Werner & Ingbar's The Thyroid: A Fundamental & Clinical Text, 9th Edition

10B.The Thyrotropin Receptor

Gilbert Vassart

Sabine Costagliola

Thyrotropin (TSH) is a glycoprotein hormone composed of two subunits, an α-subunit that is common to TSH, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and chorionic gonadotropin (CG), and a unique β-subunit. The β-subunits of these hormones, although differing in biologic activity (when combined with an α-subunit), are encoded by genes that have a common ancestor but have evolved by gene duplication (paralogous genes) and, as such, have structural similarity (Fig. 10B.1). The corresponding receptors for TSH, FSH, and LH/CG also have structural similarity and are encoded by paralogous genes.

FIGURE 10B.1. Both the β-subunits of the glycoprotein hormones and the glycoprotein hormone receptors are encoded by paralogous genes. A: Similarity of the amino acid sequences of the β-subunits of human chorionic gonadotropin (hCG), luteinizing hormone (LH), thyrotropin (TSH), and follicle-stimulating hormone (FSH). Inset: Diagram of the general structure of the receptor for these hormones, showing the ectodomain (extracellular domain), serpentine domain (transmembrane domain), and endodomain (intracellular domain). B: Similarity of the amino-acid sequences of the receptors (r) for LH/CG, TSH, and FSH. Sequence identities are indicated separately for the extracellular and serpentine domains of the three receptors. The pattern of shared similarities suggests coevolution of the hormones and the extracellular domain of their receptors, resulting in specificity barriers. The high similarity of the serpentine domains of the receptors is compatible with a conserved mechanism of intramolecular signal transduction. (Reproduced from Vassart G, Pardo L, Costagliola S. Molecular dissection of the glycoprotein hormone receptors. Trends Biochem Sci 2004;29:119, with permission.)


The receptors for TSH, FSH, and LH/CG are members of the rhodopsin-like G protein–coupled receptor family. As such, the TSH receptor has a “serpentine” domain containing seven transmembrane regions with many (but not all) of the features typical of this receptor family. In addition, and this is a hallmark of the subfamily of glycoprotein hormone receptors (1,2,3), it has a large (about 400 amino-acid residues) amino-terminal extracellular domain that contains sites that selectively bind TSH with high affinity (4).

The higher sequence identity of the serpentine domains of the glycoprotein hormone receptors (~70%) as compared with the extracellular domains (~40%) (Fig. 10B.1) suggest that the former are interchangeable modules capable of activating guanine nucleotide-binding (G) proteins (mainly Gαs) after specific binding of the individual hormones to their receptors (5). Unlike other rhodopsin-like G-protein-coupled receptors, the glycoprotein hormones bind to their respective extracellular domains with high affinity in the absence of the serpentine domain (6,7,8). The intramolecular transduction of the signal between these two portions of the receptors involves mechanisms specific to the glycoprotein hormone receptor family. The relatively high sequence identity between the hormone-binding domains of the TSH and LH/CG receptors opens the possibility of CG stimulation of thyroid secretion during normal and especially molar or twin pregnancies, when serum CG concentrations are several orders of magnitude higher than are serum TSH concentrations. This provides an explanation for cases of gestational thyrotoxicosis (see Chapters 26 and 80).

The TSH receptor contains six sites for N-glycosylation, of which four are effectively glycosylated (8). The functional role of the individual carbohydrate chains is still debated. It is likely that they contribute to the routing and stabilization of the receptor as it passes through the membrane system of the cell and is inserted into the cell membrane. Alone among the glycoprotein hormone receptors, the extracellular domain of the TSH receptor is cleaved, severing it from the serpentine domain (see reference 4 for review). This phenomenon has been related to the presence in the extracellular domain of the receptor of a 50-amino-acid insertion for which there is no counterpart in the FSH receptor or LH/CG receptor. The initial cleavage step, due to the action of a metalloprotease, takes place at about position 314 (within the 50-amino-acid insertion) from the amino terminus of the receptor, and is followed by removal of approximately 50 amino acids from the amino-terminal end of the serpentine-containing portion of the receptor (9,10). The amino-terminal end of the receptor is bound to the extracellular end of the serpentine domain by disulfide bonds. The functional importance of this TSH receptor-specific postranslational modification remains unclear. Whereas all wild-type TSH receptors on the surface of thyroid follicular cells seem to be in cleaved form, noncleavable mutant constructs are functionally undistinguishable from cleaved receptors, when expressed in transfected cells (4). When transiently or permanently transfected in nonthyroid cells, wild-type human TSH receptors are present at the cell surface as a mixture of monomers and cleaved dimers.

