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TSH Receptor Mutations and Thyroid Disease
REVIEWS TSH Receptor Mutations and Thyroid Disease Laurence Duprez, Jasmine Parma, Jacqueline Van Sande, Patrice Rodien, Jacques E. Dumont, Gilbert Vassart and Marc Abramowicz
Mutations of the thyrotropin receptor (TSHr) can be loss of function or gain of function. Loss-of-function mutations can affect a variety of loci in the TSHr gene. Their most common manifestation is resistance to TSH; they may also be the cause of a subset of cases of congenital hypothyroidism. Gain-of-function mutations are of greater theoretical interest. Somatic mutations constitutively activating the TSHr are the major cause of benign toxic thyroid adenomas, and of some cases of multinodular goiters. They underlie hereditary toxic thyroid hyperplasia, and have been found in cases of sporadic congenital non-autoimmune hyperthyroidism. A role for TSHr polymorphisms in Graves’ disease has not been documented.
Over the past few years, mutations in the thyrotropin receptor (TSHr) gene have been identified as a cause of acquired, hereditary or congenital thyroid diseases (Tonacchera et al. 1996a). Depending on their nature, the mutations cause either loss of function or gain of function, leading to hypoor hyperthyroidism, respectively. The loss-of-function mutations are relatively trivial events interfering with normal receptor function – there are hundreds of different ways to destroy the precise functional architecture of L. Duprez, J. Van Sande, P. Rodien and J.E. Dumont are at the Institut de Recherche Interdisciplinaire, Faculty of Medicine, University of Brussels, Belgium; J. Parma is at the Service de Génétique Médicale, Hôpital Erasme, University of Brussels, Belgium; and G. Vassart and M. Abramowicz are at the Institut de Recherche Interdisciplinaire and the Service de Génétique Médicale.
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a protein. However, the identification of a large series of gain-of-function mutations in the TSHr gene was one of the first examples of a new pathophysiological mechanism, in which diseases are caused by mutations that increase the basal activity of a receptor and make it constitutively active, in the absence of its normal agonist. In addition, the study of the structure– function relationships of these constitutively active mutants has led to a better understanding of the mechanisms implicated in the normal activation of the receptor by TSH, and it provides a rationale for the understanding of its activation by autoantibodies in Graves’ disease. The aim of this short review is to summarize what has been learnt over recent years by the identification and functional analyses of the different types of TSHr mutations.
•
Background
The TSHr belongs to the superfamily of seven transmembrane domain receptors coupled to G proteins (G-protein-coupled receptors; GPCRs) (Nagayama and Rapoport 1992, Vassart and Dumont 1992). More precisely, it is a member of a subfamily whose prototypic members are rhodopsin and the adrenergic receptors (Helmreich and Hofmann 1996). A common characteristic of GPCRs is the presence of seven transmembrane segments, subdividing the molecules into an extracellular domain (aminoterminal segment plus three extracellular loops), a transmembrane barrel (seven α helices) and an intracytoplasmic domain (three intracytoplasmic loops plus the carboxyl-terminal segment). In the prototypic GPCRs, the ligand binds to structures within the pocket between transmembrane helices (e.g. biogenic amine receptors) and/or to residues of the extracellular domain (neuropeptide receptors) (Strader et al. 1994). Together with the other glycoprotein hormone receptors [luteinizing hormone/chorionic gonadotropin receptor (LH/CGr) and follicle-stimulating hormone receptor (FSHr)], the TSHr is characterized by a particularly long amino-terminal segment (398 residues), which is responsible for high-affinity binding of TSH (Parmentier et al. 1989). This portion of the receptor is glycosylated and essentially made of ‘leucine repeat’ motifs. The availability of the tridimensional structure of one prototypic protein made of leucine repeats (the ribonuclease inhibitor) has allowed the building of models of the amino-terminal hormone binding segment of glycoprotein receptors (Kobe and Deisenhofer 1993 and 1995, Kajava et al. 1995). Whereas the amino-terminal domain of the LH/CG receptors is capable of high-affinity
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by its normal agonist or autoantibodies, or secondary to an activating mutation, is expected to cause concomitant hyperplasia and hyperfunction (Roger et al. 1995). If the activation is taking place in a single cell, it is expected to promote clonal expansion of hyperfunctional thyrocytes. The structural and functional dichotomy of the TSHr poses the problem of understanding its activation mechanism in the light of what is known of other GPCRs. Clearly, the bulky TSH cannot fit within the transmembrane pocket of the receptor; how then does binding of the hormone to the extracellular amino terminus result in activation of the serpentine portion? As will become apparent, the distribution of activating mutations along the receptor’s primary sructure provides clues for the elaboration of a model of receptor activation. • Figure 1. Schematic representation of the TSH receptor with an indication of the amino-acid substitutions or deletions responsible for its constitutive activation.
