Effect of TRH on TSH Glycosylation and Biological Action

zyxw zy zyx zyxw Effect of TRH on TSH Glycosylation and Biological Action BRUCE D. WEINTRAUB, NEIL GESUNDHEIT, TERRY TAYLOR, AND PETER W. GYVES Molecular, Cellular and Nutritional Endocrinology Branch National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892 zyxwv zyxwvu Thyroid-stimulating hormone (TSH) is a glycoprotein comprised of two noncovalently linked subunits, or and p. The structure of TSH from a variety of species has been elucidated, including the amino acid sequence and carbohydrate composition.'Y2 Bovine TSH has been particularly well characterized and its a subunit has a molecular weight of 14,000, of which 11,000 is composed of a protein core of 96 amino acids and 3,000 represents two oligosaccharide units linked to asparagine residues. Bovine TSHP has a molecular weight of 15,000 of which 13,000 is comprised of a protein core of 113 amino acids and 2,000 represents one asparagine-linked oligosaccharide unit. TSH is structurally related to the pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well to the placental hormone chorionic gonadotropin (CG). Within a single species the a subunits from each of these glycoprotein hormones are virtually identical, whereas the p subunits are unique and confer immunologic, receptorbinding, and biologic specificity. Attainment of the conformation necessary for hormonal activity is dependent on proper assembly and carbohydrate processing of the TSH subunits, whereas the free subunits are essentially devoid of receptor binding and biologic activity.2 TSH BIOSYNTHESIS AND PROCESSING zyx The a and /3 subunits of TSH are synthesized from separate messenger RNAs encoded by DNA from genes located on separate chromosomes that may differ between The separate chromosomal location of two TSH subunit genes raises interesting questions about how their synthesis is coordinated in various physiological states. Preliminary studies suggest that there are 5' regulatory elements upstream of the DNA coding regions for both of these genes, and these common regulatory elements may ultimately prove to be responsible for coordinate r e g ~ l a t i o n . The ~ , ~ nucleotide sequence for TSHa and TSHP has confirmed for each subunit the presence of an amino-terminal signal peptide, a sequence of 24 (for or) or 20 (for p) amino acids that is important for the binding of ribosomes containing incomplete polypeptide chains to the rough endoplasmic reticulum. Moreover, the hydrophobic nature of the signal peptide permits insertion of the chains through the lipid bilayer of the membrane and into the lumen of the endoplasmic reticulum. Each signal peptide is cleaved from the growing polypeptide before completion of messenger RNA translation, and these signal peptides are not found in subunits in intact 205 206 zyxwvutsrq zy ANNALS NEW YORK ACADEMY OF SCIENCES Initial glycosylation of TSH subunits occurs in the endoplasmic reticulum by cotranslational transfer en bloc of a precursor oligosaccharide unit, attached to a dolichol-phosphate carrier, within the rough endoplasmic reticulum.13J4The precursor carbohydrate chains contain nine mannose residues and are therefore termed “high-mannose.” Subsequent processing of these carbohydrate chains leads to elimination of all but three mannose residues and addition of other sugars such as N-acetylglucosamine, N-acetylgalactosamine, galactose, fucose, and N acetylneuraminic acid (sialic acid). In addition, certain TSH carbohydrate chains may terminate in an unusual sulfate moiety, which is found in pituitary glycoprotein hormones, but not the placental glycoprotein hormone hCG.ISBecause of the variety of carbohydrate moieties found in the final chains, these are termed “complex’’ oligosaccharide units. The most common complex oligosaccharides contain two branches and are called biantennary, but structures with three branches (triantennary) or four antennas (tetraantennary) as well as those with one complex and one high-mannose antenna (hybrid) can also occur. During glycoprotein processing, therefore, molecules originating from a common precursor obtain a spectrum of heterogeneous structures with many potential differences in their conformations and possibly in their biologic activities. Finally, there appear to be at least two intracellular routes that secretory glycoproteins follow: one that is nonregulated and uses transport or secretory vesicles, and one that is regulated, secretogogue-dependent, and uses secretory granules. l6 zy zyx zy zyxwvu ROLES