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).
GENES ENCODING THE SUBUNITS OF THYROTROPIN
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).
PRETRANSLATIONAL REGULATION OF THYROTROPIN BIOSYNTHESIS
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).
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.
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).
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.
POSTTRANSLATIONAL PROCESSING OF THYROTROPIN
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).
DEVELOPMENTAL AND ENDOCRINE REGULATION OF THYROTROPIN GLYCOSYLATION
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.
BIOACTIVITY AND METABOLIC CLEARANCE OF THYROTROPIN
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).
THREE-DIMENSIONAL CONFORMATION OF THYROTROPIN
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 THYROTROPIN
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).
1. Pierce JG, Parsons TF. Glycoprotein hormones: structure and function. Annu Rev Biochem 1981;50:465.
2. Lapthorn AJ, Harris DC, Littlejohn A, et al. Crystal structure of human chorionic gonadotropin. Nature 1994;369:455.
3. Wu H, Lustbader JW, Liu Y, et al. Structure of human chorionic gonadotropin at 2.6A resolution from MAD analysis of the selenomethionyl protein. Structure 1994;2:545.
4. Magner JA. Thyroid-stimulating hormone: biosynthesis, cell biology, and bioactivity. Endocr Rev 1990;11:354.
5. Green ED, Baenziger JU. Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. I. structural elucidation of the sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. J Biol Chem 1988;263:25.
6. Green ED, Baenziger JU. Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. II. Distributions of sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. J Biol Chem 1988; 263:36.
7. Persani L. Hypothalamic thyrotropin-releasing hormone and thyrotropin biological activity. Thyroid 1998;8:941.
8. Cole ES, Lee K, Lauziere K, et al. Recombinant human thyroid stimulating hormone: development of a biotechnology product for detection of metastatic lesions of thyroid carcinoma. Biotechnology 1993;11:1014.
9. Szkudlinski MW, Thotakura NR, Bucci I, et al. Purification and characterization of recombinant human thyrotropin (TSH) isoforms produced by Chinese hamster ovary cells: the role of sialylation and sulfation in TSH bioactivity. Endocrinology 1993; 133:1490.
10. Braverman LE, Pratt BM, Ebner S, et al. Recombinant human thyrotropin stimulates thyroid function and radioactive iodine uptake in the rhesus monkey. J Clin Endocrinol Metab 1992; 74:1135.
11. Ladenson PW, Braverman LE, Mazzaferri EL, et al. Comparison of administration of recombinant human thyrotropin with withdrawal of thyroid hormone for radioactive iodine scanning in patients with thyroid carcinoma. N Engl J Med 1997;337: 888.
12. Goodwin RG, Moncman CL, Rottman FM, et al. Characterization and nucleotide sequence of the gene for the common α-subunit of the bovine pituitary glycoprotein hormones. Nucleic Acids Res 1983;11:6873
13. Gordon DF, Wood WM, Ridgway EC. Organization and nucleotide sequence of the mouse α-subunit gene of the pituitary glycoprotein hormones. DNA 1988;7:679
14. Burnside J, Buckland PR, Chin WW. Isolation and characterization of the gene encoding the α-subunit of the rat pituitary glycoprotein hormones. Gene 1988;70:67.
15. Fiddes JC, Goodman HM. The gene encoding the common alpha subunit of the four human glycoprotein hormones. J Mol Appl Genet 1981;1:3.
16. Breathnach R, Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem 1981;50:349.
17. Deutsch PJ, Jameson JL, Habener JF. Cyclic AMP responsiveness of human gonadotropin α gene transcription is directed by a repeated 10-bp enhancer. J Biol Chem 1987;262:12169.
18. Silver BJ, Bokar JA, Virgin JB, et al. Cyclin AMP regulation of the human glycoprotein hormone α-subunit is mediated by an 18-bp element. Proc Natl Acad Sci U S A 1987;84: 2198.
19. Bokar JA, Keri RA, Farmerie TA, et al. Expression of the glycoprotein hormone α-subunit gene in the placenta requires a functional cyclic AMP response element, whereas a different cis-acting element mediates pituitary-specific expression. Mol Cell Biol 1989;9:5113.