The TSH rece ptor is specifically inserted into the basolateral membrane of thyroid follicular cells. This phenomenon involves signals encoded in the primary structure of the protein, because it is conserved when the receptor is expressed in MDCK cells, a polarized cell of nonthyroid origin (11).

The possibility that TSH receptors are present on the cell surface as dimers of cleaved dimers was raised after demonstration that most rhodopsin-like G protein–coupled receptors do dimerize (12). Functional complementation of receptors with mutations in the extracellular and the serpentine domains has been observed after cotransfection of FSH receptor constructs into cells (13), demonstrating the possibility of dimerization for glycoprotein hormone receptors. Preliminary data have been provided for dimerization of the TSH receptor (14), but definitive demonstration of the functional importance of dimerization of this receptor is lacking.


The gene coding for the human TSH receptor has been localized on the long arm of chromosome 14 (14q31) (15, 16). It is more than 60 kilobases (kb) long and is organized into 10 exons. The extracellular domain is encoded by a series of 9 exons, each of which corresponds to one or an integer number of leucine-rich repeat segments. The carboxyl-terminal part of the extracellular domain, the serpentine domain, and the intracellular carboxyl-terminal end of the receptor are encoded by a single large exon (17), in keeping with the fact that the genes for many G protein–coupled receptors have no introns. A likely evolutionary scenario derives from this gene organization: the glycoprotein hormone receptor genes would have evolved from the condensation of an intronless classic G protein–coupled receptor with a mosaic gene encoding a protein with leucine-rich repeat segments (17). Triplication of this ancestral gene and subsequent divergence led to the receptors for TSH, FSH, and LH/CG. The existence of 10 exons in both the TSH and FSH receptor genes (as opposed to the 11-exon LH/CG-receptor gene) suggests the following evolutionary steps: first, duplication of an ancestral glycoprotein hormone receptor gene, yielding the LH/ CG-receptor gene and the ancestors of the TSH- and FSH-receptor genes. After losing one intron, the latter duplicated subsequently into the TSH and FSH receptor genes.

The promoter region of the human and rat TSH receptor gene has been cloned and sequenced (17,18). It has the characteristics of “housekeeping” genes in that it is GC-rich and devoid of TATA boxes; in rats it stimulates transcription from multiple start sites (18) and contains a functional recognition site for thyroid transcription factor (TTF)-1 (19). Expression of the TSH receptor gene is largely thyroid specific. Constructs made of a chloramphenicol acetyltransferase reporter gene under control of the 5′-flanking region of the rat TSH receptor gene are expressed when transfected into FRTL5 cells and FRT cells, but not into nonthyroid cells such as HeLa or rat liver (BRL) cells (18). However, TSH receptor messenger RNA (mRNA) has been clearly demonstrated in adipose tissue of guinea pigs (20) and after differentiation of preadipocytes into adipocytes (21,22). TSH receptors may also be present in lymphocytes, extraocular tissue, cartilage, and bone, but their functional importance in these tissues is uncertain (23). Expression of the TSH receptor in thyroid cells is extremely robust. It is moderately up-regulated by TSH in vitro and down-regulated by iodide in vivo (24).


