The thyrotropin receptor (TSHR) is a seven-transmembrane protein that belongs to the family of G-protein-coupled receptors (GPCR). The receptor is located on the surface of thyroid follicular cells, and its activation leads to thyroid hormone production and growth and proliferation of thyrocytes.
The primary activator of the TSHR is the thyroid-stimulating hormone (TSH) or thyrotropin, although the receptor may also be activated by stimulating antibodies as occurs in autoimmune hyperthyroidism, known as Graves’ disease. The TSHR is expressed in many non-thyroidal tissues and may be involved in the systemic manifestations of thyroid disease, such as thyroid-associated ophthalmopathy, pretibial myxedema, and metabolic bone disease.
Structurally, the TSHR can be divided into a large extracellular domain (ECD), a seven-transmembrane domain (TMD) with its connecting intracellular loops (ICL) and extracellular loops (ECL), and an intracytoplasmic tail. It shares structural similarities with the other glycoprotein hormone receptors (GPHR) including luteinizing hormone (LH), follicular-stimulating hormone (FSH), and human chorionic gonadotropin (hCG) receptors.
The TSHR gene is located on chromosome 14q13 and contains 10 exons. The ECD is encoded by exons 1–9, while the TMD and the cytoplasmic carboxy tail are encoded by exon 10. Cloning of the TSHR gene in 1989 (Parmentier et al. 1989) was followed by the discovery of multiple mutations of the TSHR gene. These mutations may lead to increased or decreased activity of the receptor and may be associated with the development of thyroid disease.
Structure and Function of the TSHR
TMD and intracytoplasmic tail made up of 349 amino acids
ECD made up of 394 amino acids
21 amino acid signal peptide at the extracellular N-terminus of the protein
The TSHR ECD carries two unique segments, residues 38–45 and 316–366, that are not shared by the other GPHR. The shorter segment contains cysteine-41 that mediates TSH binding, and the larger segment is likely to be lost during intramolecular cleavage of the TSHR into two disulfide-linked subunits, a 394 alpha subunit, and a 349 beta subunit. It is thought that TSH binding to the TSHR may stimulate cleavage of the receptor. However, cleavage of the receptor may not be an absolute necessity for receptor function, as single-chain uncleaved receptors have been reported (Latif et al. 2004; Chen et al. 2006).
While not yet demonstrated in vivo, the alpha subunit is likely to be shed from the cell surface into the circulation following cleavage and may contribute to the development of thyroid autoimmunity and the generation of anti-TSHR autoantibodies (Vassart and Costagliola 2004). Of note, activity of the beta subunit is enhanced after cleavage suggesting that the ECD suppresses constitutive activity of the receptor.
In addition to intramolecular cleavage, other posttranslational changes such as polymerization, glycosylation, and sulfation may impact the function of the TSHR. Polymerization, or the formation of TSHR-TSHR complexes on the cell surface, increases receptor heterogeneity and influences TSH binding to the TSHR. Similarly, glycosylation of the ECD at up to six glycosylation sites is essential for TSH binding and plays a role in the folding and surface expression of the receptor, while sulfation of tyrosine residues in the hinge region is of paramount importance for TSH binding.
Following ligand binding at the LRR of the ECD, the TMD and its ICL carry the signal from the ECD to the intracytoplasmic G-proteins, leading to activation of the latter and a complex cascade of intracellular events. Gs protein activation stimulates the production of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) with subsequent activation of the cAMP response element binding (CREB) and other transcription factors. The net result is thyroid follicular cell growth, proliferation, and the production of thyroid hormones (Latif et al. 2009).
Higher levels of TSH are likely to stimulate the Gq protein located at the second ICL of the TMD. This leads to the activation of the phospholipase C (PLC)-dependent pathway with the production of hydrogen peroxide and iodination, mediated by inositol trisphosphate (IP3) and diacylglycerol (DAG). Furthermore, the PLC pathway activates the Akt pathway that is thought to increase cell proliferation and survival (Allgeier et al. 1994).
Of note, protein kinases A and C that are produced by the cAMP and PLC pathways, respectively, directly impact gene transcription and have become therapeutic targets in the treatment of thyroid cancer.
Stimulation of the TSHR can activate additional pathways that are involved in cell proliferation and differentiation, such as the murine target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) pathways.
Recent evidence suggests that the insulin-like growth factor-1 (IGF-1) receptor (IGF-1R) may regulate the function of the TSHR through cross talk between the two receptors (Morgan et al. 2016). It was reported that IGF-1 and TSH have synergistic effects on the expression of sodium iodide symporter mRNA and additive effects on the production of thyroglobulin, thyroid peroxidase, and deiodinase type 2 mRNA in cultured human thyrocytes. By blocking the IGF-1R activity, the authors demonstrated inhibition of TSH-stimulated upregulation of NIS expression. This process is thought to occur through the ERK1/2 and Akt pathways.