20. Carr FE, Need LR, Chin WW. Isolation and characterization of the rat thyrotropin β-subunit gene: differential regulation of two transcriptional start sites by thyroid hormone. J Biol Chem 1987;262:981.
21. Wondisford FE, Radovick S, Moates JM, et al. Isolation and characterization of the human thyrotropin β-subunit gene. J Biol Chem 1988;262:12538
22. Guidon PT, Whitfield GK, Porti D, et al. The human thyrotropin β-subunit gene differs in 5′ structure from murine TSHβ genes. DNA 1988;7:691.
23. Tatsumi K, Hayashizaki Y, Hiraoka Y, et al. The structure of the human thyrotropin β-subunit gene. Gene 1988;73:489
24. Wolf D, Kourides IA, Gurr JA. Expression the gene for the β-subunit of mouse thyrotropin results in multiple mRNAs differing in their 5′ untranslated region. J Biol Chem 1987;262: 16596.
25. Gordon DF, Wood WM, Ridgway EC. Organization and nucleotide sequence of the gene encoding the beta subunit of murine thyrotropin. DNA 1988;7:17.
26. Samuels MH, Wood WM, Gordon DF, et al. Clinical and molecular studies of a thyrotropin-secreting pituitary adenoma. J Clin Endocrinol Metab 1989;68:1211.
27. Hayashizaki Y, Hiraoka Y, Endo Y, et al. Thyroid-stimulating hormone (TSH) deficiency caused by a single base substitution in the CAGYC region of the β-subunit. EMBO J 1989; 8:2291.
28. Dacou-Voutetakis C, Feltquate DM, Drakopoulou M, et al. Familial hypothyroidism caused by a nonsense mutation in the thyroid-stimulating hormone β-subunit gene. Am J Hum Genet 1990;16:988.
29. Vuissoz JM, Deladoey J, Buyukgebiz A, et al. New autosomal recessive mutation of the TSH-beta subunit gene causing central isolated hypothyroidism. J Clin Endocrinol Metab 2001; 86:4468.
30. Pohlenz J, Dumitrescu A, Aumann U, et al. Congenital secondary hypothyroidism caused by exon skipping due to a homozygous donor splice site mutation in the TSH beta subunit gene. J Clin Endocrinol Metab 2002; 87:336.
31. Medeiros-Neto G, Herodotou DT, Rajan S, et al. A circulating, biologically inactive thyrotropin caused by a mutation in the beta subunit gene. J Clin Invest 1996;97:1250.
32. Deladoey J, Vuissoz JM, Domene HM, et al. Congenital secondary hypothyroidism due to a mutation C105Vfs114X thyrotropin-beta mutation: genetic study of five unrelated families from Switzerland and Argentina. Thyroid 2003;13:553.
33. Cohen LE, Radovick S. Molecular basis of combined pituitary hormone deficiencies. Endocr Rev 2002;23:431.
34. Drolet DW, Scully KM, Simmons DM, et al. TEF, a transcription factor expressed specifically in the anterior pituitary during embryogenesis, defines a new class of leucine zipper proteins. Genes Dev 1991;5:1739.
35. Lin SC, Li S, Drolet DW, et al. Pituitary ontogeny of the Snell dwarf mouse reveals Pit-1-independent and Pit-1-dependent origins of the thyrotrope. Development 1994;120:515.
36. Cohen LE, Wondisford FE, Radovick S. Role of Pit-1 in the gene expression of growth hormone, prolactin, and thyrotropin. Endocrinol Metab Clin North Am 1996; 25:523.
37. Li S, Crenshaw EB, Rawson EJ, et al. Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain pit-1. Nature 1990;347:528.
38. Radovick S, Nations M, Du Y, et al. A mutation in the POU-homeodomain of Pit-1 responsible for combined pituitary hormone deficiency. Science 1992;257:1115.
