Chapter 16a - TSH RECEPTOR MUTATIONS AND DISEASES

The thyroid-stimulating hormone (TSH) receptor (TSHR) is a member of the glycoprotein hormone receptors (GPHRs), a sub-group of class A G protein-coupled receptors (GPCRs). TSHR and its ligand thyrotropin are of essential importance for growth and function of the thyroid gland. The TSHR activates different G-protein subtypes and signaling pathways, of which Gs and Gq induced signaling are of highest importance in the thyroid gland. A proper interplay between TSH and TSHR is pivotal for thyroid growth and regulated production and release of thyroid hormones (TH). Autoimmune (antibody binding) or non-autoimmune (occurrence of mutants) TSHR dysfunctions are the underlying cause of several pathologies, including rare cancer-development. The sequential processes of TSHR binding, signal transduction across the cell-membrane and activation of intracellular effectors involve elaborate specific structural properties of the receptor and several interacting proteins. In consequence, different pathogenic mutations at TSHR or TSH may have diverse impact on particular molecular functions, but finally result in either hypoor hyperthyroid states accompanied or not by various growth anomalies. We here summarize current knowledge regarding naturally occurring TSHR mutations, associated diseases and related molecular pathogenic mechanisms at the level of TSHR structure and function. For complete coverage of this and related areas in Endocrinology, visit our free webbooks, www.endotext.org and www.thyroidmanager.org GAIN-OF-FUNCTION MUTATIONS On a theoretical basis, for a hormone receptor, “gain-of-function” may have several meanings: (i) activation in the absence of ligand (constitutively); (ii) increased sensitivity to its normal agonist; (iii) increased, or de novo sensitivity to an allosteric modulator; (iv) broadening of its specificity. When the receptor is part of a chemostat, as is the case for the TSHR, the first situation is expected to cause tissue autonomy, whereas the second would simply cause adjustment of TSH to a lower value. In the third and fourth cases, inappropriate stimulation of the target will occur because the illegitimate agonists or modulators are not expected to be subject to the normal negative feedback. If a gain-of-function mutation of the first category occurs in a single cell normally expressing the receptor (somatic mutation), it will become symptomatic only if the regulatory cascade controlled by the receptor is mitogenic in this particular cell type or, during development, if the mutation affects a progenitor contributing significantly to the final organ. Autonomous activity of the receptor will cause clonal expansion of the mutated cell. If the regulatory cascade also positively controls function, the resulting tumor may progressively take over the function of the normal tissue and ultimately result in autonomous hyperfunction. If the mutation is present in all cells of an organism (germline mutation) autonomy will be displayed by the whole organ. From what we know of thyroid cell physiology it is easy to predict the phenotypes associated with gain-of-function of the cAMP-dependent regulatory cascade. Two observations provide pertinent models of this situation. Transgenic mice made to express ectopically the adenosine A2a receptor in their thyroid display severe hyperthyroidism associated with thyroid hyperplasia (1). As the A2a adenosine receptor is coupled to Gs and displays constitutive activity due to its continuous stimulation by ambient adenosine (2), this model mimics closely the situation expected for a gain-of-function germline mutation of the TSHR. Patients with the McCune-Albright syndrome are mosaïc for mutations in the Gs protein (Gsp mutations) leading to the constitutive stimulation of adenylyl-cyclase (3). Hyperfunctioning thyroid adenomas develop in these patients from cells harboring the mutation, making them a model for gain-of-function somatic mutations of the TSHR. A transgenic model in which Gsp mutations are targeted for expression in the mouse thyroid has been constructed. Though with a less dramatic phenotype this represents also a pertinent model for a gain-of-function of the cAMP regulatory cascade (4). Since the TSHR is capable of activating both Gs and Gq (though with lower potency) the question arises whether mutations with a different effect on the two cascades would be associated with different phenotypes. Studies in mice (5) and rare patients (6) suggests that activation of Gq may be required to observe goitrogenesis in patients with non-autoimmune familial hyperthyroidism. However, when tested in transfected non-thyroid cells, all identified gain-of-function mutations of the TSHR stimulate constitutively Gs, with only a minority capable of stimulating both Gs and Gq (7,8). Also, thyroid adenomas or multinodular goitre are frequent in McCune Albright syndrome, which is characterized by pure Gs stimulation (9). Familial non-autoimmune hyperthyroidism or hereditary toxic thyroid hyperplasia The major cause of hyperthyroidism in adults is Graves' disease in which an autoimmune reaction is mounted against the thyroid gland and auto-antibodies are produced that recognize and stimulate the TSHR (10,11). This may explain why the initial description by the group of Leclère of a family showing segregation of thyrotoxicosis as an autosomal dominant trait in the absence of signs of autoimmunity was met with skepticism (12). Reinvestigation of this family together with another family from Reims identified two mutations of the TSHR gene, which segregated in perfect linkage with the disease (13). A series of additional families have been studied since (14-28) [Figure 1 and Table 1, For a completed list of naturally occurring TSHR single amino acid substitutions with