Three-dimensional structures are available for CG and FSH (25,26,27), but only structural models are available for TSH (see preceding section of this chapter) and for the extracellular domains of the TSH and other glycoprotein hormone receptors. The extracellular domains of all three glycoprotein hormone receptors are composed of a central portion containing leucine-rich repeats flanked by cysteine-rich domains (Fig. 10B.2). The leucine-rich repeat segments are composed of 20 to 25 amino acids formed into a β strand and an α helix, connected by a turn. When assembled sequentially in a protein, the leucine-rich repeat segments determine a horseshoe-like structure with the β strands forming a concave inner surface (Fig. 10B.2). This surface constitutes the binding interface in the first protein containing leucine-rich repeat segments that was crystallized: the ribonuclease inhibitor (28). The leucine-rich repeat-containing portion of the TSH receptor contains nine such segments, and it has been modeled on the known three-dimensional structure of the ribonuclease inhibitor (29). The model predicts that nonleucine residues (X1,2,3,4,5) (Fig. 10B.2) point outward and are available for interaction with TSH, immediately suggesting that they might be implicated in recognition specificity.

FIGURE 10B.2. Schematic representations of the structure of the thyrotropin (TSH) receptor. A: Two-dimensional representation with indication of the various domains. The two shaded boxes correspond to the amino-terminal and the cysteine-rich carboxyl-terminal portions of the extracellular domain, flanking nine leucine-rich repeats. B: Repeats are composed of 20 to 24 amino acids forming a β strand followed by an α helix. In proteins that contain leucine-rich repeat segments, the repeat segments are arranged with their β strands and α helices parallel to a common axis and organized spatially to form a horseshoe-shaped molecule, with the β strands and α helices making the concave and convex surfaces of the horseshoe, respectively. N, amino terminus; C, carboxyl terminus. C: Representation of a single leucine-rich repeat segment. The inner surface of the horseshoe is composed of seven residues: X1X2L1X3L2X4X5. The side chains of the leucine residues are pointing inside the hydrophobic core of the protein and are important for its stability. The side chains of the X residues are predicted to be exposed, making the surface available for interaction with the ligands. (Reproduced from Smits G, Campillo M, Govaerts C, et al. Glycoprotein hormone receptors: determinants in leucine-rich repeats responsible for ligand specificity. EMBO J 2003;22:2692, with permission.)

Replacement by site-directed mutagenesis of the Xi residues in the leucine-rich repeat portion of the TSH receptor with their counterparts in the LH/CG receptor provide strong support for the above model (30). Exchanging eight amino acids of the TSH receptor for the corresponding amino acids in the LH/CG receptor resulted in a mutant receptor that bound human CG as well as the wild-type LH/CG receptor. While gaining sensitivity to human CG, the mutant receptor retained normal sensitivity to TSH, making it a dual-specificity receptor. It is necessary to exchange 12 additional amino-acid residues to transform fully this mutant receptor into a bona fide LH/CG receptor (30). From an evolutionary point of view, these observations indicate that the specificity of hormone receptors is based on both attractive and repulsive residues, and that residues at different homologous positions have been selected to achieve this result in the different receptors.

Inspection of electrostatic surface maps of models of the wild-type TSH and LH/CG receptors and some of the mutants is revealing in this respect (30). The LH/CG receptor has an acidic groove in the middle of its horseshoe, extending to the lower part of it (corresponding to the carboxyl-terminal ends of the β strands). Generation of a similar distribution of charges in the dual-specificity and reverse-specificity TSH receptor mutants suggests that this is important for recognition of human CG. Attempts to correlate charge distributions in the wild-type and mutant TSH receptors with those of residues in TSH and human CG suggest that the “seat-belt” portion of the β subunits of the hormones, known to play a key role in recognition specificity (see preceding section and references 31,32,33), might face the bottom border of the horseshoe. The importance of electrostatic bonds in TSH–TSH receptor interactions has long been known, from the observation that efficient binding in vitro required low salt concentrations.

In addition to the hormone-specific interactions genetically encoded in the primary structure of glycoprotein hormone receptors and their ligands, there are important non-hormone-specific ionic interactions involving sulfated tyrosine residues present in the extracellular domains of all three receptors (34). In the TSH receptor, both tyrosine residues of a conserved Tyr-Asp-Tyr motif located close to the border between the extracellular domain and the first transmembrane helix are sulfated (Fig. 10B.3). Sulfation of the first tyrosine of the motif contributes importantly to the affinity of the receptor for TSH, without interfering with specificity (34).