OF TSH CARBOHYDRATE IN BIOSYNTHESIS, SECRETION, AND ACTION The carbohydrate of TSH apparently plays multiple roles in hormone assembly, secretion, and action. These conclusions have been reached by observing the effects of inhibition of subunit glycosylation or carbohydrate processing during biosynthesis, the effects of chemical deglycosylation of TSH on bioactivity and metabolic clearance, as well as the bioactivity and clearance of naturally occurring TSH forms differing in carbohydrate content. The precursor high-mannose carbohydrate unit of TSH permits a-P subunit combination and protects against intracellular proteolysis and aggregation. These conclusions were reached by experiments with the antibiotic tunicamycin, an inhibitor of asparagine-linked glycosylation.11,17 Recently we have also employed another antibiotic, deoxynojirimycin, that inhibits the processing of the precursor high-mannose sugar chains to the final complex chains at a very early step.ls This agent did not inhibit a-P subunit combination, but did inhibit TSH secretion, implying that maturation of sugar chains is related to intracellular TSH transport. The final complex structure of secreted TSH carbohydrate apparently is important in determining the intrinsic biologic activity and the metabolic clearance rate of the secreted, circulating hormone. Various forms of TSH from different intrapituitary or secreted sources were fractionated by gel chromatography and found to have components with widely different biological to immunological (B/1) ratios.I9 These forms were found to bind differently to various lectin columns, suggesting that differences in B/I ratios were primarily related to changes in carbohydrate composition. These data suggest that the complex carbohydrate moieties of TSH modulate its bioactivity. Recently wem and others21,22 demonstrated that after deglycosylation with anhydrous hydrogen fluoride, TSH demonstrates markedly reduced bioactivity despite normal or increased receptor binding zyxwvu zyxwvutsrq zyxwvu zyxzy WEINTRAUB et al.: TRH AND TSH GLYCOSYLATION 207 properties and also acts as a competitive antagonist to normally glycosylated TSH. When such deglycosylation was performed with newly available endoglycosidases, such as endoglycosidase F, the effects on in uitro activity were not as marked as were those previously noted with the use of hydrogen fluoride.23However, it is not yet clear whether enzymatic removal of sugars is as complete as that achieved with chemical methods. Nonetheless, even with such possible incomplete enzymatic methods, a reduction in bioactivity of up to 6040% was observed. Moreover, after intravenous injection into normal rats the deglycosylated hormone had a more rapid metabolic clearance rate than did the native hornone.^^ Various naturally occumng forms of intrapituitary and secreted TSH derived from different physiological states also showed differences in their metabolic clearance rate, presumably on the basis of differences in carbohydrate structure.24 zy zyxwvu PRETRANSLATIONALREGULATION OF TSH BY THYROID HORMONE AND TRH There are many potential points for regulation of TSH synthesis and glycosylation. The effects of alterations in thyroid hormone levels in uiuo have been elucidated in great detail, and these studies primarily show effects at a pretranslational It was recently demonstrated that the effects of thyroid hormone deficiency on TSH biosynthesis are mediated by increased levels of a and fl subunit messenger RNA. After administration of thyroid hormone to hypothyroid animals, TSH subunit messenger RNA levels decreased within hours, with the effects on /3 being more rapid and greater than those on a. Direct inhibition of a! and /? subunit gene transcription by thyroid hormone administration also was recently demonstrated. It should be stressed that these in uiuo studies are probably causing major changes in endogenous TRH secretion, which may play a significant role in synergizingwith thyroid hormone to achieve the final changes in gene transcription. For example, it is now established that primary hypothyroidism is associated with increased hypothalamic TRH biosynthesisB and that administration of thyroid hormone reduces endogenous TRH synthesis. Therefore, future studies with in uiuo models in which hypothyroidism has been achieved in the setting of TRH deficiency, or in uifro models of dispersed pituitary cells in which TRH is absent will be necessary to separate thyroid hormone from TRH effects. The direct effects of TRH on TSH protein biosynthesis have been studied in both in uiuo and in uitro models. TRH administered in uiuo does not appear to have a major effect on TSH biosynthesis. Neither we30nor others6have observed significant changes in pituitary a! or p messenger RNA levels after in uiuo TRH administration to normal or hypothyroid animals or in animals in which the pituitary was transplanted to the renal capsule, producing a state of hypothalamic hormone deficiency. However, under certain conditions, it has been possible to demonstrate that TRH administered in uitro to pituitary cells from normal or hypothyroid rats31,32 or from dispersed thyrotropic tumor ~ e l l scauses ~~.