39. Pfaffle RW, DiMattia GE, Parks JS, et al. Mutation of the POU-specific domain of Pit-1 and hypopituitarism without pituitary hypoplasia. Science 1992;257:1118.
40. Cohen LE, Wondisford FE, Salvatoni A, et al. A “hot spot” in the Pit-1 gene responsible for combined pituitary hormone deficiency: clinical and molecular correlates. J Clin Endocrinol Metab 1995; 80:679.
41. Cohen LE, Zanger K, Brue T, et al. Defective retinoic acid regulation of the Pit-1 gene enhancer: a novel mechanism of combined pituitary hormone deficiency. Mol Endocrinol 1999;13: 476.
42. Sornson MW, Wu W, Dasen JS, et al. Pituitary lineage determination by the prophet of Pit-1 homeodomain factor defective in Ames dwarfism. Nature 1996;384:327.
43. Wu W, Cogan JD, Pfaffle RW, et al. Mutations in PROP1 cause familial combined pituitary hormone deficiency. Nat Genet 1998;18:147.
44. Fluck C, Deladoey, Rutishauser K, et al. Phenotypic variability in familial combined pituitary hormone deficiency caused by a PROP1 gene mutation resulting in the substitution of Arg–Cys at Codon 120 (R120C). J Clin Endocrinol Metab 1998;83:3727.
45. Cogan JD, Wu W, Phillips JA, et al. The PROP1 2-base pair deletion is a common cause of combined pituitary hormone deficiency. J Clin Endocrinol Metab 1998;83:3346.
46. Lazar M.A. Thyroid hormone receptors: multiple forms, multiple possibilities. Endocr Rev 1993 14:184.
47. Bradley DJ, Towle HC, Young WS 3rd. Alpha and beta thyroid hormone receptor (TR) gene expression during auditory neurogenesis: evidence for TR isoform-specific transcriptional regulation in vivo. Proc Natl Acad Sci U S A 1994;91:439.
48. Yen PM. Physiological and molecular basis of thyroid hormone action. Physiol Rev 2001;81:1097.
49. Jepsen K, Rosenfeld MG. Biological roles and mechanistic actions of co-repressor complexes. J Cell Sci 2002;115:689.
50. Shupnik MA, Chin WW, Habener JF, et al. Transcriptional regulation of the thyrotropin subunit genes by thyroid hormone. J Biol Chem 1985;260:2900.
51. Gurr JA, Kourides IA. Thyroid hormone regulation of thyrotropin α and β-subunit gene transcription. DNA 1985;4:301.
52. Segerson TP, Kauer J, Wolfe HC, et al. Thyroid hormone regulates TRH biosynthesis in the paraventricular nucleus of the rat hypothalamus. Science 1987;238:78.
53. Taylor T, Wondisford FE, Blaine T, et al. The paraventricular nucleus of the hypothalamus has a major role in thyroid hormone feedback regulation of thyrotropin synthesis and secretion. Endocrinology 1990;126:317.
54. Ridgway EC, Kourides IA, Chin WW, et al. Augmentation of pituitary thyrotropin response to TRH during subphysiological triiodothyronine therapy in hypothyroidism. Clin Endocrinol (Oxf) 1979;10:343.
55. Wondisford FE. Thyroid hormone action: insight from transgenic mouse models. J Investig Med 2003;51:215.
56. Cohen O, Flynn TR, Wondisford FE. Ligand-dependent antagonism by retinoid X receptors of inhibitory thyroid hormone response elements. J Biol Chem 1995;270:13899.
57. Hollenberg AN, Monden T, Flynn TR, et al. The human thyrotropin-releasing hormone gene is regulated by thyroid hormone through two distinct classes of negative thyroid hormone response elements. Mol Endocrinol 1995;9:540.
58. Shibusawa N, Hollenberg AN, Wondisford FE. Thyroid hormone receptor DNA binding is required for both positive and negative gene regulation. J Biol Chem 2003 278:732.
59. Shibusawa N, Hashimoto K, Nikrodhanond AA, et al. Thyroid hormone action in the absence of thyroid hormone receptor DNA-binding in vivo. J Clin Invest 2003;112:588.