FIGURE 10B.3. Linear representation of the thyrotropin (TSH) receptor. Sequences common to all rhodopsin-like G protein–coupled receptors and sequences specific to the glycoprotein hormone receptor gene family are both implicated in activation of the TSH receptor. Key residues are indicated (dots), as are conserved motifs: SO3- denotes postranslational sulfation of the indicated tyrosine residues (34). The boxes containing Roman numerals denote transmembrane helices; I1-I3 and E1-E3, intracellular and extracellular loops, respectively; and LRR, leucine-rich repeats.


Being a member of the G protein–coupled receptor family, the serpentine domain of the TSH receptor is likely to share with rhodopsin common mechanisms of activation (35,36). However, sequence variations in this domain of the glycoprotein hormone receptors suggest the existence of idiosyncrasies associated with hormone-specific mechanisms of activation (Fig. 10B.3). In addition, many gain-of-function somatic mutations have been found in this domain of the TSH receptor (37,38,39) (see Chapter 25); according to the available data, mutations of more than 30 residues result in constitutive activation of the TSH receptor. Many somatic mutations affecting a given residue have been found repeatedly; therefore, it is likely that we are getting close to having a saturation map for spontaneous gain-of-function mutations. Attempts have been made to translate this map into mechanisms of transition between inactive and active conformations of the receptor, in the light of structural data for rhodopsin. Three sequence patterns affected by gain-of-function mutations deserve special mention and may help in understanding how the TSH receptor is activated.

The first pattern is centered on an aspartate in position 6.44 (Asp633), at the cytoplasmic side of transmembrane helix VI. [The standardized numbering system of Ballesteros and Weinstein is used to identify residues in the transmembrane segments of different receptors. Each residue is identified by two numbers: the first, 1 through 7, corresponds to the helix in which it is located; the second indicates its position relative to the most conserved residue in that helix, arbitrarily assigned to 50 (40)]. When mutated to a variety of amino acids the result is constitutive activation (38,41). This suggests that the gain of function results from the breakage of one or more bonds, rather than the creation of novel interactions, by the mutated residue, and the main partner of Asp6.44 was identified as Asn7.49 in transmembrane helix VII. The results of site-directed mutagenesis studies suggested that, in the inactive conformation of the TSH receptor, the side chain of Asp7.49 is normally “sequestered” by both Thr6.43 and Asp6.44, and that the active conformations require establishment of novel interactions of N7.49, probably involving Asp in position 2.50 (41,42).

In the second pattern, glutamate 3.49 and arginine 3.50 of the highly conserved “D/ERY/W” motif at the bottom of transmembrane helix III form an ionic lock with aspartate 6.30 at the cytoplasmic end of transmembrane helix VI. Disruption of this ionic lock (e.g., by mutations affecting Asp6.30) leads to constitutively active mutant receptors (42). Thus, the movements of transmembrane helix III and transmembrane helix VI at the cytoplasmic side of the membrane is necessary for receptor activation (43).

The third pattern involves serine 281, which belongs to a YPSHCCAF sequence located at the carboxyl-terminal end of the leucine-rich repeat segment in the extracellular domain of the receptor (Fig. 10B.3). Mutation of this serine residue activates the TSH receptor constitutively (44), and this segment, sometimes referred to as the “hinge” motif, plays an important role in activation of all three glycoprotein hormone receptors (45). The functional effect of substitutions of S281 in the TSH receptor likely results in a local loss of structure, because the more destructuring the substitutions, the stronger the activation (45,46). This observation, together with the finding that mutation of specific residues in the extracellular loops of the TSH receptor cause constitutive activation (47), led to the hypothesis that activation of the receptor could involve the rupture of an inhibitory interaction between the extracellular domain and the serpentine domain (44).