~ small ~ but consistent increases in TSH biosynthesis. Similarly, it was shown recently that TSH gene transcription and messenger RNA levels can be increased significantly by TRH administration to dispersed pituitary cells from hypothyroid rats34 and to a lesser extent to cells derived from normal rats.35Thus, although TRH apparently modulates TSH biosynthesis, this effect may require concomitant thyroid hormone deficiency. 208 zyxwv zy ANNALS NEW YORK ACADEMY OF SCIENCES POSTTRANSLATIONAL REGULATION OF TSH BY TRH zyxwv zyxwvut zyxwv zyxwvut zyxwvut The best documented and most significant effects of TRH apparently are those involving TSH secretion and carbohydrate processing. Early biosynthetic studies suggested that TRH, in addition to promoting secretion of TSH, increased the relative incorporation of [3H]glucosamineinto TSH from whole36and dispersed3’ rat pituitaries. Using subunit-specific analytical methods and electrophoresis of TSH on SDS-polyacrylamide gels, we showed that normal rat pituitaries stimulated with TRH for 24 hours in uitro demonstrated a threefold stimulation of labeled glucosamine incorporated into secreted TSH.31The increase in relative glucosamine incorporation in the presence of TRH was observed equally in the cy and p subunits, suggesting parallel alterations in their glycosylation. In uiuo administration of TRH into newly thyroidectomized rats resulted in a specific increase in certain high-mannose species of oligosaccharides, particularly in the /3 subunit of TSH.38This species contained one residue of glucose and nine residues of mannose. Thus, TRH apparently causes changes in the kinetics of early carbohydrate processing and may actually stimulate the addition of glucose residucs to the oligosaccharide chain posttranslationally during high-mannose processing. To explore the structural basis for the apparent increase in TSH incorporation of labeled sugar precursors in the presence of TRH, hypothyroid mouse pituitaries were incubated with r3H]mannose with or without TRH for 18 hours.39Secreted TSH was precipitated, and TSH glycopeptides were prepared for analysis by concanavalin A (conA)-agarose affinity chromatography. Glycopeptides eluted in three general classes on con A, depending on the specific sugar structures: unbound glycopeptides consisting of multiantennary complex structures; weakly bound glycopeptides corresponding to biantennary complex structures; and strongly bound glycopeptides corresponding to high-mannose or hybrid carbohydrate structures. Analysis of both intracellular and secreted TSH glycopeptides revealed a 2.5fold increase by TRH in one specific class: secreted glycopeptides that were weakly bound to con A. No change was noted in any intracellular glycopeptide class or in the other two secreted glycopeptide classes. These data suggest that TRH affects the final structure of secreted TSH carbohydrate; however, it is not known if this is due to activation or inhibition of specific carbohydrate processing enzymes or to stimulation of specific routes of secretion that result in altered glycosylation. For example, it is possible that TRH specifically stimulates the regulated route of secretion via classic secretory granules and that these forms of TSH may have different structures from those of the type of TSH that is secreted in the basal state by other subcellular transport routes. To determine if the TRH-induced changes in TSH carbohydrate structure are specific effects of this secretogogue, we compared TRH to other agents known to stimulate TSH secretion.40 Mouse thyrotropic tumors were enzymatically dispersed and incubated with [3H]mannosefor 36 hours, washed, and then incubated for 24 hours in serum-free media containing additional [3H]mannose,as well as a variety of secretogogues including TRH, TPA (a phorbol ester that stimulates protein kinase C), or 60 mM KCI which causes membrane