their functional characteristics, see the glycoprotein hormone receptor information resource “SSFA” (29,30) available under: http://www.ssfa-gphr.de]. The functional characteristics of these mutant receptors confirm that they are constitutively stimulated (see below). This nosological entity, hereditary toxic thyroid hyperplasia (HTTH), sometimes called Leclère’s disease, is characterized by the following clinical characteristics: autosomal dominant transmission; hyperthyroidism with a variable age of onset (from infancy to adulthood, even within a given family); hyperplastic goiter of variable size, but with a steady growth; absence of clinical or biological stigmata of auto-immunity. An observation common to most cases is the need for drastic ablative therapy (surgery or radioiodine) in order to control the disease, once the patient has become hyperthyroid (13,31). The autonomous nature of the thyroid tissue from these patients has been elegantly demonstrated by grafting in nude mice (32). Contrary to tissue from Graves' disease patients, HTTH cells continue to grow in the absence of stimulation by TSH or TSAb. The prevalence of hereditary toxic thyroid hyperplasia is difficult to estimate. It is likely that many cases are still mistaken for Graves' disease. This may be explained by the high frequency of thyroid auto-antibodies (anti-thyroglobulin, anti-thyroperoxidase) in the general population. It is expected that wider knowledge of the existence of the disease will lead to better diagnosis. This is not a purely academic problem, since presymptomatic diagnosis in children of affected families may prevent the developmental or psychological complications associated with infantile or juvenile hyperthyroidism (for a review, see (33)). A country-wide screening for the condition has been performed in Denmark. It was found in one out of 121 patients with juvenile thyrotoxicosis (0.8%; 95% CI: 0.02-4.6%), which corresponds to one in 17 patients with presumed non-autoimmune juvenile thyrotoxicosis (6%; 95% CI:0.15-28.69) (34). Sporadic toxic thyroid hyperplasia Cases with toxic thyroid hyperplasia have been described in children born from unaffected parents (35-39). Conspicuously, congenital hyperthyroidism was present in most of the cases and required aggressive treatment. Mutations of one TSHR allele were identified in the children, but were absent in the parents. As paternity was confirmed by minior microsatellite testing, these cases qualify as true neo-mutations. When comparing the amino acid substitutions implicated in hereditary and sporadic cases, for the majority, they do not overlap (see Table 1). Whereas most of the sporadic cases harbor mutations that are also found in toxic adenomas, most of the hereditary cases have "private" mutations. Although there may be exceptions, the analysis of the functional characteristics of the individual mutant receptors in COS cells, and the clinical course of individual patients, suggest an explanation for this observation: "sporadic" and somatic mutations seem to have a stronger activating effect than "hereditary" mutations (40). From their severe phenotypes, it is likely that newborns with neo-mutations would not have survived, if not treated efficiently. On the contrary, from inspection of the available pedigrees, it seems that the milder phenotype of patients with “hereditary" mutations has only limited effect on reproductive fitness. The fact that "hereditary" mutations are rarely observed in toxic adenomas is compatible with the suggestion that they would cause extremely slow tissue growth and, accordingly, would rarely cause thyrotoxicosis if somatic. There is, however, no a priori reason for neo-mutations to cause stronger gain-of-function than hereditary mutations. Accordingly, an activating mutation of the TSHR gene has been found in a six month child with subclinical hyperthyroidism presenting with weight loss as the initial symptom (41). Somatic mutations: autonomous toxic adenomas Soon after mutations of Gαs had been found in adenomas of the pituitary somatotrophs (42), similar mutations (also called Gsp mutations) were found in some toxic thyroid adenomas and follicular carcinomas (43-46). The mutated residues (Arg201, Glu227) are homologous to those found mutated in the ras proto-oncogenes: i.e. the mutations decrease the endogenous GTPase activity of the G protein, resulting in a constitutively active molecule. Toxic adenomas were found to be a fruitful source of somatic mutations activating the TSHR, probably because the phenotype is very conspicuous and easy to diagnose (47). Whereas mutations are distributed all along the serpentine portion of the receptor and even in the extracellular amino-terminal domain (48-54), there is clearly a hotspot at the cytoplasmic end of the sixth transmembrane segment (see Figure 1). The clustering reflects the pivotal role of this portion in the activation mechanism observed in the TSHR and in class A GPCRs generally [e.g. (55-61)]. Despite some dispute about the prevalence of TSHR mutations in toxic adenomas (which may be due to different origin of patients (62,63) or different sensitivity of the methodology) the current consensus is that activating mutations of the TSHR are the major cause of solitary toxic adenomas and account for about 60 to 80% of cases (15,7,64-66). Contrary to initial suggestions (63), the same percentage of activating TSHR mutations is observed in Japan, an iodine-sufficient country with low prevalence of toxic adenomas (67). In some patients with a multinodular goiter and two zones of autonomy at scintigraphy, a different mutation of the TSHR was identified in each nodule (68,36,69,70). This indicates that the pathophysiological mechanism responsible for solitary toxic adenomas can be at work on a background of multinodular goiter.