Role in Thyroid Disease
While the TSHR is crucial for thyroid function and growth, it plays an important role in various thyroid disorders including autoimmune thyroid disease (AITD), autonomously functioning thyroid nodules (AFTN), differentiated thyroid cancer (DTC), thyroid hormone resistance, and hCG-induced hyperthyroidism (Kopp 2001).
AITD refers to autoimmune hyperthyroidism and hypothyroidism, although the role of the TSHR in autoimmune hypothyroidism is not well understood particularly as the majority of patients with autoimmune atrophic thyroiditis do not have elevated Thyroid stimulation blocking antibodies (TSBAbs).
In contrast, the TSHR is the main autoantigen in autoimmune hyperthyroidism which is better known as Graves’ disease and is associated with the presence of thyroid-stimulating antibodies (TSAbs) in the majority of patients. The TSAbs stimulate the TSHR in the thyroid gland leading to the development of goiter and hyperthyroidism and may play a role in the development of thyroid-associated ophthalmopathy and dermopathy (Weetman 2000).
Thyroid autoantibodies bind to the LRR of the ECD, at the same site of TSH binding. While TSH binding may stimulate cleavage of the receptor, it is postulated that antibody binding to the TSHR increases expression of the receptor at the cell surface by inhibiting posttranslational cleavage, thereby increasing the antigenic load and facilitating propagation of AITD. This applies to the TSBAbs, TSAbs, and possibly to neutral TSHR antibodies which are not thought to affect TSH binding to the receptor (Latif et al. 2009). The latter antibodies bind at the cleavage region and may influence thyroid cell signaling and induce apoptosis.
AITD poses a risk of thyroid dysfunction to the fetus in pregnant women with AITD. Transplacental passage of stimulating and/or blocking antibodies may lead to fetal hyper- and hypothyroidism, respectively. Fetal thyroid function is usually restored within 3–6 months following delivery as the antibodies are cleared from the neonate’s circulation.
Stimulation of the TSHR by hCG can lead to hyperthyroidism as occurs in patients with hydatidiform moles and choriocarcinoma and during the first trimester of pregnancy. The latter condition is transient and may be associated with hyperemesis gravidarum. Point mutations in the TSHR gene are associated with hypersensitivity to hCG and a familial form of gestational hyperthyroidism (Coulon et al. 2016), thus TSHR gene sequencing is recommended for persistent and severe gestational thyrotoxicosis with normal serum hCG concentrations and negative TSAbs.
Mutations of the TSHR
There are two main types of mutations, somatic and germline. Somatic mutations lead to activation of the TSHR as occurs in up to 80% of autonomously functioning thyroid nodules (AFTNs) and toxic multinodular goiters (TMNG) and less commonly in differentiated thyroid carcinoma (DTC).
Somatic mutations are more likely to involve the TMD rather than the ECD, leading to constitutive activation of TSHR through the cAMP pathway as opposed to the IP3 cascade. Potential mechanisms leading to activation of the mutated TSHR include conformational changes that mimic ligand binding, disinhibition of receptor activation in the absence of a ligand, and increased activity of the cAMP pathway. Of note, activating mutations of the alpha subunit of the stimulating guanine nucleotide-binding protein (Gs) have been reported to occur in AFTNs, usually as part of the McCune-Albright syndrome which is associated with cafe au lait skin pigmentation, polyostotic fibrous dysplasia, and precocious puberty.
Unlike somatic mutations, germline mutations of the TSHR may be activating or inactivating. Non-autoimmune hyperthyroidism, subclassified as familial (FNAH) or sporadic (SNAH), is an autosomal dominant condition associated with mutations in exon 10 and affects the TMD resulting in increased TSHR activity. SNAH tends to present shortly after birth with severe hyperthyroidism, whereas FNAH presents at any age in an individual with a family history of hyperthyroidism affecting two or more generations and may be associated with extrathyroidal manifestations such as developmental delay, cerebral palsy, and cardiac valvular disease (Gozu et al. 2010). However, these mutations are not common, and the majority of hyperthyroid patients with diffuse goiter and negative TRAb do not have mutations of the TSHR gene, as was shown in a Japanese study of 89 patients of which only 4.5% had germline mutations of the TSHR (Nishihara et al. 2014).
