Introduction
Over time it has become clear that the fascination with the TSH receptor (TSHR) is
not only its complexity and its relationship to human disease but the fact that it
keeps teaching us fundamental biology at all levels; cellular, molecular, and genetic.
There are good examples of each of these facets in this cutting edge collection of
papers. This contribution provides a brief and broad overview highlighting those areas
of active progress by briefly eluding to some of the contributions in this collection.
The TSHR is a member of the class A family of G-protein coupled receptors (GPCR) with
seven transmembrane helices traversing the plasma membrane and a large extracellular
ectodomain. The ectodomain (ECD) is linked to a distal signal-specific domain—the
hinge region—which is attached to a transmembrane domain (TMD) consisting of extracellular
(ECL) and intracellular (ICL) loops (Figure 1). A partial TSHR ectodomain (residues
1–260) has been crystallized either bound to a stimulating TSHR antibody and/or a
blocking TSHR antibody (1, 5) and recently in an unbound native state with stabilizing
mutations. Like other GPCRs, the TSH receptor can also not exist in an ensemble of
conformational states which can lead to its varied signaling potential. The review
by Kleinau et al. in this collection takes a comprehensive look at the structure-function
relationship of the TSHR via modeling and mutational approaches. It is now well-known
that the full-length TSHR undergoes complex post translational processing (6, 7) inclusive
of common protein modifications such as glycosylation and phosphorylation and even
whole receptor modifications such as cleavage and multimerization (7, 8) thus resulting
in a surprising variety of receptor configurations, many of which are expressed on
the cell surface (9) and in some cases even shed from the cell surface (10). Although
the shed receptor forms have not been conclusively demonstrated in the serum of patients
with Graves' disease (GD), probably secondary to degradation, the evidence that these
and other receptor structures are critical to the immunopathogenesis of GD has been
well-covered in the review by Inaba et al.
Figure 1
Homology model of the entire TSH holoreceptor. This model highlights the tripartite
structure of the TSHR. The ectodomain, shown in gray/black, is made up of 10 leucine-rich
repeat domains (LRD) characterized as a “scythe-blade” shaped structure with loops
and β pleated sheets obtained from the published crystal structure (1) (PDB:3G04).
The region connecting the LRD and transmembrane domain (TMD), known as the “hinge”
region, has recently been crystallized for the FSH receptor (2) (PDB:4AY9) and is
shown as a looped structure (orange) with a helix conformation close to the carboxyl
end of the LRD. The hinge in the TSHR has an additional sequence insert and is larger
than in the FSH receptor. Therefore, amino acids 305-381 are missing in the illustrated
model (3) and this insert is depicted as a closed dotted loop. The TMD (yellow), with
its seven helices, is depicted as cylindrical structures connected to each other by
the specific TSHR intra and extracellular loops. The TMD is the region that harbors
the allosteric binding pockets for the SMLs. LRD, leucine-rich domain; TMD, transmembrane
domain; ECL, extracellular loops; and ICL, intracellular loops [Figure adapted from
(4)].
Signal transduction at the TSHR is complex because of the promiscuous nature of the
TSHR in engaging with different G proteins (11). In addition, the TSHR signals can
be both G protein dependent and G protein independent. The TSHR has been shown to
engage predominantly β-arrestin-2 for internalization (12) and arrestin-1, in human
osteoblast cells, for differentiation, and MAP kinase signaling (13). In addition,
it has long been known that the TSHR is involved with the IGF1/insulin receptor in
thyroid cells and the “marriage” of these two receptors in fibroblasts has suggested
their involvement in Graves' eye disease pathophysiology as well-reviewed by Smith
et al.. The complex life cycle of GPCRs such as the TSHR (Figure 2) has also begun
to be revealed showing that these types of GPCRs, after being sequestered via clathrin-coated
pits or caveolin scaffolding proteins, are still able to signal after internalization.
New evidence points out that these internalized receptors can lead to a “second wave”
of signals from the TSHR (14). The result is that not only does the receptor come
in multiple configurations but there are also multiple signal pathways that may or
may not be initiated as the receptor conformation changes on ligand binding and this
may continue after the receptors are internalized. The days of thinking simply of
the TSH induced cyclic AMP response coming only from the surface receptors have long
gone. Single-particle electron microscopy has confirmed the presence of intracellular
megaplexes which consist of a GPCR bound to β-arrestin at its C terminus and a G protein
complex at its core (15). The crystallization of a GPCR bound to G proteins has enhanced
our understanding that ligands can stabilize different receptor conformations and
that these ligand bound receptor complexes can stabilize different effector conformations
leading to diversified signaling. However, such full-length receptor and G protein
crystallized conformation(s) have not yet been achieved for the TSHR.
