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

10A.Chemistry and Biosynthesis of Thyrotropin

Ronald N. Cohen

Fredric E. Wondisford

Thyrotropin (thyroid-stimulating hormone, TSH) is the main regulator of thyroid hormone biosynthesis and secretion. Therefore, its availability and actions are critical determinants of thyroid function.


TSH is one of four related glycoprotein hormones synthesized either by the anterior lobe of the pituitary gland or by the placenta. TSH, pituitary luteinizing hormone (LH), pituitary follicle-stimulating hormone (FSH), and chorionic gonadotropin (CG) consist of two noncovalently linked α- and β-subunits (1). The amino acid sequence of the α-subunit is common to all four of these hormones within any mammalian species. The β-subunit of each hormone has a different amino acid sequence, and this subunit carries the specific information relating to receptor binding and expression of hormonal activity; however, free β-subunits are devoid of bioactivity and require noncovalent combination with the common α-subunit to express this information.

Thyroid-stimulating activity has been extracted from virtually all mammalian pituitary glands, as well as in those of lower vertebrates. The best chemical information has been derived from a study of bovine, porcine, and human TSH (1). TSH preparations purified from pituitary glands by various chromatographic procedures contain heterogeneous but closely related components, which have variable biologic activity. These components can be separated by gel electrophoresis, isoelectric focusing, or chromatofocusing. The molecular weights of these various components of mammalian TSH are in the range of 28,000 to 30,000 daltons. Differences in molecular weight are attributed to heterogeneity of the oligosaccharide chains, heterogeneity at the amino terminus, and the extent of amidation of glutamic and aspartic acid residues.

The human α-subunit contains a protein core of 92 amino acid residues, while the bovine α-subunit contains 96 amino acids. The heterogeneity of the amino terminus of the α-subunits of the different glycoprotein hormones is probably artifactual, since in vitro studies (see later in the chapter) suggest that processing of the precursor protein yields a single product. The α-subunits contain 10 half-cystine residues, all of which are in disulfide linkage, as are those in the β-subunits. Knowledge of the specific location of the disulfide bonds in both subunits has been advanced by X-ray crystallographic analysis of the closely related glycoprotein, human CG (2,3). The three-dimensional structure of TSH likely contains the cysteine knot motif and basic folding patterns found in human CG.

The β-subunits dissociated from intact human TSH prepared from pituitaries have a protein core of 112 amino acids; however, the complementary DNA for human TSH-β predicts a protein of 118 amino acids, 6 more than present in the isolated protein. When the linear sequences of β-subunits from different species are aligned to juxtapose half-cystine residues (12 in number), many regions of similar or identical sequences are apparent. From such relationships, and the fact that each can recombine with the common α-subunit, it seems probable that the three-dimensional structures of different β-subunits are similar. Indeed, evidence from chemical modification studies indicate that the regions around amino acid residues 51 to 57 and 75 to 80 of the β-subunit are among those involved in interaction with the common α-subunit. Based on the similarities in sequences between the β-subunits, all probably evolved from a single gene precursor.

Both the α- and β-subunits of TSH, as well as of LH, FSH, and CG, contain covalently linked carbohydrate chains. For TSH, LH, and FSH, these chains are linked to asparagine residues (N-linked), and for the β-subunit of human CG there are additional linkages to serine residues (O-linked). Moreover, the free α-subunit that is secreted also contains an additional site of O-glycosylation (4). In human TSH the sugar residues include mannose, fucose, N-acetylglucosamine, galactose, N-acetylgalactosamine, and sialic acid. Moreover, TSH and LH contain an unusual sulfate group that terminates certain chains; such sulfation is found only to a small extent in FSH, and not at all in CG (5,6). The sugar residues are three oligosaccharide units, all of which are heterogeneous and whose specific structures may vary with the developmental and endocrine state (7). Recombinant human TSH has been produced in large amounts (see later in the chapter), and the biologic properties of particular isoforms of TSH and clinical use of TSH are being investigated (8,9,10,11).


Chromosome Localization

The gene encoding the common α-subunit of human CG, FSH, LH, and TSH, and the genes encoding their respective β-subunits are all located on different chromosomes. The location of the common α-subunit and specific β-subunit on different chromosomes raises interesting questions about how their expression is coordinately regulated during the synthesis of each hormone. Moreover, the gene for the β-subunit of CG is the only β-subunit gene of this family that exists in more than one copy; at least two of the seven copies on chromosome 19 are actively transcribed.

Structure of the Common α-Subunit Gene

The α-subunit gene has been isolated from a variety of species, including cows (12), mice (13), rats (14), and humans (15). The organization of each gene is similar in that it contains four exons and three introns, and all are of approximately the same size. The human gene is 9.4 kilobases (kb) in length and contains three introns of 6.4 kb, 1.7 kb, and 0.4 kb. Intron 1 is located between the 5′-untranslated region contained in exons 1 and 2; intron 2 interrupts the α-subunit peptide-coding region in codon 6; and intron 3 is between codons 67 and 68 of the peptide (Fig. 10A.1). In rats and cows, intron 2 interrupts the peptide-coding region within amino acid 10, resulting in a mature α-subunit peptide that is four amino acids longer than the human α-subunit peptide (96 amino acids vs. 92 amino acids).

FIGURE 10A.1. Schematic representation of the genes for the subunits of human thyrotropin (TSH). The genes for the α- and β-subunits have four and three exons (numerals), respectively, represented by boxes separated by introns (thin lines). The coding exons are denoted by black boxes, and the untranslated regions are represented by white boxes. The translation start (AUG) and stop (TAA) codons are shown in their relative position below each gene; the start of transcription is denoted by a bent arrow.

A single transcription start site has been found in each of these genes by mapping studies of pituitary RNA. Upstream of these start sites are consensus TATA boxes, thought to be important for correct and efficient transcription by RNA polymerase II (16). In the human α-subunit gene, the TATA box is -26 base pairs (bp) from the transcription start site (15). In addition, there is considerable homology among these species in other 5′-flanking regions, including a palindromic sequence of TGACGTCA in the human α-subunit gene that confers responsiveness to cyclic adenosine monophosphate (cyclic AMP) (17,18). However, the palindrome is altered in the other species to TGATGTCA; at least in cows, this change reduces its cyclic AMP responsiveness dramatically (19).

