may lie broadly upstream of the ␣-subunit or downstream to the
␤-subunit (54). To help determine their functional consequences,
we characterized signaling molecules in exposed rat thyroid cells.
Most striking was that both TSHR-blocking antibodies (Tab-8
and RSR-B2) that we examined not only resulted in signal molecule activation but they also showed different pathway dominance. RSR-B2 activated the c-Raf/ERK/CREB pathway but not
p90RSK, whereas Tab-8 did not activate c-Raf/ERK but activated p90RSK. Although both of these antibodies recognize conformational epitopes, these findings may arise from differences
in their binding domains. Alternatively, the differences may be
due to interference with the constitutive activity of the TSHR.
Interestingly, one of the blocking TSHR-Abs (RSR-B2) induced
a small increase in cAMP levels that was paralleled by increased
cell proliferation, indicating that c-Raf/ERK and cAMP/CREB
activation may have contributed to this proliferation. Recently,
two independent groups identified blocking antibodies that suppress the constitutive activity of TSHR and cAMP generation
(52, 55). Indeed, these findings together with ours are in agreement with the hypothesis that the blocking antibodies may have
influenced thyrocyte functions by altering multiple signaling
mechanisms. Hence, these varieties of blocking antibodies could
be considered as heterotopic allosteric modulators causing either
activation or inhibition of TSHR activities. More study is necessary to correlate the effector mechanisms used by these antibodies with their different epitopes.
A third type of TSHR-Ab, the neutral TSHR-Abs, has also
been recognized (51, 56). Such antibodies have no influence on
TSH binding (57). It has, therefore, been assumed that neutral
TSHR-Abs should have no influence on TSHR action and are of
no clinical import. With the aim of examining the role these
neutral antibodies may play in thyroid biology, we characterized
their influence on signaling cascades and downstream effectors.
We identified two separate mechanisms working in the rat thyroid cells when exposed to two neutral antibodies (7G10 and
Tab-16). 7G10 suppressed multiple signaling modules, including
cell proliferation, whereas Tab-16 caused activation of many of
528
Morshed et al.
TSHR Signal Transduction
them paralleled with cell proliferation. However, both failed to
inhibit TSH binding (by definition), and both failed to activate
cAMP production. The amino acid sequences for these neutral
antibody binding sites have already been described by us and
other groups (30). 7G10 recognized 322-341 and Tab-16 identified 337-356 amino acid residues. Both of them failed to recognize CHO-NC35 cells expressing a TSHR variant lacking the
cleaved region (30, 58). Because an overlap of five amino acids
(337-341 residues) in these two antibody binding sequences exists, we suggest that the last 16 amino acids (341-356) may be
critical to signal activating events. These findings raise the possibility that multiple domains in this molecular stretch of the
TSHR may exist that suppress or stimulate the TSHR. Certainly,
neutral antibodies that bind to the uncleaved region appear capable of initiating a signal.
In conclusion, stimulating antibodies used signaling pathways similar to the TSH activation modules contributing to cell
activation and growth. Both TSH-blocking and neutral TSHR
antibodies distinguished different signaling networks and resulted in variable signal responses, indicating that some may be
weak agonists or inverse agonists. These observations help explain how TSHR-Abs may contribute to different clinical phenotypes in autoimmune thyroid disease.
Endocrinology, January 2009, 150(1):519 –529
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Acknowledgments
Address all correspondence and requests for reprints to: Dr. Syed A.
Morshed, Room 2F-26, Veterans Affairs Medical Center, 130 West
Kingsbridge Road, Bronx, New York 10468. E-mail: syed.morshed@
mssm.edu.
This work was supported in part by DK069713 from National Institute of Diabetes and Digestive and Kidney Diseases and in part by the
Veterans Affairs Merit Award program and the David Owen Segal
Endowment.
Disclosure Statement: S.A.M. and R.F. have nothing to declare.