Figure 2
Generic life-cycle of the TSH receptor. The TSHR residing on the plasma membrane of
thyrocytes on binding with its cognate ligand, TSH, is activated and in turn undergoes
conformational changes to recruit and activate a G protein complex leading to a predominate
wave of Gs generated cAMP (Signal 1). The activated receptor, after signal 1, is phosphorylated
and then moved to clathrin-coated pits where β-arrestin is bound to the activated
receptor. At this stage it is believed that the receptor can signal via arrestin leading
to β-arrestin-associated signals. Furthermore, this activated receptor in the invaginated
pits is pinched-off to form the early and late endosomes. It is described that within
the endosome the receptor with its associated ligand and second messengers is capable
of giving out a second wave cAMP signal (Signal 2). Following this the receptor can
be either degraded or it enters recycling vesicles and is recycled to the plasma membrane
whereas the ligand is transported to the lysosome and is degraded. This is the life
that the TSHR lives on the surface of thyrocytes or any other cell where it is expressed.
TSHR Stimulators
The TSHR can be activated by TSH itself, or by autoantibodies which can bind to the
orthosteric site(s) on the large ectodomain. In fact, activation of the TSHR has been
in clinical use for many years. Semi-purified bovine TSH was originally used for short-term
thyroid testing of TSHR function but proved to have too many immune related side effects
in clinical practice. The clinical use of TSH was not widely adopted until the introduction
of recombinant human TSH in the 1990's. This is now used for detecting thyroglobulin
release from metastatic thyroid cancer and for enhancing RAI uptake into thyroid glands
(16–18). The discovery of stimulating TSHR antibodies by Adams and Purves (19) demonstrated
the cause of Graves' disease and helped open up the entire field of autoimmune disease.
Since the discovery of TSHR autoantibodies there has been the development of clinical
assays to effectively detect these antibodies in Graves' patients with improving accuracy
and sensitivity. The reviews by Giuliani et al. tracing the development of TSH bioassays
and by Kahaly et al. on functionality and nomenclature are interesting and important
in this regard. Although the current assays for detecting these antibodies are relatively
robust the solid phase assays cannot detect bioactivity and the cell based bioassays
are also not ideal where high concentrations of blocking antibodies may decrease the
TSHR response to stimulating antibodies. Such problems arise due to the plethora of
antibodies with variable bioactivities seen in GD indicative of a wide spectrum of
variable activities as discussed further below.
In recent years it been shown by several investigators that selected small molecule
ligands (SML) can easily permeate the plasma membrane and allosterically activate
or inhibit TSHR signals. High throughput functional screening methods led to their
identification and has opened up new therapeutic potentials (20–22). Furthermore,
the concept that various effectors can stabilize the TSHR in a particular conformation
has opened the possibility of biased TSHR signaling as achieved with other GPCR's
(23, 24).
TSHR Antagonists
A major clinical need is for potent TSHR antagonists that can block the TSHR antibodies
of hyperthyroid Graves' disease allowing us to dispense with the side effects of the
common antithyroid drugs (methimazole and PTU) which deter many physicians from their
long term use. A blocking human monoclonal TSHR antibody has been proposed as one
method of achieving this aim (25) and results of a Phase II clinical trial are awaited.
Although therapeutic antibodies have the theoretical advantage of specificity so do
potential small molecule TSHR antagonists. Several groups, including our own laboratory
as described by Latif et al. included in this collection (26, 27), have shown that
allosteric inhibition of TSHR G protein signaling can silence the TSHR receptor. However,
low potency and inadequate specificity of these SML antagonists indicate that more
hurdles have to be crossed for the advancement of this approach. Peptide mimetics
and aptamers to the TSHR that can either disrupt signaling via preventing G protein
binding or by interfering with TSHR antigen processing are also under development
and in early stage clinical trials and further data are awaited.