60. Forrest D, Erway LC, Ng L, et al. Thyroid hormone receptor beta is essential for development of auditory function. Nat Genet 1996;13:354.
61. Weiss RE, Forrest D, Pohlenz J, et al. Thyrotropin regulation by thyroid hormone in thyroid hormone receptor beta-deficient mice. Endocrinology 1997;138:3624.
62. Abel ED, Boers ME, Pazos-Moura C, et al. Divergent roles for thyroid hormone receptor beta isoforms in the endocrine axis and auditory system. J Clin Invest 1999;104:291.
63. Gauthier K, Chassande O, Plateroti M, et al. Different functions for the thyroid hormone receptors TR alpha and TR beta in the control of thyroid hormone production and post-natal development. EMBO J 1999;18:623.
64. Gothe S, Wang Z, Ng L, et al. Mice devoid of all known thyroid hormone receptors are viable but exhibit disorders of the pituitary-thyroid axis, growth, and bone maturation. Genes Dev 1999;13:1329.
65. Abel ED, Ahima R, Boer ME, et al. Critical role for thyroid hormone receptor β-2 in the regulation of TRH neurons in the paraventricular hypothalamus. J Clin Invest 2001;107: 1017.
66. Weiss RE, Refetoff S. Resistance to thyroid hormone. Rev Endocrinol Metab Disord 2000;1:97.
67. Hashimoto K, Curty FH, Borges PP, et al. An unliganded thyroid hormone receptor causes severe neurological dysfunction. Proc Natl Acad Sci U S A 2001;98:3998.
68. Kaneshige M, Kaneshige K, Zhu X, et al. Mice with a targeted mutation in the thyroid hormone beta receptor gene exhibit impaired growth and resistance to thyroid hormone. Proc Natl Acad Sci U S A 2000;97:13209.
69. Ando S, Sarlis NJ, Krishnan J, et al. Aberrant alternative splicing of thyroid hormone receptor in a TSH-secreting pituitary tumor is a mechanism for hormone resistance. Mol Endocrinol 2001;15:1529.
70. Re RN, Kourides IA, Ridgway EC. The effect of glucocorticoid administration on human pituitary secretion of thyrotropin and prolactin. J Clin Endocrinol Metab 1976;43:338.
71. Ross DS, Ellis MF, Milbury P, et al. A comparison of changes in plasma thyrotropin β and α-subunits, and mouse thyrotropic tumor thyrotropin β and α-subunit mRNA concentrations after in vivo dexamethasone or T3 administration. Metabolism 1987;36:799.
72. Ahlquist JAO, Franklyn JA, Wood DF, et al. Hormonal regulation of thyrotropin synthesis and secretion. Horm Metab Res Suppl 1987;17:86.
73. Shupnik MA, Greenspan SL, Ridgway EC. Transcriptional regulation of thyrotropin subunit genes by thyrotropin-releasing hormone and dopamine in pituitary cell culture. J Biol Chem 1986; 261:12675.
74. Perrone MH, Hinkle PM. Regulation of pituitary receptors for thyrotropin-releasing hormone by thyroid hormones. J Biol Chem 1978;253:5168.
75. Weintraub BD, Wondisford FE, Farr EA, et al. Pre-translational and post-translational regulation of TSH synthesis in normal and neoplastic thyrotrophs. Horm Res 1989;32:22.
76. Steinfelder HJ, Radovick S, Wondisford FE. Hormonal regulation of the thyrotropin β subunit gene by phosphorylation of the pituitary-specific transcription factor Pit-1. Proc Natl Acad Sci U S A 1992;89:5942.
77. Zanger K, Cohen LE, Hashimoto K, et al. A novel mechanism for cyclic adenosine 3′,5′-monophosphate regulation of gene expression by CREB-binding protein. Mol Endocrinol 1999; 13: 268.
78. Hashimoto K, Zanger K, Hollenberg AN, et al. cAMP response element-binding protein-binding protein mediates thyrotropin-releasing hormone signaling on thyrotropin subunit genes. J Biol Chem 2000;275:33365.