T.F.D. is 1) a Member of the Board of Kronus Inc., which markets
diagnostic kits including those for thyroid autoantibodies and 2) a lecturer and consultant for Abbott Corp., manufacturers of L-T4.
20.
21.
22.
23.
24.
25.
26.
27.
References
1. Kimura T, Van KA, Golstein J, Fusco A, Dumont JE, Roger PP 2001 Regulation of thyroid cell proliferation by TSH and other factors: a critical evaluation of in vitro models. Endocr Rev 22:631– 656
2. Medina DL, Santisteban P 2000 Thyrotropin-dependent proliferation of in
vitro rat thyroid cell systems. Eur J Endocrinol 143:161–178
3. Bachrach LK, Eggo MC, Mak WW, Burrow GN 1985 Phorbol esters stimulate
growth and inhibit differentiation in cultured thyroid cells. Endocrinology
116:1603–1609
4. Roger PP, Reuse S, Servais P, Van HB, Dumont JE 1986 Stimulation of cell
proliferation and inhibition of differentiation expression by tumor-promoting
phorbol esters in dog thyroid cells in primary culture. Cancer Res 46:898 –906
5. Morrison DK, Cutler RE 1997 The complexity of Raf-1 regulation. Curr Opin
Cell Biol 9:174 –179
6. Marshall CJ 1994 MAP kinase kinase kinase, MAP kinase kinase and MAP
kinase. Curr Opin Genet Dev 4:82– 89
7. McKay MM, Morrison DK 2007 Integrating signals from RTKs to ERK/
MAPK. Oncogene 26:3113–3121
8. Dremier S, Milenkovic M, Blancquaert S, Dumont JE, Doskeland SO,
Maenhaut C, Roger PP 2007 Cyclic adenosine 3⬘,5⬘-monophosphate (cAMP)dependent protein kinases, but not exchange proteins directly activated by
28.
29.
30.
31.
32.
33.
34.
cAMP (Epac), mediate thyrotropin/cAMP-dependent regulation of thyroid
cells. Endocrinology 148:4612– 4622
Saavedra AP, Tsygankova OM, Prendergast GV, Dworet JH, Cheng G,
Meinkoth JL 2002 Role of cAMP, PKA and Rap1A in thyroid follicular cell
survival. Oncogene 21:778 –788
Frodin M, Gammeltoft S 1999 Role and regulation of 90 kDa ribosomal S6
kinase (RSK) in signal transduction. Mol Cell Endocrinol 151:65–77
Yang SH, Sharrocks AD 2006 Convergence of the SUMO and MAPK pathways on the ETS-domain transcription factor Elk-1. Biochem Soc
Symp121–129
Stoyanov B, Volinia S, Hanck T, Rubio I, Loubtchenkov M, Malek D,
Stoyanova S, Vanhaesebroeck B, Dhand R, Nurnberg B 1995 Cloning and
characterization of a G protein-activated human phosphoinositide-3 kinase. Science 269:690 – 693
Berridge MJ, Bootman MD, Roderick HL 2003 Calcium signalling: dynamics,
homeostasis and remodelling. Nat Rev Mol Cell Biol 4:517–529
Kasri NN, Holmes AM, Bultynck G, Parys JB, Bootman MD, Rietdorf K,
Missiaen L, McDonald F, De SH, Conway SJ, Holmes AB, Berridge MJ,
Roderick HL 2004 Regulation of InsP3 receptor activity by neuronal Ca2⫹binding proteins. EMBO J 23:312–321
Vanhaesebroeck B, Leevers SJ, Ahmadi K, Timms J, Katso R, Driscoll PC,
Woscholski R, Parker PJ, Waterfield MD 2001 Synthesis and function of
3-phosphorylated inositol lipids. Annu Rev Biochem 70:535– 602
Toker A, Newton AC 2000 Cellular signaling: pivoting around PDK-1. Cell
103:185–188
Balendran A, Hare GR, Kieloch A, Williams MR, Alessi DR 2000 Further
evidence that 3-phosphoinositide-dependent protein kinase-1 (PDK1) is required for the stability and phosphorylation of protein kinase C (PKC) isoforms. FEBS Lett 484:217–223