Structure of the Thyrotropin β-Subunit Gene

The TSH-β-subunit gene has been isolated from rats (20), humans (21,22,23), and mice (24,25). The rat and human genes contain three exons. The mouse gene contains five exons: the two additional exons are 5′-untranslated regions; these are unique due to changes in the sequence of the gene in mice (21). With this exception, the organization of these genes is quite similar in that there are 5′-untranslated exons(s) separated from the TSH-β coding region by a large first intron (Fig. 10A.1). The first exon of the human TSH-β gene is 37 bp and is untranslated, while the second exon encodes the leader peptide and first 34 amino acids of the mature TSH-β peptide; the third exon contains the remaining coding region (amino acids 35 to 118) and 3′-untranslated sequences.

The start of transcription has been determined in the rat, mouse, and human TSH-β genes. Both the rat and mouse genes contain two transcriptional start sites approximately 40 base pairs (bp) apart, as assessed by primer extension or S1 nuclease analysis of pituitary RNA (20,24,25). Most transcription initiates from the downstream site (90% to 99%), and both sites are preceded by consensus TATA box sequences. Transcription from the downstream site is dramatically increased in hypothyroid rats and mice, while transcription from the upstream site is either unaffected or reduced by thyroid hormone (20,24,25).

The TSH-β gene in humans contains only one transcriptional start site in a location similar to that of the downstream site in the TSH-β gene in rats and mice (21,22,23). This difference may be due to an alteration in the upstream TATA box, which is changed from TATATAA in the rat and mouse gene to TGTATAA in the human gene. There may be an additional start site in the human gene (26). However, based on RNA mapping studies done in one TSH-secreting adenoma, this site does not correspond to the upstream TATA box in the human TSH-β gene.

Central Hypothyroidism Due to Thyrotropin-β-Subunit Gene Abnormalities or Alterations in Thyrotroph Development

Several kindreds with central hypothyroidism and undetectable or at least inappropriately low serum TSH concentrations due to mutations in the TSH β-subunit gene have been described (27,28). Two affected members of a Japanese family had a point mutation in exon 2 that changed a glycine residue to an arginine residue (G29R) (27). This mutation is in a region that is highly conserved among glycoprotein subunit genes (CAGY) and is thought to be important in subunit combination (Fig. 10A.2). This mutation resulted in synthesis of a TSH-β-subunit that could not associate with the α-subunit, and therefore no TSH heterodimer was produced.

FIGURE 10A.2. Point mutations of the human thyrotropin (TSH) β-subunit gene in patients with familial TSH deficiency. Three point mutations in this gene have been described (see text). Two of these mutations (E12X and G29R) occur in exon 2, and predict either a truncated TSH-β subunit or one with an amino-acid change in the CAGY region. The single letters define the wild-type amino acid (preceding the number of the amino acid) and the substituted amino acid (after the number of the amino acid); fs, frame shift.

Two related Greek families have been described in which affected family members had a mutation in the β-subunit of TSH that caused premature termination of synthesis of the subunit (E12X) (28). A Turkish family was found to have a different mutation that also resulted in premature termination of synthesis of the subunit (Q49X) (29). In another family a mutation was found in the second intron of the β-subunit gene (G29R) that predicts skipping of exon 2 and generation of a 25 amino-acid nonsense peptide from an aberrant start site in exon 3 (30). A Brazilian kindred with central hypothyroidism was described in which there was a frame-shift deletion in codon 105 (C105Vfs114X) of the β-subunit gene (31). This mutation substantially reduced TSH secretion, although some TSH was detected in the serum of the affected patients. Based on the crystallographic structure of the related hormone, CG, the mutation interferes with a disulfide bond that stabilizes the αβ heterodimer of TSH (2). This mutation may be the most frequent β-subunit mutation (32). All of these disorders are autosomal recessive in inheritance, and affected family members do not produce TSH with biologic activity (Fig. 10A.2) (see Chapter 51).

Central hypothyroidism can also be caused by defects in pituitary development, which results in impaired formation or survival of thyrotrophs and expression of TSH (see Chapter 2). In general, pituitary organogenesis is characterized by the orderly expression of cell-specific transcription factors (Fig. 10A.3), including Pit-1, Prophet of Pit-1 (Prop-1), P-Lim (Lhx-3), Ptx, Hesx-1 (Rpx), and thyrotroph embryonic factor (TEF) (for review see reference 33).

FIGURE 10A.3. Schematic representation of development of the anterior pituitary gland. The sequential expression of transcription factors leads to cell-specific expression of pituitary hormones. ACTH, corticotropin; CUTE, corticotroph upstream transcription binding protein; GH, growth hormone; Prl, prolactin; Prop-1, Prophet of Pit-1; SF-1, steroidogenic factor-1; and TEF, thyrotroph embryonic factor.

TEF is expressed in the region of developing thyrotrophs, at the time of onset of expression of the β-subunit of TSH (34). TEF binds to sites in the promoter region of the β-subunit gene to regulate expression of the gene. In contrast, Pit-1 is expressed after the initiation of expression of the TSH-β-subunit gene (35). However, Pit-1 is required for thyrotroph survival and expression of the TSH-β-subunit gene. While there are Pit-1–dependent and Pit-1–independent thyrotrophs in the developing anterior pituitary, the latter population disappears before birth (35,36). Pit-1 also regulates the expression of growth hormone and prolactin, and is required for normal somatotroph and lactotroph function. In mice, Pit-1 mutations result in TSH, growth hormone, and prolactin deficiency and pituitary hypoplasia (37). Pit-1 mutations in humans cause analogous defects, a syndrome termed combined pituitary hormone deficiency (Fig. 10A.4) (38,39). A well-characterized Pit-1 mutation implicated in hypopituitarism in humans is the R271W mutation (40), which leads to production of a mutant Pit-1 that binds DNA but does not transactivate appropriately. These patients have hypothyroidism, but may have inappropriately normal serum TSH concentrations. They have a blunted serum TSH response to thyrotropin-releasing hormone (TRH), suggesting the importance of Pit-1 in the TRH signaling pathway (see later in the chapter).