79. Yamada M, Saga Y, Shibusawa N, et al. Tertiary hypothyroidism and hyperglycemia in mice with targeted disruption of the thyrotropin-releasing hormone gene. Proc Natl Acad Sci U S A 1997;94:10862.
80. Collu R, Tang J, Castagne J, et al. A novel mechanism for isolated central hypothyroidism: inactivating mutations in the thyrotropin-releasing hormone receptor gene. J Clin Endocrinol Metab 1997;82:1561.
81. Collu R. Genetic aspects of central hypothyroidism. J Endocrinol Invest 2000;23:125.
82. Xu L, Lavinsky RM, Dasen JS, et al. Signal-specific co-activator domain requirements for Pit-1 activation. Nature 1998; 395:301.
83. Cooper DS, Klibanski A, Ridgway EC. Dopaminergic modulation of TSH and its subunits; in vivo and in vitro studies. Clin Endocrinol (Oxf) 1983;18:265.
84. Scanlon WF, Weightman DR, Shale DJ, et al. Dopamine is a physiological regulator of thyrotropin secretion in man. Clin Endocrinol (Oxf) 1979;10:7.
85. Lee E, Chen P, Rao H, et al. Effect of acute high dose dobutamine administration on serum thyrotrophin (TSH). Clin Endocrinol (Oxf) 1999;50:487.
86. Chin WW, Habener JF, Kieffer JD, et al. Cell-free translation of the messenger RNA coding for the α-subunit of thyroid-stimulating hormone. J Biol Chem 1978;253:7985.
87. Kourides IA, Weintraub BD. mRNA directed biosynthesis of α subunit of thyrotropin: translation in cell-free and whole-cell systems. Proc Natl Acad Sci U S A 1979;76:298.
88. Giudice LC, Weintraub BD. Evidence for conformational differences between precursor and processed forms of TSH-β-subunit. J Biol Chem 1979;254:12679.
89. Chin WW, Maloof F, Habener JF. Thyroid-stimulating hormone biosynthesis. J Biol Chem 1981;256:3059.
90. Weintraub BD, Stannard BSD, Magner JA, et al. Glycosylation and post-translational processing of thyroid-stimulating hormone: clinical implications. Recent Prog Horm Res 1985;41: 577.
91. Magner JA, Weintraub BD. Thyroid-stimulating hormone subunit processing and combination in microsomal subfractions of mouse pituitary tumor. J Biol Chem 1982;257:6709.
92. Parsons TF, Bloomfield GA, Pierce JG. Purification of an alternative form of the α subunit of the glycoprotein hormones from bovine pituitaries and identification of its O-linked oligosaccharides. J Biol Chem 1983;258:240.
93. Ronin C, Stannard BS, Weintraub BD. Differential processing and regulation of thyroid-stimulating hormone subunit carbohydrate chains in thyrotropic tumors and in normal and hypothyroid pituitaries. Biochemistry 1985;24:562.
94. Parsons TF, Pierce JG. Oligosaccharide moieties of glycoprotein hormones: bovine lutropin resists enzymatic deglycosylation because of terminal O-sulfated N-acetylhexosamines. Proc Natl Acad Sci U S A 1980;77:7089.
95. Hortin G, Natowicz M, Pierce J, et al. Metabolic labeling of lutropin with [35S]sulfate. Proc Natl Acad Sci U S A 1981;78: 7468.
96. Anumula KR, Bahl OP. Biosynthesis of lutropin in ovine pituitary slices: incorporation of [35S]sulfate in carbohydrate units. Arch Biochem Biophys 1993;220:645.
97. Gesundheit N, Magner JA, Chen T, et al. Differential sulfation and sialylation of secreted mouse thyrotropin (TSH) subunits: regulation by TSH-releasing hormone. Endocrinology 1986; 119:455.
98. Gesundheit N, Gyves PW, DeCherney GS, et al. Characterization and charge distribution of the asparagine-linked oligosaccharides on secreted mouse thyrotropin and free α subunits. Endocrinology 1989;124:2967.