Mutant TSH receptor constructs devoid of the extracellular domain are partially activated, confirming the inhibitory effect of the extracellular domain on the serpentine domain (48,49). However, cyclic AMP production in cells transfected with truncated constructs was increased much less than after full stimulation of the wild-type receptor by TSH or after mutation of Ser281 (48). These results led to the following model for activation of the TSH receptor (48) (Fig. 10B.4). In the resting state, the extracellular domain inhibits the activity of an inherently active rhodopsin-like serpentine domain. Upon activation, by binding of TSH, or secondary to mutation of S281 in the hinge region, the extracellular domain switches from inverse agonist to full agonist of the serpentine domain. The ability of the strongest S281 mutants to activate the receptor fully in the absence of TSH suggests that the ultimate agonist of the serpentine domain would be the “activated” extracellular domain, with no need for a direct interaction between TSH and the serpentine domain.

FIGURE 10B.4. Model for activation of the thyrotropin (TSH) receptor. Interactions between the extracellular domain and the serpentine domain are implicated in the activation mechanism. The TSH receptor is represented with its extracellular domain containing a concave, hormone-binding structure facing upward and a transmembrane serpentine domain. The basal state of the receptor is characterized by an inhibitory interaction between the extracellular domain and the serpentine domain [indicated by the (-) sign]. In the absence of the agonist, the extracellular domain would function as a tethered inverse agonist of the serpentine domain. Mutation of Ser281 in the extracellular domain into leucine switches the extracellular domain from an inverse agonist into a full agonist of the serpentine domain [indicated by the (+) sign]. Binding of TSH (indicated by the αβ dimeric structure) to the extracellular domain is proposed to have a similar effect, converting it into a full agonist of the serpentine domain. The serpentine domain in the basal state is shown as a compact structure. The fully activated serpentine domain is shown as a relaxed structure with arrows indicating activation of Gα within the cell. Wt TSHr, wild-type TSH receptor.


The sequence similarity between TSH and human CG, and between their receptors, allows for some degree of promiscuous activation of the TSH receptor by CG during the first trimester of pregnancy, when serum CG concentrations are highest. The inverse relationship between serum TSH and CG concentrations in most pregnant women is a clear indication that their thyroid gland is stimulated by CG (50) (see Chapter 80). Although most pregnant women are euthyroid, thyrotoxicosis may occur if CG production is excessive (as occurs in twin pregnancies or chorionic tumors (see Chapter 26), or in rare women who have a mutant TSH receptor with increased sensitivity to CG (51).


The serum antibodies found in most patients with Graves' thyrotoxicosis and some patients with hypothyroidism caused by chronic autoimmune thyroiditis can stimulate or block the TSH receptor, respectively (see Chapters 15, 23, and 47). The epitopes recognized by TSH receptor–stimulating antibodies and the mechanisms leading to activation of the receptor are still ill defined. This situation should change shortly, now that monoclonal antibodies with thyroid-stimulating activity have been produced in mice and hamsters and isolated from the serum of patients with Graves' thyrotoxicosis (52,53,54,55). Although most TSH receptor–stimulating antibodies do compete with TSH for binding to the receptor (56), the precise targets of TSH and the antibodies are likely to be different. The sulfated tyrosine residues, which are important for TSH binding, are not implicated in recognition of TSH receptor by TSH receptor–stimulating antibodies (34). Also, most TSH receptor–stimulating antibodies stimulate cyclic AMP accumulation in cells transfected with TSH receptors more slowly than does TSH (57). The recent availability of highly purified TSH receptor–stimulating antibody preparations from individual patients should allow these differences between the antibodies and TSH to be explored directly (56).


Desensitization of some G protein–coupled receptors involves phosphorylation of specific residues by G protein receptor kinases (homologous desensitization) or protein kinase A (heterologous desensitization) (58). Acute desensitization of the receptor in the presence of TSH, presumably by phosphorylation, is weak and delayed (59). When compared with other G protein–coupled receptors, the TSH receptor contains few serine or threonine residues in its intracellular loops and intracellular carboxyl-terminal domain that can be phosphorylated, which probably accounts for the limited desensitization after stimulation by TSH. Weak down-regulation, confounded by the long life of both TSH receptor mRNA and protein, does occur but has little functional role (60). The persistence of thyrotoxicosis in patients with TSH-secreting pituitary adenomas and in patients with Graves' disease is in vivo evidence of the weakness of down-regulation of the TSH receptor.


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