Williams MR, Arthur JS, Balendran A, van der KJ, Poli V, Cohen P, Alessi DR
2000 The role of 3-phosphoinositide-dependent protein kinase 1 in activating
AGC kinases defined in embryonic stem cells. Curr Biol 10:439 – 448
Belham C, Wu S, Avruch J 1999 Intracellular signalling: PDK1—a kinase at the
hub of things. Curr Biol 9:R93–R96
Downward J 1998 Mechanisms and consequences of activation of protein
kinase B/Akt. Curr Opin Cell Biol 10:262–267
Downward J 1998 Signal transduction. New exchange, new target. Nature
396:416 – 417
Cao X, Kambe F, Seo H 2005 Requirement of thyrotropin-dependent complex
formation of protein kinase A catalytic subunit with inhibitor of ␬B proteins
for activation of p65 nuclear factor-␬B by tumor necrosis factor-␣. Endocrinology 146:1999 –2005
Kikumori T, Kambe F, Nagaya T, Funahashi H, Seo H 2001 Thyrotropin
modifies activation of nuclear factor ␬B by tumour necrosis factor ␣ in rat
thyroid cell line. Biochem J 354:573–579
Baeuerle PA, Henkel T 1994 Function and activation of NF-␬B in the immune
system. Annu Rev Immunol 12:141–179
Baeuerle PA, Baltimore D 1996 NF-␬B: ten years after. Cell 87:13–20
Pawson T, Raina M, Nash P 2002 Interaction domains: from simple binding
events to complex cellular behavior. FEBS Lett 513:2–10
Schlessinger J 2000 Cell signaling by receptor tyrosine kinases. Cell 103:
211–225
Iacovelli L, Capobianco L, Salvatore L, Sallese M, D’Ancona GM, De BA 2001
Thyrotropin activates mitogen-activated protein kinase pathway in FRTL-5 by
a cAMP-dependent protein kinase A-independent mechanism. Mol Pharmacol
60:924 –933
Ando T, Latif R, Pritsker A, Moran T, Nagayama Y, Davies TF 2002 A
monoclonal thyroid-stimulating antibody. J Clin Invest 110:1667–1674
Ando T, Latif R, Daniel S, Eguchi K, Davies TF 2004 Dissecting linear and
conformational epitopes on the native thyrotropin receptor. Endocrinology
145:5185–5193
Ando T, Latif R, Davies TF 2004 Concentration-dependent regulation of
thyrotropin receptor function by thyroid-stimulating antibody. J Clin Invest
113:1589 –1595
Sanders J, Jeffreys J, Depraetere H, Richards T, Evans M, Kiddie A, Brereton
K, Groenen M, Oda Y, Furmaniak J, Rees SB 2002 Thyroid-stimulating monoclonal antibodies. Thyroid 12:1043–1050
Sanders J, Jeffreys J, Depraetere H, Evans M, Richards T, Kiddie A, Brereton
K, Premawardhana LD, Chirgadze DY, Nunez MR, Blundell TL, Furmaniak
J, Rees SB 2004 Characteristics of a human monoclonal autoantibody to the
thyrotropin receptor: sequence structure and function. Thyroid 14:560 –570
Sanders J, Allen F, Jeffreys J, Bolton J, Richards T, Depraetere H, Nakatake
N, Evans M, Kiddie A, Premawardhana LD, Chirgadze DY, Miguel RN,
Blundell TL, Furmaniak J, Smith BR 2005 Characteristics of a monoclonal
Endocrinology, January 2009, 150(1):519 –529
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
antibody to the thyrotropin receptor that acts as a powerful thyroid-stimulating autoantibody antagonist. Thyroid 15:672– 682
Nagy EV, Burch HB, Mahoney K, Lukes YG, Morris III JC, Burman KD 1992
Graves’ IgG recognizes linear epitopes in the human thyrotropin receptor.