99. Baenziger JU, Green ED. Pituitary glycoprotein hormone oligosaccharides: structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin. Biochim Biophys Acta 1988;947:287.
100. Smith PL, Baenziger JU. A pituitary N-acetylgalactosamine transferase that specifically recognizes glycoprotein hormones. Science 1988;242:930.
101. Hiyama J, Weisshaar G, Renwick AG. The asparagine-linked oligosaccharides at individual glycosylation sites in human thyrotropin. Glycobiology 1992;2:401.
102. Magner JA, Novak W, Papagiannes E. Subcellular localization of fucose incorporation into mouse thyrotropin and free α subunits: studies employing subcellular fractionation and inhibitors of the intracellular translocation of proteins. Endocrinology 1986;119:1315.
103. Magner JA, Kane J, Chou ET. Intravenous thyrotropin (TSH)-releasing hormone releases human TSH that is structurally different than basal TSH. J Clin Endocrinol Metab 1992;74:1306.
104. Gesundheit N, Fink DL, Silverman LA, et al. Effect of thyrotropin-releasing hormone on the carbohydrate structure of secreted mouse thyrotropin: analysis by lectin affinity chromatography. J Biol Chem 1987;262:5197.
105. Taylor T, Gesundheit N, Gyves PW, et al. Hypothalamic hypothyroidism caused by lesions in rat paraventricular nuclei alters the carbohydrate structure of secreted thyrotropin. Endocrinology 1988;122:283.
106. Taylor T, Gesundheit N, Weintraub BD. Effects of in vivo bolus versus continuous TRH administration on TSH secretion, biosynthesis, and glycosylation in normal and hypothyroid rats. Mol Cell Endocrinol 1986;46:253.
107. Taylor T, Weintraub BD. Altered thyrotropin (TSH) carbohydrate structures in hypothalamic hypothyroidism created by paraventricular nuclear lesions are corrected by in vivo TSH-releasing hormone. Endocrinology 1989;125:2198.
108. Taylor T, Wondisford FE, Blaine T, et al. The paraventricular nucleus of the hypothalamus has a major role in thyroid feedback regulation of thyrotropin synthesis and secretion. Endocrinology 1990;126:317.
109. Gyves PW, Gesundheit N, Stannard BS, et al. Alterations in the glycosylation of secreted thyrotropin during ontogenesis: analysis of sialylated and sulfated oligosaccharides. J Biol Chem 1989;264:6104.
110. Gyves PW, Gesundheit N, Thotakura NR, et al. Changes in the sialylation and sulfation of secreted thyrotropin in congenital hypothyroidism. Proc Natl Acad Sci U S A 1990;87: 3792.
111. DeCherney GS, Gesundheit N, Gyves PW, et al. Alterations in the sialylation and sulfation of secreted mouse thyrotropin in primary hypothyroidism. Biochem Biophys Res Commun 1989; 159:755.
112. Papandreou M-J, Persani L, Asteria C, et al. Variable carbohydrate structures of circulating thyrotropin as studied by lectin affinity chromatography in different clinical conditions. J Clin Endocrinol Metab 1993;77:393.
113. Papandreou M-J, Asteria C, Pettersson K, et al. Concanavalin A affinity chromatography of human serum gonadotropins: evidence for changes of carbohydrate structure in different clinical conditions. J Clin Endocrinol Metab 1993;76:1008.
114. Lee HL, Suhl J, Pekary AE, et al. Secretion of thyrotropin with reduced concanavalin-A-binding activity in patients with severe nonthyroidal illness. J Clin Endocrinol Metab 1987;65:942.
115. Magner JA, Klibanski A, Fein H, et al. Ricin and lentil lectin-affinity chromatography reveals oligosaccharide heterogeneity of thyrotropin secreted by 12 human pituitary tumors. Metabolism 1992;41:1009.
116. Sergi I, Medri G, Papandreou M-J, et al. Polymorphism of thyrotropin and alpha subunit in human pituitary adenomas. J Endocrinol Invest 1993;16:45.