FIGURE 10A.4. Point mutations in the human Pit-1 gene in patients with combined pituitary hormone deficiency, as defined using the single-letter code for amino acids. POU-specific and POU-homeodomains, and the corresponding α-helices, are outlined in relation to indicated mutations in the Pit-1 gene. ++, lysine- and arginine-rich domains; N, the amino terminus of the Pit-1 protein; and C, the carboxyl terminus of the Pit-1 protein.

Not all Pit-1 mutations leading to combined pituitary hormone deficiency impair transactivation of the TSH subunit, growth hormone, and prolactin genes. The K216E mutation binds to DNA and transactivates normally (41) but binds poorly to retinoic acid receptors (RAR), leading to defective retinoic acid induction of the Pit-1 gene enhancer. This defect, in turn, causes impaired pituitary development (41).

Prop-1 gene expression precedes the expression of Pit-1, and appears to be required for the development of Pit-1–dependent cell lines. Ames mice, which have hormonal defects similar to those in Snell and Jackson dwarf mice, have a mutation in the alpha helix of the homeodomain of the Prop-1 gene (42). Similar to Snell and Jackson dwarf mice, analogous defects have been found in some patients with congenital combined pituitary hormone deficiency (43,44,45). These patients have central hypothyroidism as well as growth hormone and prolactin deficiency, similar to patients with Pit-1 mutations. However, patients with mutations in Prop-1 also tend to have LH and FSH deficiency. One common Prop-1 mutation is a 2-bp deletion (A301G302) (45), which results in impaired DNA binding (43).


Thyroid Hormone

Thyroid hormone receptors (TR) are cellular homologues of the viral erythroblastic leukemia oncogene (v-erbA) (Fig. 10A.5). TRs are derived from two separate gene loci in mammals: α and β (see Chapter 8) (46). The α locus in humans is located on chromosome 17 (17q11.2) and the β locus on chromosome 3 (3p24.3). The TR-α gene undergoes alternative splicing, generating two isoforms: TR-α1 and TR-α2. TR-α1 is a bona fide TR, whereas alternative splicing of the C-terminus of TR-α2 generates a non–triiodothyronine (T3)-binding isoform. The TR-β gene generates two major isoforms due to alternative promoter utilization: TR-β1 and TR-β2. This results in two β receptors, each with a different amino terminus.

FIGURE 10A.5. Schematic drawing of the human thyroid hormone receptors. DBD, DNA-binding domain; LBD, ligand-binding domain.

TRs are variably expressed in mammals. In general, TR-α1 and TR-β1 are ubiquitously expressed. TR-α1 is most highly expressed in the brain and heart, whereas TR-β1 is most highly expressed in the liver. In contrast, TR-β2 expression is limited principally to the hypothalamus, pituitary, cochlea, and retina (47). Differences in the expression patterns as well as structure of TRs suggest that one or both of these characteristics may determine thyroid hormone action in particular tissues.

TRs are members of the nuclear hormone receptor superfamily. TRs share in common with other members of this superfamily a conserved DNA-binding region, which contains two coordinated zinc molecules. Carboxy-terminal to the DNA-binding domain is the ligand-binding domain, which determines ligand-binding specificity. This domain is not highly conserved among different members of this superfamily. Similarly, the amino terminus domain, N-terminal to the DNA-binding domain, is not conserved in the superfamily. In addition to known ligands for members of this superfamily, other receptors known as orphan receptors have been described for which no ligands have been identified.

TRs bind to DNA as a homodimer or a heterodimer with the retinoid X receptor, also known as RXR (48). TR homodimers dissociate after binding T3, whereas TR-RXR heterodimers do not. This has led to the suggestion that TR-RXR heterodimers are the most important determinants of T3 action. In the absence of T3, TRs bind constitutively to a class of proteins termed nuclear corepressors. These proteins share the characteristic of recruiting histone deacetylase enzymes to the transcription complex, which results in histone deacetylation and increased chromatin packing (49). When T3 binds to its receptors, the nuclear corepressors are released and nuclear coactivators are recruited. These include principally the p160 class of coactivators, named because of their molecular weight, as well as the cointegrator class of cofactors (CBP and p300). These proteins have intrinsic histone acetylase activity, which results in histone acetylation and unwrapping of the chromatin. Presumably, this looser configuration of chromatin allows easier access to RNA polymerase II, and transcription is increased. TRs are among the few members of the nuclear receptor superfamily that bind nuclear corepressors in the absence of ligand. This unique property suggests that it may control gene expression in a way that is unique from receptors that do not bind corepressors in this manner.

Thyroid hormone inhibits the synthesis and secretion of TSH at the level of the pituitary and indirectly at the level of the hypothalamus by reducing the secretion of TRH (50,51,53). In animals, thyroid hormone administration results in a dramatic decrease in the levels of messenger RNA (mRNA) for the common α- and TSH-β-subunits due to a reduction in transcription of the respective genes (50,51). However, the magnitude and rapidity of suppression varies among the subunits. In mouse TSH-secreting tumor cells exposed to T3, the level of TSH-β mRNA fell more rapidly (50% inhibition at 4 hours) and to a greater extent (>95% suppression at 4 hours) than that of the common α-subunit. In addition, after prolonged T3 exposure TSH-β-subunit mRNA was undetectable, whereas the level of α- subunit mRNA remains at approximately 25% of control levels (see section on regulation of thyrotropin secretion in this chapter) (50).

In addition to the expected suppression of TSH secretion by thyroid hormone, paradoxical increases in TSH secretion have been noted soon after initiation of thyroid hormone replacement, followed by later suppression of TSH secretion. In one study, administration of a low dose of T3 to patients with hypothyroidism resulted in an increase in the serum TSH response to TRH (54). This effect may be due to a generalized defect in protein synthesis, corrected by low doses of T3, or stimulation of expression of TSH subunit genes, perhaps by T3 activation of a stimulatory cis-acting element.