Biochem Biophys Res Commun 188:28 –33
Ambesi-Impiombato FS, Parks LA, Coon HG 1980 Culture of hormone-dependent functional epithelial cells from rat thyroids. Proc Natl Acad Sci USA
77:3455–3459
Cass LA, Summers SA, Prendergast GV, Backer JM, Birnbaum MJ, Meinkoth JL
1999 Protein kinase A-dependent and -independent signaling pathways contribute to cyclic AMP-stimulated proliferation. Mol Cell Biol 19:5882–5891
Morshed SA, Parveen S, Leckman JF, Mercadante MT, Bittencourt Kiss MH,
Miguel EC, Arman A, Yazgan Y, Fujii T, Paul S, Peterson BS, Zhang H, King
RA, Scahill L, Lombroso PJ 2001 Antibodies against neural, nuclear, cytoskeletal, and streptococcal epitopes in children and adults with Tourette’s syndrome, Sydenham’s chorea, and autoimmune disorders. Biol Psychiatry 50:
566 –577
Lenzner C, Morgenthaler NG 2003 The effect of thyrotropin-receptor blocking antibodies on stimulating autoantibodies from patients with Graves’ disease. Thyroid 13:1153–1161
Schwarz-Lauer L, Chazenbalk GD, McLachlan SM, Ochi Y, Nagayama Y,
Rapoport B 2002 Evidence for a simplified view of autoantibody interactions
with the thyrotropin receptor. Thyroid 12:115–120
Stork PJ, Schmitt JM 2002 Crosstalk between cAMP and MAP kinase signaling
in the regulation of cell proliferation. Trends Cell Biol 12:258 –266
Dremier S, Vandeput F, Zwartkruis FJ, Bos JL, Dumont JE, Maenhaut C 2000
Activation of the small G protein Rap1 in dog thyroid cells by both cAMPdependent and -independent pathways. Biochem Biophys Res Commun
267:7–11
Gilbert JA, Gianoukakis AG, Salehi S, Moorhead J, Rao PV, Khan MZ,
McGregor AM, Smith TJ, Banga JP 2006 Monoclonal pathogenic antibodies
to the thyroid-stimulating hormone receptor in Graves’ disease with potent
thyroid-stimulating activity but differential blocking activity activate multiple
signaling pathways. J Immunol 176:5084 –5092
Suh JM, Song JH, Kim DW, Kim H, Chung HK, Hwang JH, Kim JM, Hwang
ES, Chung J, Han JH, Cho BY, Ro HK, Shong M 2003 Regulation of the
phosphatidylinositol 3-kinase, Akt/protein kinase B, FRAP/mammalian target
of rapamycin, and ribosomal S6 kinase 1 signaling pathways by thyroid-stimulating hormone (TSH) and stimulating type TSH receptor antibodies in the
thyroid gland. J Biol Chem 278:21960 –21971
Mei FC, Qiao J, Tsygankova OM, Meinkoth JL, Quilliam LA, Cheng X 2002
Differential signaling of cyclic AMP: opposing effects of exchange protein
directly activated by cyclic AMP and cAMP-dependent protein kinase on protein kinase B activation. J Biol Chem 277:11497–11504
Kim H, Suh JM, Hwang ES, Kim DW, Chung HK, Song JH, Hwang JH, Park
KC, Ro HK, Jo EK, Chang JS, Lee TH, Lee MS, Kohn LD, Shong M 2003
Thyrotropin-mediated repression of class II trans-activator expression in thyroid cells: involvement of STAT3 and suppressor of cytokine signaling. J Immunol 171:616 – 627
Kim JH, Kim SW, Jung PJ, Yon C, Kim SC, Han JS 2002 Phosphatidylcholine-
endo.endojournals.org
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
529