117. Chanson P, Weintraub BD, Harris AG. Octreotide therapy for thyroid-stimulating hormone-secreting pituitary adenomas: a follow-up of 52 patients. Ann Intern Med 1993;119: 236.
118. Francis TB, Smallridge RC, Kane J, et al. Octreotide changes serum thyrotropin (TSH) glycoisomer distribution as assessed by lectin chromatography in a TSH macroadenoma patient. J Clin Endocrinol Metab 1993;77:183.
119. Amir SM, Kubota K, Tramontano D, et al. The carbohydrate moiety of bovine thyrotropin is essential for full bioactivity but not for receptor recognition. Endocrinology 1987;120: 345.
120. Amr S, Menezes-Ferreira MM, Shimohigashi Y, et al. Activities of deglycosylated thyrotropin at the thyroid membrane receptor adenylate cyclase system. J Endocrinol Invest 1986;8:537.
121. Thotakura NR, LiCalzi L, Weintraub BD. The role of carbohydrate in thyrotropin action assessed by a novel method of enzymatic deglycosylation. J Biol Chem 1990;265:11527.
122. Endo Y, Tetsumoto T, Nagasaki H, et al. The distinct roles of α and β subunits of human thyrotropin in the receptor-binding and postreceptor events. Endocrinology 1990;127:149.
123. Thotakura NR, Szkudlinski MW, Weintraub BD. Structure-function studies of oligosaccharides of recombinant human thyrotrophin by sequential deglycosylation and resialylation. Glycobiology 1994;4:525.
124. Beck-Peccoz P, Persani L. Variable biological activity of thyroid-stimulating hormone. Acta Endocrinol (Copenh) 1994;131:331.
125. Menezes-Ferreira MM, Petrick PA, Weintraub BD. Regulation of thyrotropin (TSH) bioactivity by TSH-releasing hormone and thyroid hormone. Endocrinology 1986;118:2125.
126. Beck-Peccoz, Amr S, Menezes-Ferreira MM, et al. Decreased receptor binding of biologically inactive thyrotropin in central hypothyroidism: effect of treatment with thyrotropin-releasing hormone. N Engl J Med 1985;312:1085.
127. Nissim M, Lee KO, Petrick PA, et al. A sensitive thyrotropin (TSH) bioassay based on iodide uptake in rat FRTL-5 thyroid cells: comparison with the adenosine 3′,5′-monophosphate response to human serum TSH and enzymatically deglycosylated bovine and human TSH. Endocrinology 1987;121:1278.
128. Persani L, Asteria C, Tonacchera M, et al. Evidence for the secretion of thyrotropin with enhanced bioactivity in syndromes of thyroid hormone resistance. J Clin Endocrinol Metab 1994; 78:1034.
129. Ridgway EC, Weintraub BD, Maloof F. Metabolic clearance and production rates of human thyrotropin. J Clin Invest 1974; 53:895.
130. Constant RB, Weintraub BD. Differences in the metabolic clearance of pituitary and serum thyrotropin (TSH) derived from euthyroid and hypothyroid rats: effects of chemical deglycosylation of pituitary TSH. Endocrinology 1986;119:2720.
131. Szkudlinski MW, Fremont V, Ronin C, et al. Thyroid-stimulating hormone and thyroid-stimulating hormone receptor structure-function relationships. Physiol Rev 2002;82:473.
132. Fairlie WD, Stanton PG, Hearn MT. The disulphide bond structure of thyroid-stimulating hormone beta subunit. Biochem J 1996;314:449.
133. Reichert Jr LE, Dattatreyamurty B, Grasso P, et al. Structure-function relationships of the glycoprotein hormones and their receptors. Trends Pharmacol Sci 1991;12:199.
134. Combarnous Y. Molecular basis of the specificity of binding of glycoprotein hormones to their receptors. Endocr Rev 1992; 13:670.