Extra-Thyroidal TSHRs
At long last it is becoming widely known that the TSHR is expressed in more places
than the thyroid gland and can even be found to be expressed in embryonic stem cells
suggesting a role in development (11). The TSHR is expressed in fibroblasts, adipocytes,
bone cells, and a variety of additional cell types (28, 29) and have, in particular,
attracted a lot of attention in the retro-orbit (30–32) and bone (33, 34). This ubiquitous
presence of the receptor clearly suggests that it has more functions than controlling
thyroid hormone production. The role of TSHR activation and its signaling influence
on adipocytes has been studied (35) and activation of the TSHR can modulate adipogenesis
and fat cell phenotype further reinforced in the article by Draman et al.. The role
of the TSHR in differentiation of preadipocytes into mature adipocytes from embryonic
stem cells has also been shown (36) although the signals that influence this differentiation
pathway are still unclear. The “Graves' Disease Triad” consists of hyperthyroidism
with a dermopathy, referred to as pre-tibial myxedema, and an orbitopathy often referred
to as Graves' Eye Disease and involves fibroblasts and adipocytes at both extra-thyroidal
sites. Retro-orbital expression of the TSHR, in combination with IGF-1 receptors (37),
expressed on the fibroblasts and adipocytes behind the eye appear to be involved in
the pathogenesis of Graves' orbitopathy GO- (see Smith et al.) and serum TSHR-Ab levels
tend to correlate with eye disease (38–40). IGF-1 is well-known to enhance TSH action
on thyroid cells and recent studies show that blockade of the IGF-1R appears to be
a useful mode of therapy for GO (41, 42) presumably by reducing stimulating TSHR-Ab-induced
adipocyte proliferation and cytokine release from retro-orbital fibroblasts. Such
cytokines contribute to glycosaminoglycan generation and disrupt the osmotic pressure
behind the eyes causing muscle fiber damage and swelling (42, 43). Similarly, our
work on TSHR expression in osteoblasts and osteoclasts has identified TSH as a potential
osteoprotective molecule (33). The identification of a TSH-β subunit splice variant
secreted by bone marrow macrophages may be the effector of this protective effect
as discussed in detail by Baliram et al. (44).
TSHR Antibodies
One of the unique characteristics of Graves' disease, not found in normal individuals
or in the rest of the animal kingdom, is the presence of TSHR antibodies (TSHR-Ab)
which are easily detectable in the vast majority of patients as discussed earlier
(45). In such patients, TSHR-reactive T cells and B cells survive central and peripheral
deletion and under appropriate circumstances the B cells secrete TSHR antibodies and
also induce T cells to secrete pro-inflammatory cytokines (46). Hence both B cells
and T cells play a central role in mediating the chronic inflammatory changes of the
autoimmune diseases seen in the thyroid gland, in the retro-orbit and in the skin
(19), and may be resistant to T regulatory cell (Treg) control or allowed to be active
secondary to inadequate Treg function (47). Although TSHR autoantibodies represent
the hallmark of GD, finding the triggers that lead to this immunological derangement
has been a challenge. Genome-wide association studies have established the association
of the TSHR gene specifically with GD and understanding the functional mechanism by
which such polymorphisms modify the physiological processes and trigger disease by
interfering with central tolerance is outlined in the review by Stefan et al.. Whatever
may be the major mechanisms for these triggers we now see three varieties of TSHR-Ab
that can be found in patients with autoimmune thyroid disease and in TSHR immunized
rodents; stimulating, blocking, and so called “neutral” antibodies; the latter often
directed at the hinge region of the TSHR ectodomain and are far from being neutral
in their biological activity. Stimulating antibodies induce cyclic AMP, thyroid cell
proliferation and thyroid hormone synthesis, and secretion. They bind exclusively
to conformational epitopes in the TSHR ectodomain leucine rich repeat region and compete
with TSH for binding. TSHR blocking antibodies compete with TSH for binding and once
bound they inhibit TSH action to a variable extent. However, the degree of blocking
may be profound enough that they may induce hypothyroidism although some blocking
TSHR antibodies may actually behave as weak TSHR agonists. In contrast, the neutral
TSHR antibodies neither block TSH binding nor block TSH action but may be involved
in aberrant signal initiation and thyroid cell apoptosis (48, 49). It is important
to also remember that TSHR antibodies have an important role to play in pregnancy
because these antibodies cross the placenta and influence both maternal and fetal
thyroid function and their biochemical and immunological aspects are well-dealt with
by Bucci et al..