The mechanisms underlying negative regulation of the TRH- and TSH-subunit genes by thyroid hormone are not clear. The thyroid hormone-inhibitory elements in these genes have unique properties, as compared with thyroid hormone-stimulatory elements (for review see reference 55). First, most of the inhibitory elements in these genes do not bind RXRs, and, unlike stimulating TR elements, RXRs antagonize TR binding to these elements (56). The exception is an important negative thyroid hormone response element in the TRH gene promoter that binds TR monomers, TR homodimers, and TR-RXR heterodimers (57). Although some models suggest that negative regulation by thyroid hormone can occur in the absence of TR binding to DNA, newer in vitro and in vivo studies have clearly demonstrated that this binding is essential for thyroid hormone inhibition (58,59)

Studies in mice in which one or several of the TR isoforms were deleted have revealed that not all TR isoforms are involved in the production of TSH (60,61,62,63,64). The most important isoform controlling TSH production is TR-β2 (62). In the hypothalamus, it is perhaps the only isoform responsible for negative regulation of the TRH gene (65). It is also the most important isoform for negative regulation in the pituitary, although the TR-α1 and TR-β1 isoforms play some role at the extremes of serum thyroid hormone concentrations. Based on these studies, it seems clear that the main locus for the set point at which serum thyroid hormone concentrations control TSH secretion is the hypothalamus, due to the ability of TRH to modify the biological activity of TSH. In contrast, the main locus for coarse control of TSH synthesis is the pituitary (Fig. 10A.6).

FIGURE 10A.6. Schematic diagram of the hypothalamic pituitary thyroid axis. Thyroid hormone inhibition of thyrotropin (TSH) secretion occurs both in the hypothalamus, where the hormones inhibit TSH secretion, and in the thyrotroph cells of the pituitary gland, where the hormones directly inhibit TSH secretion (central feedback). The role of individual thyroid hormone receptor (TR) isoforms in each compartment is summarized on the right side of the figure.

In contrast to many mouse TR knockout models of resistance to thyroid hormone, human forms of this syndrome are caused by point mutations in the TR-β gene, except for one case (reviewed in 66) (see Chapter 81). This syndrome is characterized by mild-to-marked elevations in serum thyroxine (T4) and T3concentrations and inappropriately normal or slightly high serum TSH concentrations resulting from resistance to thyroid hormone action in the hypothalamus and pituitary. Point mutations in the hinge region and ligand-binding domain of the gene for TR-β have been found in several hundred families; in contrast, no patients with mutations in the TR-α gene have been identified. Studies of mice in which the TR-β gene was knocked out provide an explanation for this curious finding in patients with thyroid hormone resistance. Mice with homozygous deletions of the TR-β gene have central resistance to thyroid hormone, indicating that the TR-α receptors cannot substitute for TR-β receptors in the regulation of TSH secretion (67,68). Thus, given the TR isoform–specific regulation of the pituitary, it is not surprising that TR mutations in patients with thyroid hormone resistance are found only in the gene for the β isoform of the TR. Moreover, a somatic mutation of the TR-β gene conferring thyroid hormone resistance was detected in a TSH-secreting pituitary tumor (69).

Steroid Hormones

TSH subunit gene expression is altered by steroid hormones, including glucocorticoids, estrogen, and testosterone. In humans, high doses of dexamethasone decrease serum TSH concentrations (70); dexamethasone also decreases TSH secretion in patients with TSH-secreting pituitary adenomas. In mice with thyrotropic tumors, dexamethasone decreased TSH secretion, but the levels of TSH-subunit mRNA in the tumor did not change (71). Therefore, dexamethasone may exert its effect on TSH-subunit biosynthesis at a translational or posttranslational level.

In animals and humans, estrogen administration does not alter basal or TRH-stimulated serum TSH concentrations (72). In hypothyroid rats, however, high doses of estradiol augment thyroid hormone-stimulated suppression of synthesis of α- and β-subunit mRNA (72). In animals, testosterone has similar effects, perhaps caused by its peripheral conversion to estrogen. Steroid hormones are not major regulators of TSH secretion under normal conditions, but they may play a role during some pathologic conditions such as hypothyroidism.

Thyrotropin-Releasing Hormone

In humans, TRH is the major positive regulator of TSH secretion. In animals, TRH stimulates the transcriptional activity of the TSH-subunit genes three- to fivefold (73). The stimulatory effect of TRH is augmented in hypothyroidism, which may be explained, at least in part, by an increase in the number of TRH receptors on the thyrotrophs (74). Alternatively, TRH may augment the effect of hypothyroidism to increase expression of the genes for the common α- and TSH β-subunits. There are cis-acting elements in the gene for the β-subunit of TSH in humans located between -128 and +8 bp in relation to the transcription start site (75). One element is located between -128 and -91 bp, and the second element is located from -28 to +8 bp and includes the TATA box and transcription start site. The upstream element in the human gene and the equivalent region in the rat gene bind the pituitary-specific transcription factor Pit-1 and mediate both TRH and cyclic AMP responsiveness of this gene through changes in the state of the Pit-1 protein (76). As noted above, mutations in Pit-1 result in loss of somatotroph, lactotroph, and thyrotroph function (combined pituitary hormone deficiency). In some families, only TRH-stimulation of TSH or prolactin secretion is impaired, and basal secretion of these hormones is normal, further implicating Pit-1 in the TRH signaling pathway. In fact, cyclic AMP-response element (CREB)-binding protein (CBP) and Pit-1 synergistically activate prolactin gene expression in response to TRH, suggesting a specific role for Pit-1 and CBP in TRH action (77,78).

In mice, deletion of the TRH gene results in central hypothyroidism (79). These animals have slightly high serum TSH concentrations, suggesting that TRH is not absolutely necessary for TSH production. However, their TSH has decreased biologic activity. Patients with TRH-receptor mutations have been described who also have hypothyroidism (see Chapter 48) (80,81).

In addition, in mice in which a mutant TR was expressed in the pituitary, thyroid hormone feedback at the pituitary, but not at the hypothalamus, was impaired (62). These mice had high serum TSH concentrations and high levels of TSH-β mRNA in the pituitary, but only a small increase in serum T4concentrations, indicating that the biological activity of the TSH was reduced. Because the mutant receptor was not expressed in the hypothalamus, T3feedback at this level was intact. In fact, in situ hybridization suggested that TRH gene expression was decreased compared with control animals. When TRH was replaced, serum T4 concentrations increased significantly, suggesting that in thyroid hormone resistance syndromes resistance occurs at the levels of both the pituitary and hypothalamus (central resistance).