depolarization. Although KCI was an even more potent stimulus than TRH for TSH secretion, it did not affect the con A binding profile. By contrast, the phorbol ester caused a change in carbohydrate composition intermediate between KCl and the specific secretogogue TRH. These results suggest that the TRH-stimulated changes in oligosaccharide structure are specific and are not solely mediated by membrane zy zyxwvuts zyxwvu zyxwv WEINTRAUB el al.: TRH AND TSH GLYCOSYLATION 209 depolarization or stimulation of protein kinase C. It is possible that a combination of multiple second messenger analogues, such as a phorbol ester and a calcium ionophore, might produce changes in TSH carbohydrate structure approaching those induced by TRH. IN VIVO MODELS OF TRH DEFICIENCY The effect of endogenous TRH as well as other hypothalamic factors was also examined by causing anterior hypothalamic deafferentation in rats.41As compared to sham cuts, hypothalamic deafferentationcaused decreased incorporation of both labeled amino acid and carbohydrate into TSH. After exogenous administration of TRH, both of these deficiencies were corrected. Recently, we have made more specific lesions in the paraventricular nuclei of the rat hypothalamus, creating central hypothyroidi~m.~~ These lesions caused a major change in the carbohydrate structure of secreted TSH as assessed by con A affinity chromatography of labeled TSH glycopeptides. Compared to sham controls, rats with paraventricular nuclear lesions demonstrated increased secretion of labeled glycopeptides that bound to con A. This effect was not solely due to the change in thyroid hormone produced by lesions, because rats with primary hypothyroidism caused by thyroidectomy with equally low thyroid hormone levels had an opposite pattern of glycopeptide binding to con A. Again, the effects of these paraventricular lesions were completely reversed by exogenous administration of TRH,43 suggesting that this peptide, rather than other hypothalamicfactors, was responsible for the change in carbohydrate structure. Interestingly, the effects of in uiuo administration of TRH to rats were different from those previously observed in uitro with mouse pituitary ex plant^.^^ This finding suggests that multiple endocrine factors, the duration of hormonal manipulation, as well as the particular animal species may be important in determining the specific posttranslational response to endocrine factors. In another model of in uiuo TRH deficiency, the perinatal rat, we also examined the effects of TRH on TSH carbohydrate s t r u ~ t u r e . It ~ .was ~ ~ previously demonstrated that hypothalamic TRH secretion in the rat does not occur until after 5 days of age.& Compared to adult rats, late fetal or perinatal rats showed a glycopeptide distribution with increased binding to con A, similar to that previously observed in animals with paraventricular nuclear lesions. Interestingly, administration of TRH in uitro to perinatal rats did not cause restoration of TSH glycopeptides to the distribution observed for mature rats, in contrast to the observations noted in adult animals with hypothalamic lesions when given TRH in uiuo. These data suggest that the pathways for TRH alteration of TSH carbohydrate may not be fully developed in the perinatal animal or that the mode of TRH administration may be important in the modulation of carbohydrate structure. z zyx EFFECTS OF TRH ON TSH BIOLOGICAL ACTION We previously reported that in uifro TRH administration to normal or hypothyroid rat pituitary explants caused a selective increase in the relative bioactivity of secreted TSH as measured by adenylate cyclase-stimulatingactivity in thyroid membranes.47Moreover, early studies suggested that certain cases of idiopathic central hypothyroidism in man might result from secretion of a biologically inac- 210 zyxwv zy zyxwvut ANNALS NEW YORK ACADEMY OF SCIENCES zyxwv zyxwv tive TSH.48To investigate this possibility and to define the mechanism of defective hormone action, we measured the adenylate cyclase-stimulating bioactivity (B) and receptor-binding(R) activity of immunoactive (I) TSH, which was aflinity purified from the serum of seven selected patients with central hyp~thyroidism.