Apoptosis in Graves' Disease
It is now apparent that apoptosis plays an important role in the development and perpetuation
of autoimmune thyroid disease. Areas of apoptosis are recognized in thyroid tissue
from patients with Hashimoto's Thyroiditis and Graves' disease (50). Subsequent studies
on apoptosis have provided insight into autoimmune target destruction, indicating
the involvement of death receptors and cytokine-regulated apoptotic pathways in the
pathogenesis, and perpetuation of thyroid autoimmunity. There is evidence that such
thyrocyte apoptosis in Graves' disease may be antibody induced (51) or T cell mediated
via defects in T regulatory cells which induce an abnormal production of cytokines
(52) or changes in the expression of apoptotic molecules (Fas/FasL and caspase 8)
on the surface of T lymphocytes and thyroid follicular cells (53, 54). In fact, all
antibody binding to the thyroid cell induces thyroid cell stress, as first shown by
our own laboratory, but we have shown that some neutral antibodies induce excessive
ROS accumulation leading to thyroid cell apoptosis in the absence of G-protein signaling
(49, 55, 56). This antibody induced apoptosis can facilitate the breakdown of self-
tolerance mechanisms in individuals with the right major histocompatibility complex
(MHC) class II background in myriad ways. It could be the release of excessive cytosolic
DNA fragments that can act as adjuvants/immune modulators and induce aberrant MHC
II expression in thyrocytes thus inducing the release of multiple inflammatory cytokines
and chemokines as seen in various animal models and well-reviewed by Luo et al. in
this collection.
The Multiplicity of TSH Receptor Forms and Responses May Explain the Graves' Disease
Phenotype
With the initial discovery of the classical G-protein–coupled-receptors (GPCR) the
essential mechanisms appeared at first to be straightforward. The ectodomain was responsible
for hormone specificity and the intracellular domain was responsible for the cyclic
AMP signal. Each receptor had a specific ligand and an expected action. The receptor
for TSH was very similar to that for FSH and LH/hCG and each activated PKA and the
cyclic AMP pathway. Such simplicity, however, was short lived. Firstly, the TSHR was
found to have two unique inserts into the ectodomain, including one which made it
subject to complex post translational processing not seen with the LHR and FSHR. Then
the phenomenon of specificity cross-over reared its head. Suddenly the concept of
high specificity of a hormone receptor was in doubt. For example, a number of ligands
are able to bind to and activate the TSHR including hCG and LH. Stimulation of the
TSHR by hCG is seen in gestational thyrotoxicosis (57) and in choriocarcinoma and
a unique TSHR mutation even more highly hCG reactive has been described. With the
burgeoning of our understanding into the structure of the TSHR by comparative modeling
and partial crystal structures the entire field of TSHR signal transduction opened
up. TSH/hCG and small molecule agonists could initiate different signals depending
on the concentration of ligand available for receptor binding, the number of receptors
activated, the forms of receptor (dimeric vs. monomeric) and also the orthosteric
vs. the allosteric sites. Hence, we have the issue of multiple specificities and multiple
signal responses indicating that an enormous number of variables are at play at just
one GPCR. If we then consider Graves' disease and its multiple clinical forms which
can vary from a highly localized thyroid disease to almost a systemic autoimmune diathesis
much of this may be explicable by the variable forms of the receptor available for
immune activation, the variable sites of TSHR expression and the multiplicity of signals
that the TSHR can employ. In addition, the presence of differing proportions of high
affinity TSHR-Abs with varied biological activity in patients with GD no doubt also
contributes to the multiple clinical phenotypes; varying from hyperthyroidism to hypothyroidism
and vice versa and with or without Graves' orbitopathy and pre-tibial myxedema.
Conclusion
The collection of papers that form part of this special issue shows the different
facets of the TSHR thus allowing us to rightly say that many roads lead from and to
this GPCR. For sure the TSHR, with its structural and signaling complexity, is going
to hold our scientific imagination and enthusiasm for many more years to come.
Author Contributions
All authors listed have made a substantial, direct and intellectual contribution to
the work, and approved it for publication.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.
The handling Editor declared a shared affiliation, though no other collaboration with
the authors.