Cyclic AMP

Elevation of intracellular cyclic AMP levels increases the levels of mRNA of the common α- and TSH- β-subunits. The hypothalamic hormone arginine vasopressin stimulates TSH release from the thyrotrophs (82). It also stimulates TSH-subunit gene expression via an increase in cyclic AMP levels; however, it is unclear at present whether the vasopressin receptor in pituitary thyrotrophs is coupled to adenylyl cyclase. The TSH- β-subunit gene in humans has DNA sequences that mediate cyclic AMP induction located predominantly between -128 and -28 bp of the gene (75). This region does not contain DNA sequences homologous to the cyclic AMP-responsive element that binds the transacting factor, CREB, which mediates activation of the gene for the common α-subunit. Pit-1 interacts with CBP (77,82), and CBP and Pit-1 synergistically activate protein kinase A-dependent prolactin gene expression, suggesting a role for Pit-1 in cyclic AMP-mediated gene expression in the pituitary. The regions of CBP responsible for this protein kinase A effect are distinct from those involved in TRH stimulation of the prolactin gene (77).


Dopamine rapidly decreases basal and TRH-stimulated TSH secretion in man by approximately 50% (83). In rat pituitary cells, dopamine decreases TSH-subunit gene transcription by about 50% in 15 minutes, and inhibition is maximal (75% inhibition) in 30 minutes (73). Dopamine may act to decrease intracellular cyclic AMP levels and thus interfere with cyclic AMP-mediated stimulation of TSH-subunit gene expression. Endogenous dopamine may exert some tonic control of TSH secretion, because dopamine antagonist drugs increase serum TSH concentrations transiently in normal subjects and patients with hypothyroidism (84,85).

Summary of Pretranslation Regulation of the Human Common α- and TSH- β-subunit Gene Expression

Figure 10A.7. is a schematic representation of a thyrotroph cell and the regulatory pathways that appear to be important in modifying TSH-subunit gene expression. Thyroid hormone is the major negative regulator of TSH-subunit gene expression; dopamine and somatostatin are less-important negative regulatory hormones. Thyroid hormone inhibits gene expression by binding to DNA cis-acting elements via a nuclear TR, and presumably interacts with the transcription initiation complex through protein–protein interactions. Somatostatin and presumably dopamine reduce intracellular cyclic AMP levels, via an inhibitory guanyl nucleotide-binding protein, and thus reduce TSH-subunit gene expression.

FIGURE 10A.7. Overview of the regulation of expression of the gene for the β-subunit of thyrotropin (TSH) within the thyrotroph cells of the pituitary gland (see text for details). AC, adenylyl cyclase; CBP, CREB-binding protein; CREB, cyclic adenosine monophosphate-response element; DA, dopamine; Gi, inhibitory guanine-nucleotide-binding protein; Gs, stimulatory guanine nucleotide-binding protein; nTRE, negative thyroid hormone-response element; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; and TR, thyroid hormone receptor.

TRH is the major positive regulator of TSH-subunit gene expression and acts through a guanyl nucleotide-binding protein to activate phospholipase C. Phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol 1,4,5 triphosphate (IP3). DAG activates protein kinase C, which, in turn, phosphorylates and presumably activates transacting nuclear factors responsible for TSH-subunit gene expression. IP3 releases Ca++from intracellular pools and raises intracellular Ca++ levels. Another positive regulator of TSH-subunit gene expression is vasopressin, which may act via a stimulatory guanyl nucleotide-binding protein to increase intracellular cyclic AMP levels. Cyclic AMP then activates protein kinase A (PKA) and cellular proteins are phosphorylated. One such protein is CREB, the cyclic AMP-response DNA-binding protein that has been shown to activate common α-subunit gene transcription. Protein kinase A activates TSH-β-subunit gene expression through a CREB-independent mechanism that is mediated, in part, by Pit-1. Human disorders that directly alter thyrotroph function are summarized in Fig. 10A.8.

FIGURE 10A.8. Overview of molecular defects in the expression of genes for the subunits of TSH within the thyrotroph cells of the anterior pituitary gland. AC, adenylyl cyclase; AVP, arginine vasopressin; CPHD, combined pituitary hormone deficiencies; CBP, CREB-binding protein; CREB, cyclic aden-osine monophosphate-response element; Gi, inhibitory guanine nucleotide-binding protein; Gs, stimulatory guanine nucleotide-binding protein; mut, mutation; nTRE, negative thyroid hormone response element; PKA, protein kinase A; PKC, protein kinase C; and TRHR, thyrotropin-releasing hormone receptor.


In Vitro Translation of Thyrotropin- β-Subunit and α-Subunit Messenger RNA

The posttranslational processing of TSH was initially studied in cell-free translation systems, which were important to define the initial translation products, termed presubunits (86,87). Such pre–α- and pre–β-subunits contain the subunit proteins as well as amino-terminal leader or signal peptides necessary for translocation across the membrane of the endoplasmic reticulum.

α-Subunit mRNA was extracted from mouse thyrotropic tumors and translated in wheat germ or reticulocyte lysate cell-free systems devoid of the enzymes necessary for the proteolytic cleavage of polypeptide precursors or glycosylation (86,87). The major translation product, pre–α-subunit, had an apparent molecular weight of 14,000 to 17,000 daltons. Even though it did not contain carbohydrate, its molecular weight was about 3,000 daltons greater than the protein portion of the standard α-subunit, suggesting the presence of a signal peptide

Detection of the TSH β-subunit precursor, pre-β, in cell-free translation mixtures of mouse tumor mRNA was also achieved, though this proved to be more difficult than detection of pre–α-subunits (88). Gel electrophoresis of the β precursor disclosed an apparent molecular weight of 15,000 (or 2,500 daltons greater than the protein portion of standard TSH-β-subunits), again consistent with the presence of a signal peptide.