specific phospholipase C and RhoA are involved in the thyrotropin-induced
activation of phospholipase D in FRTL-5 thyroid cells. Mol Cells 14:272–280
Urso L, Muscella A, Calabriso N, Ciccarese A, Fanizzi FP, Migoni D, Di JB,
Storelli C, Marsigliante S 2005 Differential functions of PKC-␦ and PKC-␨ in
cisplatin response of normal and transformed thyroid cells. Biochem Biophys
Res Commun 337:297–305
Sanders J, Bolton J, Sanders P, Jeffreys J, Nakatake N, Richards T, Evans M,
Kiddie A, Summerhayes S, Roberts E, Miguel RN, Furmaniak J, Smith BR
2006 Effects of TSH receptor mutations on binding and biological activity of
monoclonal antibodies and TSH. Thyroid 16:1195–1206
Sanders J, Chirgadze DY, Sanders P, Baker S, Sullivan A, Bhardwaja A, Bolton
J, Reeve M, Nakatake N, Evans M, Richards T, Powell M, Miguel RN,
Blundell TL, Furmaniak J, Smith BR 2007 Crystal structure of the TSH
receptor in complex with a thyroid-stimulating autoantibody. Thyroid 17:
395– 410
Rapoport B, Chazenbalk GD, Jaume JC, McLachlan SM 1998 The thyrotropin (TSH) receptor: interaction with TSH and autoantibodies. Endocr Rev
19:673–716
Sanders J, Evans M, Betterle C, Sanders P, Bhardwaja A, Young S, Roberts E,
Wilmot J, Richards T, Kiddie A, Small K, Platt H, Summerhayes S, Harris R,
Reeve M, Coco G, Zanchetta R, Chen S, Furmaniak J, Smith BR 2008 A human
monoclonal autoantibody to the thyrotropin receptor with thyroid-stimulating blocking activity. Thyroid 18:735–746
Minich WB, Lenzner C, Morgenthaler NG 2004 Antibodies to TSH-receptor
in thyroid autoimmune disease interact with monoclonal antibodies whose
epitopes are broadly distributed on the receptor. Clin Exp Immunol 136:
129 –136
Schwarz-Lauer L, Pichurin PN, Chen CR, Nagayama Y, Paras C, Morris JC,
Rapoport B, McLachlan SM 2003 The cysteine-rich amino terminus of the
thyrotropin receptor is the immunodominant linear antibody epitope in mice
immunized using naked deoxyribonucleic acid or adenovirus vectors. Endocrinology 144:1718 –1725
Chen CR, McLachlan SM, Rapoport B 2007 Suppression of thyrotropin receptor constitutive activity by a monoclonal antibody with inverse agonist
activity. Endocrinology 148:2375–2382
Tonacchera M, Costagliola S, Cetani F, Ducobu J, Stordeur P, Vassart G,
Ludgate M 1996 Patient with monoclonal gammopathy, thyrotoxicosis,
pretibial myxedema and thyroid-associated ophthalmopathy: demonstration
of direct binding of autoantibodies to the thyrotropin receptor. Eur J Endocrinol 134:97–103
Minich WB, Loos U 2000 Detection of functionally different types of pathological autoantibodies against thyrotropin receptor in Graves’ patients sera by
luminescent immunoprecipitation analysis. Exp Clin Endocrinol Diabetes
108:110 –119
Kakinuma A, Chazenbalk GD, Tanaka K, Nagayama Y, McLachlan SM,
Rapoport B 1997 An N-linked glycosylation motif from the noncleaving luteinizing hormone receptor substituted for the homologous region (Gly367 to
Glu369) of the thyrotropin receptor prevents cleavage at its second, downstream site. J Biol Chem 272:28296 –28300