Inactivating mutations of the TSHR were first described in 1995, with approximately 51 mutations reported at the time of writing this manuscript. Most loss-of-function mutations are located in the ECD as opposed to activating mutations which are predominantly located in the transmembrane and intracellular domains of the receptor. These mutations lead to impaired receptor function through one or more of several mechanisms including reduced ligand binding, inefficient TSH-mediated activation of the cAMP and IP3 pathways, and decreased cell surface expression of the receptor (Persani et al. 2010). These mutations may be the result of frameshift, missense and nonsense mutations, or altered intron-exon boundaries. Patients may present with congenital hypothyroidism or subclinical hypothyroidism. The inheritance is recessive and compared to heterozygous patients; homozygous patients tend to present with more severe hypothyroidism and are more likely to require thyroid hormone replacement (Tenenbaum-Rakover et al. 2015). Interestingly, The TSHR mutations may co-exist with inactivating mutations of the Gs and thyroid peroxidase genes. Recent studies have reported small molecule TSHR agonists that are capable of overcoming the resistance to endogenous TSH and can activate the TSHR (Allen et al. 2011). Unlike TSH, small molecule agonists bind to the TMD and can bypass the mutated ECD of the receptor as described in the next section.
The TSHR as a Diagnostic and Therapeutic Target in Thyroid Disease
The TSHR plays a central role in many thyroid disorders, and modifying its activity in certain conditions may be clinically beneficial (El-Kaissi and Wall 2012). This view represents a novel approach to the conventional management of thyroid disorders, whereby overactivity of the thyroid is often treated with antithyroid medications (thionamides) or thyroid ablative therapy, while hypothyroidism is managed with thyroid hormone replacement.
In this regard, recent attention has shifted toward TSHR monoclonal antibodies that can stimulate or block the activity of the TSHR and small molecule (SM) TSHR effectors. The drug-like SM compounds can be designed to function as TSHR agonists, antagonists, or inverse agonists. SM agonists stimulate the TMD of the TSHR and may be useful in patients with TSH resistance due to deactivating mutations of the ECD. They may also prove valuable in the diagnosis of thyroid cancer remnants in post-thyroidectomy DTC patients by stimulating radioactive iodine uptake and thyroglobulin production from thyroid cancer foci, without having to discontinue thyroid hormone replacement (Emerson 2011).
In contrast, SM antagonists may have a multitude of clinical benefits where blocking the activity of the TSHR is clinically indicated, as in patients with primary or secondary hyperthyroidism, and in those with DTC. In the latter condition, SM antagonists could be theoretically combined with levothyroxine in a “block and replace” regimen, thereby allowing patients with DTC to maintain suppression of residual disease activity while avoiding the potential side effects of TSH suppression such as palpitations, atrial fibrillation, and detrimental effects on bone mineral density.
From a diagnostic standpoint, the detection of circulating TSHR-mRNA may prove to be a useful tool in the confirmation of thyroid malignancy in indeterminate nodules preoperatively (Aliyev et al. 2016), as well as the detection of residual or recurrent DTC in patients with positive anti-thyroglobulin antibodies, which limit the utility of serum thyroglobulin in thyroid cancer surveillance.
The TSHR is a large seven-transmembrane G-protein-coupled receptor that is made up of the ECD which contains the ligand-binding LRR and is connected to the TMD by the hinge region. The TSHR is activated predominantly by TSH but can also be activated by stimulating autoantibodies and to a lesser extent by hCG. The TSHR undergoes intramolecular cleavage into two subunits, alpha and beta, and it is argued that the extracellular alpha subunit is shed into the circulation following cleavage although this remains to be demonstrated in vivo. As cleavage is stimulated by TSH binding at the LRR, shedding of the alpha subunit into the circulation may prevent further activation of the receptor by TSH, thereby representing a rate-limiting step in receptor activation.
The TSHR gene is located on chromosome 14q13 and contains ten exons. The first 9 exons encode the ECD while the TMD and the cytoplasmic carboxy tail are encoded by exon 10. Somatic mutations of the TSHR gene affect the TMD and lead to increased constitutive activity of the receptor, presenting clinically as AFTNs. Germline mutations may lead to increased or decreased activity of the receptor, the latter presenting as TSH resistance.
Given the central role of the TSHR in the development of thyroid dysfunction not only in patients with TSHR gene mutations but also those with stimulating and blocking thyroid autoantibodies, recent research has focused on drug-like small molecules that can modulate the function of the receptor and treat thyroid dysfunction in a more efficient manner than conventional thyroid hormonal manipulation. With these groundbreaking discoveries, the future of thyroid disease management looks equally bright and exciting.
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