Biosynthesis of Thyrotropin in Intact Cells

Cell-free mRNA translation studies were important to define the initial precursor forms of TSH subunits, because these forms are rapidly processed in vivo.Studies of TSH biosynthesis in intact cells, however, were necessary to elucidate the steps of posttranslational processing of TSH, including the  glycosylation and combination of TSH subunits, and the subsequent processing of the oligosaccharides (89).

After incubation of mouse thyrotropic tumor tissue with 35S-methionine for 10 minutes, most α-subunits had a molecular weight of 18,000 daltons, while a few had a molecular weight of 21,000 daltons (90). When the pulse incubation was followed by chase incubations, the 18,000-dalton form of the α-subunit was progressively converted to the 21,000-dalton form. Treatment with endoglycosidase H converted both the 18,000- and 21,000-dalton molecular weight forms to an 11,000-dalton form, consistent with the weight of the protein portion of standard α-subunits, suggesting that the 11,000-, 18,000-, and 21,000-dalton forms of the α-subunit have 0, 1, and 2 asparagine-linked oligosaccharide chains, respectively.

35S-methionine-labeled β-subunits accumulated as an 18,000-dalton form, which was then converted to an 11,000-dalton form after endoglycosidase H treatment. Thus, it appeared that the 11,000- and 18,000-dalton forms had 0 and 1 asparagine-linked oligosaccharide units, respectively (90). Approximately 20% of the β-subunits combined with excess α-subunits after a 20-minute pulse incubation. Analyses of subcellular fractionations disclosed that combination of α- and β-subunits began in the rough endoplasmic reticulum, and that the combining subunits had high-mannose, endoglycosidase H–sensitive oligosaccharides (91). TSH-subunit precursors were processed slowly to forms with mature complex oligosaccharides that were resistant to endoglycosidase H. TSH and excess free α-subunits, but no free β-subunits, were released into the medium after a 60- to 240-minute chase, and most had endoglycosidase H–resistant oligosaccharide chains.

The free α-subunits that were secreted had a slightly higher molecular weight than the form of α-subunit that combined with β-subunits. Free α-subunits from bovine pituitaries were found to be glycosylated at an additional site: the threonine at position 43 (92). This residue is located in a domain of the α-subunit thought to contact the β-subunit during heterodimer formation (1). Apparently, free α-subunits bearing this O-linked oligosaccharide are no longer able to bind to β-subunits. The physiologic importance of such noncombining forms of α-subunits is unclear.

Processing of High-Mannose Precursor Oligosaccharides

The subunits of TSH are cotranslationally glycosylated with oligosaccharides containing three glucose and nine mannose residues, termed high-mannose precursors. The oligosaccharides are preassembled in the rough endoplasmic reticulum, in which they are linked by phosphates at the reducing terminus to a long organic molecule containing approximately 20 units of polyprene, the dolichol phosphate carrier. Asparagine residues in nascent peptides destined to become glycosylated in an N-linked fashion are present in the sequence: asparagine-(X)-serine or asparagine-(X)-threonine, where X is any amino acid. There is a cotranslational en bloc transfer of the oligosaccharide from the dolichol carrier to the asparagine in the nascent α- or β-subunit. Two glucose residues are then quickly trimmed by a glucosidase, followed by a slower cleavage of the third glucose by another glucosidase. Mannose residues are then progressively cleaved by two mannosidases until a three-unit “core” remains, followed by addition of N-acetylglucosamine (GlcNAc) and other sugars by specific glycosyltransferases, to form complex oligosaccharides. For the pituitary glycoprotein hormones, TSH and LH, such complex oligosaccharides may terminate in either sulfate or sialic acid residues and yield heterogeneous forms with one sulfate (S1), two sulfates (S2), one sialic acid (N1), two sialic acids (N2), three sialic acids (N3), or one sulfate and one sialic acid (S-N). Processing of the high-mannose oligosaccharides of the TSH subunits is relatively slow compared with other glycoproteins (90,93). The rate of trimming of mannose residues appears to be much faster for free α-subunits than for TSH β-subunits.

Terminal Sulfation and Sialylation of Complex Oligosaccharides

The presence of an unusual terminal constituent on TSH oligosaccharides was suggested by studies showing that TSH, unlike CG, was partially resistant to neuraminidase digestion. Subsequently, sulfate was detected as a component of certain complex oligosaccharides of α-subunits of bovine TSH and LH and human LH, but not human CG (94). The negatively charged sulfate was thought to play some functional role comparable to that of the negatively charged sialic acid. Later studies revealed metabolic incorporation of 35S-sulfate into the oligosaccharides of the α- and β-subunits of bovine and ovine LH (95,96). Similarly, subunits of mouse TSH could be metabolically labeled with sulfate and sialic acid, there was differential sulfation of α- and β-subunits of TSH, and the sulfate moieties proved to be entirely linked to carbohydrate chains (97,98).

The sulfate moiety in TSH and LH oligosaccharides is covalently linked to N-acetylgalactosamine residues, in contrast to the usual terminal structure of complex oligosaccharides, in which sialic acid is bound to galactose residues (99). Bovine pituitary tissue contains a N-acetylgalactosamine transferase that specifically recognizes the β-subunits of TSH and LH, as well as the common α-subunit (100). A pituitary sulfotransferase is responsible for transfer of the sulfate moiety to the oligosaccharide and does not require subunit peptide determinants for recognition. This enzyme, like the N-acetylgalactosamine transferase, is absent from the placenta, which explains why only pituitary glycoprotein hormones contain the unusual N-acetylgalactosamine sulfate terminal residues.

The structures and distributions of heterogeneous forms of sulfated and sialyated oligosaccharides on TSH as well as various other glycoprotein hormones have been determined (98,101). For bovine TSH, 48% of the oligosaccharides contained S2, 32% contained S1, 18% were neutral, and 2% contained S-N; no complex oligosaccharides contained sialic acid residues exclusively. In contrast, the complex oligosaccharide structure of human TSH was somewhat different: 25% contained S1, 21% contained S-N, 18% were neutral, 12% contained N2, and 5% contained N1.