135. Keutmann HT. Receptor-binding regions in human glycoprotein hormones. Mol Cell Endocrinol 1992;186:C1.
136. Reed DK, Ryan RJ, McCormick DJ. Residues in the subunit of human choriotropin that are important for interaction with the lutropin receptor. J Biol Chem 1991;266:14251.
137. Leinung MC, Reed DK, McCormick DJ, et al. Further characterization of the receptor-binding region of the thyroid-stimulating hormone α subunit: A comprehensive synthetic peptide study of the α subunit 26–46 sequence. Biochemistry 1991;88: 9707.
138. Liu C, Roth KE, Shepard BAL, et al. Site-directed alanine mutagenesis of Phe33, Arg35, and Arg42-Ser43-Lys44 in the human gonadotropin α subunit. J Biol Chem 1993;268: 21613.
139. Xia H, Chen F, Puett D. A region in the human glycoprotein hormone α subunit important in holoprotein formation and receptor binding. Endocrinology 1994;134:1768.
140. Yoo J, Zeng H, Ji I, et al. COOH-terminal amino acids of the α subunit play common and different roles in human choriogonadotropin and follitropin. J Biol Chem 1993;28: 13034.
141. Leinung MC, Bergert ER, McCormick DJ, et al. Synthetic analogs of the carboxyl-terminus of β-thyrotropin: The importance of basic amino acids in receptor binding activity. Biochemistry 1992;31:10094.
142. Freeman SL, McCormick DJ, Ryan RJ, et al. Inhibition of TSH bioactivity by synthetic TSH beta peptides. Endocr Res 1992;18:1.
143. Grossman M, Szkudlinski MW, Wong R, et al. Substitution of the seat-belt region of the thyroid-stimulating hormone (TSH) β subunit with the corresponding regions of choriogonadotropin or follitropin confers luteotropic but not follitropic activity to chimeric TSH. J Biol Chem 1997;272: 15532.
144. Szkudlinski MW, Teh NG, Grossman M, et al. Engineering human glycoprotein hormone superactive analogues. Nat Biotechnol 1996;14:1257.
145. Grossman M, Szkudlinski MW, Dias JA, et al. Site-directed mutagenesis of amino acids 33–44 of the common α subunit reveals different structural requirements for heterodimer expression among the glycoprotein hormones and suggests that cyclic adenosine 3′,5′-monophosphate production and growth promotion are potentially dissociable functions of human thyrotropin. Mol Endocrinol 1996;10:769.
146. Grossman M, Szkudlinski MW, Zeng H, et al. Role of the carboxy-terminal residues of the α subunit in the expression and bioactivity of human thyroid-stimulating hormone. Mol Endocrinol 1995;9:948.
147. Sato A, Perlas E, Ben-Menahem D, et al. Cystine knot of the gonadotropin α-subunit is critical for intracellular behavior but not for in vitro biological activity. J Biol Chem 1997;272: 18098.
148. Grossman M, Wong R, Szkudlinski MW, et al. Human thyroid-stimulating hormone (hTSH) subunit gene fusion produces hTSH with increased stability and serum half-life and compensates for mutagenesis-induced defects in subunit association. J Biol Chem 1997;272:21312.
149. Fares FA, Yamabe S, Ben-Menahem D, et al. Conversion of thyrotropin heterodimer to a biologically active single-chain. Endocrinology 1998;139:2459.
150. Garcia-Campayo V, Sato A, Hirsch B, et al. Design of stable biologically active recombinant lutropin analogs. Nat Biotech 1997;15:663.
151. Wondisford FE, Usala SJ, DeCherney SG, et al. Cloning of the human thyrotropin beta subunit gene and transient expression of biologically active human thyrotropin after gene transfection. Mol Endocrinol 1988;2:32.
152. Watanabe S, Hayashizaki Y, Endo Y, et al. Production of human thyroid-stimulating hormone in Chinese hamster ovary cells. Biochem Biophys Res Commun 1989;149:1149.
153. Thotakura NR, Desai RK, Bates LG, et al. Biological activity and metabolic clearance of a recombinant human thyrotropin produced in Chinese hamster ovary cells. Endocrinology 1991; 128:341.