~~ These patients (five idiopathic, two with tumor) displayed normal or increased levels of immunoactive TSH that was highly responsive to TRH. We found a strikingly decreased R/I ratio (<0.15) in patients compared to controls (0.6-2.7) and a similarly decreased B/I ratio (<0.2 vs. 2.8-5.6). After acute TRH injection, the R/I ratio increased in two of three patients, whereas the B/I ratio normalized in only one patient. After chronic TRH administration (40&day orally for 20 days), both ratios normalized in all but one patient who showed apparent desensitization. The increased bioactivity of the secreted TSH after chronic TRH therapy resulted in increased secretion of serum thyroid hormones in all patients studied, with restoration of clinical euthyroidism. We conclude that in certain cases of central hypothyroidism the secreted TSH lacks biological activity because of impaired binding to its receptor; TRH treatment can correct both of these defects. These data imply that TRH regulates not only TSH secretion, but also its specific molecular and conformational features required for hormone action. In view of the results just presented showing that TRH causes selective changes in TSH glycosylation, it seems likely that these conformational changes result from alterations in carbohydrate structure. However, other alterations in apoprotein structure or other unknown posttranslational modifications cannot be excluded. It will be interesting in future studies to examine TSH purified from patients with various other states of TRH deficiency or hypersecretion. It is to be hoped that characterization of the carbohydrate structure as well as the receptor binding and biologic properties of these hormones will provide new insights into the role of TRH in the posttranslational regulation of TSH. REFERENCES 1. PIERCE, J. G. & T. F. PARSONS.1981. Glycoprotein hormones: structure and function. Annu. Rev. Biochem. 50: 465-495. 2. WEINTRAUB, B. D., B. S. STANNARD, J. A. MAGNER, C. RONIN,T. TAYLOR, L. JOSHI, R. B. CONSTANT, M. M. MENEZES-FERREIRA, P. A. PETRICK& N. GESUNDHEIT. 1985. 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GESUNDHEIT, ing hormone on the carbohydrate structure of secreted mouse thyrotropin: Analysis by lectin aflinity chromatography. J. Biol. Chem. 262: 5197-5203. 40. BUTLER,J. B., N. GESUNDHEIT & B. D. WEINTRAUB. 1986. Thyrotropin-releasing hormone (TRH) and phorbol ester, but not potassium chloride (KCI) alter the structure of secreted thyrotropin (TSH). Presented at the 66th Annual Meeting of the American Thyroid Association (Abstract No. 13). 41. TAYLOR,T., S. W. SCOUTEN,D. M. JACOBOWITZ & B. D. WEINTRAUB. 1986. The effects of anterior hypothalamic deafferentation on thyrotropin (TSH) biosynthesis and response to TSH-releasing hormone. Endocrinology 118: 2417-2424. 42. TAYLOR, T., N. GESUNDHEIT, P. W. GYVES, D. M. JACOBOWITZ & B. D. WEINTRAUB. 1988. Hypothalamic hypothyroidism caused by lesions in rat paraventricular nuclei alters the carbohydrate structure of secreted thyrotropin. Endocrinology 122. In press. 43. TAYLOR,T. & B. D. WEINTRAUB. 1987. Altered TSH carbohydrate structures in hypothalamic hypothyroidism created by paraventricular nuclear lesions are corrected by in viuo thyrotropin-releasing hormone administration (abstract). Clin. Res. 35(3): 402A. 44. GYVES,P. W., N. GESUNDHEIT, T. TAYLOR, J. B. BUTLER & B. D. WEINTRAUB. 1987. Changes in thyrotropin (TSH) carbohydrate structure and response to TSH-releasing hormone during postnatal ontogeny: Analysis by concanavalin-A chromatography. Endocrinology 121: 133-140. 45. GYVES, P. W., N. GESUNDHEIT, B. S. STANNARD & B. D. WEINTRAUB.1987. Alterations in the sulfation and sialylation of secreted rat thyrotropin carbohydrate struc- zyxwvu zyxwvuts zyxwvu zyx zy zyxw WEINTRAUB er al.: TRH AND TSH GLYCOSYLATION 46. 47. 48. 49. 213 ture during ontogeny. Presented at the 67th Annual Meeting of the American Thyroid Association (Abstract No. 83). FISHER,D. A., J. H. DUSSAULT, J. SACKAND & I. J. CHOPRA.1977. Ontogenesis of hypothalamic-pituitary-thyroid function and metabolism in man, sheet and rat. Recent Prog. Horm. Res. 33: 59-116. 1986. Regulation of MENEZES-FERREIRA, M. M., P. A. PETRICK& B. D. WEINTRAUB. thyrotropin bioactivity by thyrotropin-releasing hormone and thyroid hormone. Endocrinology 118: 2125-2130. A. PINCHERA, C. FERRARI,A. PARACCHI, P. BECK-PECCOZ, FAGLIA,G., L. BITENSKY, B. AMBROSI & A. SPADA.1979. Thyrotropin secretion in patients with central hypothyroidism: Evidence for reduced biological activity of immunoreactive thyrotropin. J. Clin. Endocrinol. Metab. 48:989-998. BECK-PECCOZ, P., S. AMR,M. M. MENEZES-FERREIRA, G. FACLIA& B. D. WEINTRAUB. 1985. Decreased receptor binding of biologically inactive thyrotropin in central hypothyroidism: Effect of treatment with thyrotropin-releasing hormone. N. Engl. J. Med. 312: 1085-1090.