In addition to terminal sulfation and sialylation, TSH oligosaccharides contain variable amounts of fucose linked to the innermost N-acetylglucosamine residue (101). In general, β-subunits of TSH contain about twice as much fucose as α-subunits. Although fucosylation is normally thought to occur primarily in the Golgi apparatus, in pituitaries from hypothyroid mice a major proportion of the fucosylation occurs in the rough endoplasmic reticulum (102). In normal subjects, intravenous infusion of TRH causes the acute release of TSH isoforms that are more highly fucosylated than the TSH present before TRH administration (103).


The high-mannose oligosaccharides of TSH are differentially processed in mouse pituitary glands, depending on the thyroid state of the animals or level of TRH stimulation (93,104). In vitro, TRH stimulates the production of TSH enriched in biantennary complex oligosaccharides, as opposed to more complicated triantennary forms. This effect is closely coupled with TSH secretion, and intracellular forms of TSH do not vary in carbohydrate structure. In vivo, rats with hypothalamic or thyroidal hypothyroidism also have changes in the oligosaccharide structure of TSH (105,106,107,108). Animals with lesions of the paraventricular nucleus of the hypothalamus, which results in TRH deficiency, secreted TSH with fewer biantennary structures, as compared with normal animals. In contrast, thyroidectomized animals secreted TSH with more complex triantennary structures. Administration of TRH to the animals with hypothalamic lesions resulted in secretion of TSH with an increase in multiantennary structures so that their TSH was similar to that of normal animals. The differences between in vitro and in vivo TRH administration on the carbohydrate structure of TSH indicates that static exposure of cultured cells to TRH results in a pattern of glycosylation that differs from the resulting pulsatile pattern of TRH secretion in vivo. In TRH-knockout mice, the TSH that is secreted has decreased biological activity, presumably as a result of alterations in glycosylation (79).

The pattern of sulfation and sialylation of the oligosaccharides of TSH that is secreted varies during maturation of the pituitary–thyroid axis in rats (109,110,111). During the neonatal period, the amounts of sialylated oligosaccharides increased, as compared with sulfated oligosaccharides, as did all forms of complex oligosaccharides containing three or more negatively charged terminal moieties. In the neonatal period, hypothyroidism of even a few days or weeks duration resulted in major increases in the sialylated as compared with sulfated oligosaccharides. This change, present in both α- and β-subunits, was particularly striking in the latter. Moreover, there was a major increase in β-subunits containing three or more charged oligosaccharide units. Similar increases in the proportion of sialylated compared with sulfated oligosaccharides in TSH were found in adult hypothyroid rats, but, as compared with neonatal rats, the appearance of these changes was slow.

In humans, the sialylation of TSH varies among those who are normal and those with various thyroid disorders, in particular primary and central hypothyroidism (112, 113). Patients with severe nonthyroidal illness secrete forms of TSH with altered binding to concanavalin-A, suggesting increased amounts of multiantennary complex chains (114). In patients with TSH-secreting pituitary adenomas, the TSH and free α-subunits that are secreted vary considerably in oligosaccharide content (115,116), which changes if tumoral secretion of TSH falls in response to octreotide therapy (117,118) (see Chapter 24).

Thus, the complex carbohydrate structure of TSH can be altered by a variety of developmental, endocrine, and cellular factors. Although the functional effect of changes in the carbohydrate structure of TSH have not been completely elucidated, it is very clear that certain structure changes are associated with changes in the biologic activity of TSH or its metabolic clearance.


Heterogeneous forms of TSH may have different degrees of biological activity in a variety of in vivo and in vitro bioassays (reviewed in 7). These differences in activity are primarily due to differences in carbohydrate composition, as noted previously. The role of the complex oligosaccharide moieties in TSH action has been determined by studies in which chemical or enzymatic deglycosylation was used to remove complex side-chains. In initial studies, bovine or human TSH preparations were deglycosylated with anhydrous hydrogen fluoride or trifluoromethane sulfonic acid (119,120,121). Although deglycosylated TSH had receptor-binding properties similar to those of the native hormone, its biological activity both in vitro and in vivo was markedly decreased. Moreover, the deglycosylated TSH competitively inhibited the action of native TSH (120). Most studies have confirmed the link between TSH oligosaccharide structure and bioactivity, and have established that the oligosaccharides of the α-subunit are particularly important for bioactivity (122,123,124).

TSH isohormones separated by isoelectric focusing have different bioactivity, but the chemical basis of the difference has remained unclear. Incubation of rat pituitary glands with TRH in vitro led to secretion of TSH with increased bioactivity (125). Because other studies (described earlier in the chapter) demonstrated that TRH alters the carbohydrate structure of TSH, it seems reasonable to conclude that such altered bioactivity is related to changes in carbohydrate. In fact, serum TSH from some patients with central hypothyroidism had a low ratio of biologic to immunologic activity, as compared with TSH from normal subjects (126). In some of the patients, chronic TRH administration resulted in secretion of TSH with more biological activity, as detected not only in vitro, but also by a rise in the patients' serum T4 concentrations (126). Increases in the ratio of biologic to immunologic activity have also been found in the serum TSH of patients with TSH-secreting pituitary adenomas (127) and patients with resistance to thyroid hormone (128). However, a change in the ratio of biologic to immunologic activity of TSH in serum does not necessarily prove that its bioactivity has changed, because the ratio would also change if the hormone's immunologic activity changed.

The oligosaccharide content of TSH not only affects its bioactivity, but also its metabolic clearance. In humans, the half-time of disappearance of TSH varies with thyroid state—being slower in patients with hypothyroidism and faster in patients with thyrotoxicosis (129). In rats, the kidney is the major organ of clearance, with the thyroid being of secondary importance, and there is little hepatic clearance; chemically deglycosylated TSH is cleared more rapidly than is native TSH (130). Moreover, serum or pituitary TSH from hypothyroid rats is cleared more slowly than that from normal rats when injected into normal rats. These results suggest that the clearance of TSH varies according to both the thyroid status of the TSH ‘donor’ and the thyroid status of the animal in which the clearance studies are done. Presumably, the slower metabolic clearance rate of the TSH of hypothyroid rats is related to its increased sialylation (110,111). With respect to recombinant human TSH, sialylation is an important determinant of the hormone's metabolic clearance rate and in vivo bioactivity (10).


While the exact structure of TSH is not known, the structure of human CG, as determined by crystallography, is known (3,4). Modeling of the structure of TSH based on the structure of CG has been extremely valuable in understanding its structure–function relationships (for review see reference 131); for example, it has allowed the location of the disulfide bonds within the subunits to be determined—crucial information for a meaningful model to be constructed. Based on a double alkylation method, TSH contains disulfide bonds analogous to human CG (132). Each TSH subunit has three loops (L1-L3) and a central cystine-knot structure (Fig. 10A.9). Two loops from the same subunit are on one side of the knot and a long loop is on the other side. The α-subunit loop 2 (αL2) loop is unique in that it contains a two-turn α helix. In the β-subunit of TSH, three disulfide bridges are involved in forming the cystine-knot, one bridge links two β-hairpin loops, and two bridges form the seat belt where the β-subunit wraps around the α-subunit. Moreover, one of the α-subunit oligosaccharides known to be more important for signal transduction, the oligosaccharide at Asn52, lies near TSH-subunit residues believed to be vital for receptor binding. However, differences in not only amino acid content but also glycosylation between human CG and TSH could affect their structures. Finally, TSH may undergo a conformational change when bound to its receptor.

FIGURE 10A.9. Diagram of the structure of human thyrotropin (TSH) based on human chorionic gonadotropin crystallography and TSH structure function studies. The seat-belt region is where the β-subunit wraps around loop 2 of the α-subunit (αL2) and stablizes the TSH heterodimer. αL, α-subunit loop; βL, β-subunit loop.

The use of computer modeling, site-directed mutagenesis, and antibodies or synthetic peptides has enabled investigators to make inferences about the three-dimensional conformation of TSH, the subunit contact domains, and the receptor-binding domains of the two subunits (133,134,135,136,137,138,139,140,141,142). For example, based on a mutagenesis approach, the seat-belt region appears to regulate the specificity of glycoprotein hormone receptor recognition (143). In particular, when the seat-belt region of TSH-β is replaced by the corresponding region of CG, the chimeric protein binds to the CG/LH receptor, but not the TSH receptor. Conversely, when the seat-belt region of CG is replaced by the corresponding region of TSH, CG/LH receptor recognition is abolished, and the chimeric protein binds to the TSH receptor. In contrast, when the seat-belt region of TSH is exchanged with that of FSH, the chimeric protein does not bind to the FSH receptor (143), suggesting that other regions also mediate the specificity of receptor interactions.

Mutagenesis experiments have also implicated domains in the α-subunit responsible for TSH action. For example, bovine TSH has greater biologic activity than human TSH, and they differ in amino acids 11 to 20 of the α-subunit. Replacement of amino acids 11, 13, 16, or 20 of the human α-subunit with lysine (all are present as lysine in the bovine α-subunit), and joining the mutant α-subunit with the β-subunit of human TSH, generates TSH molecules with greater receptor-binding affinity and biological activity than human TSH (144).

Mutagenesis of individual residues within amino acids 33 to 44 of the α-subunit has demonstrated the importance of this region for glycoprotein hormone assembly and action (145). In particular, Pro38 is important for heterodimerization with TSH-β and FSH-β. In contrast, Phe33 and Arg35 mediate heterodimerization with FSH-β, but not TSH-β, and Ala36 mediates heterodimeriztion with CG-β. Mutations of Arg35 impair the ability of TSH to stimulate cyclic AMP production in thyroid tissue, and residues Arg42-Ser43-Lys44 are important for TSH-receptor binding. Therefore, changes in the amino-terminal portion of the α-subunit generate mutants with altered capacity for heterodimerization and receptor binding, and altered in vitro bioactivity (144,145).

Mutations in the carboxyl-terminal portion of the α-subunit, residues 89 to 92, also cause decreased TSH receptor binding and bioactivity (146). In contrast, mutations in the cystine knot region of the α-subunit impaired secretion and heterodimer formation, but did not decrease in vitro bioactivity (147). Fusion of the α- and β-subunits created a single protein with longer half-life than native TSH, but normal receptor binding (148,149). Fusion of LH-β with the α-subunit also generated a mutant protein with increased stability (150). These data suggest that heterodimer dissociation is important in determining the in vivo half-life of glycoprotein hormones (148). These mutant hormones may be useful in generating other glycoprotein hormone analogs with novel properties.


Recombinant human TSH has been produced by transfection of the gene for the common α-subunit and the gene for the β-subunit of human TSH into human embryonic kidney cells or Chinese hamster ovary cells (151,152,153). The recombinant TSH was more highly sialylated than TSH extracted from human pituitary glands, and it also contained no N-acetylgalactosamine, implying the absence of terminal sulfate moieties [both are present in pituitary TSH (9)]. The absence of N-acetylgalactosamine and sulfate was expected, since only the pituitary contains the specific enzymes for transfer of these moieties to carbohydrate chains. The maximum stimulatory activity of recombinant human TSH was similar to that of pituitary TSH in two different in vitro bioassays (153); however, the recombinant preparation was slightly less potent as judged by the concentration required for half-maximal response. This decreased potency was clearly related to the increased sialic acid content, because the activity of the TSH increased after treatment with neuraminidase. In rats, the rate of metabolic clearance of recombinant human TSH was slower, by half, than that of pituitary TSH, which resulted in greater than 10-fold higher serum concentrations 3 hours after intravenous TSH administration (153). The slower metabolic clearance rate was again related to the increased sialic acid content, since neuraminidase-treated TSH was cleared considerably faster. All these differences can be related to the higher degree of sialylation of recombinant human TSH, as compared with standard pituitary TSH.

It is possible to produce recombinant human TSH in large quantities (10). Unlike natural human TSH extracted and purified derived from pools of human pituitaries obtained at autopsy, recombinant TSH is more homogeneous and free of other contaminating pituitary hormones and growth factors as well as artifactual proteolytic cleavage products. The heterogeneity of the recombinant hormone is primarily related to six to nine different isoforms, differing in the number of terminal sialic acid residues present in the carbohydrate chains (10).

Recombinant TSH is also valuable clinically and has provided a new diagnostic agent for the evaluation of patients with thyroid carcinoma (see Chapter 14 and section on radioiodine and other treatments and outcomes in Chapter 70).


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