hormone binding when expressed alone, no convincing data of this sort have been obtained for the TSHr. Also, the TSHr is unique because of its dimeric structure: a proportion of the molecules, synthesized as a single polypeptidic chain, undergoes a still poorly defined maturation step, leading to a cleavage near the border between the amino terminus and the first transmembrane helix (Couet et al. 1996a, Chazenbalk et al. 1997). As suggested initially (Vassart and Dumont 1992), a 50 amino-acid residue segment located in this region and with no counterpart in the LH/CG or FSH receptors, probably plays a key role in this maturation step (Chazenbalk et al. 1997). After cleavage, the amino terminus remains mostly attached to the ‘serpentine’ portion by disulfide bond(s), but there is evidence of continuous shedding of the amino terminus by a mechanism involving protein disulfide isomerase (PDI) (Couet et al. 1996b). Similar to the protein, the TSHr gene (located on chromosome 14q31) can be subdivided into two segments with
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different functional significance: a large exon encodes the carboxy-terminal portion of the ectodomain, the serpentine and intracellular domains (residues 294–764), while a series of nine exons code for the remainder of the ectodomain (Gross et al. 1991). The intracytoplasmic domain of GPCRs contains the site(s) of interaction with G proteins (O’Dowd et al. 1988), the transducers between activated receptors and the various effectors (Neer 1995). The human TSHr interacts functionally with both the Gs and Gq proteins, the former interaction mediating the effects of TSH on tissue growth and hormone secretion, the latter being responsible for stimulation of hormone synthesis (Dumont et al. 1992). Contrary to many GPCRs, including the LH/CG receptor, the TSHr exhibits very limited homologous desensitization. This should be remembered when interpreting the effects of activating mutations and the stimulatory immunoglobulins of Graves’ disease. The continuous stimulation of the receptor, whether
Gain-of-function Mutations
From our knowledge of thyroid cell physiology, constitutive activation of the cAMP-dependent regulatory cascade in human thyrocytes is expected to cause unregulated growth of autonomous hyperfunctioning tissue (Dumont et al. 1989). Transgenic mouse models of this situation have been constructed (Ledent et al. 1992, Michiels et al. 1994). Up until now, mutations in two molecules of the cAMP regulatory cascade have been associated with thyroid hyperfunction: G␣s and the TSH receptor. Mutations in G␣s resulting in decrease of GTPase activity have been identified in a proportion of somatotropic adenoma (Lyons et al. 1990) and in McCune– Albright syndrome (Patten et al. 1990). The possibility that spontaneous mutations activating the TSHr exist was predicted by earlier studies with the adrenergic and melanocortin receptors, as well as with rhodopsin (Cotecchia et al. 1992, Robinson et al. 1992, Robbins et al. 1993). Somatic Mutations Activating the TSHr Constitutively are the Major Cause of Benign Toxic Thyroid Adenomas Solitary autonomous adenoma and multinodular goiter are the two major TEM Vol. 9, No. 4, 1998
clinical presentations of thyroid autonomy in countries with low or borderline iodine supply. Thyroid adenomas are encapsulated benign tumors, characterized mainly by autonomy, a variable degree of hyperfunction and usually a slow growth. This definition can be extended to at least some adenomatous nodules lacking a capsule, provided their autonomy is documented. Eight years after the identification of the first mutation in Gs (Lyons et al. 1990), and five years after the demonstration of TSHr receptor mutations in autonomous adenomas (Parma et al. 1993), the situation can be summarized as follows. In our series of 33 adenomatous nodules, 82% were found to harbor a somatic mutation in the TSHr gene and 6% in the G␣s gene (Parma et al. 1997). All these mutations were found in the heterozygous state in the nodular tissue and not in the adjacent tissue or the peripheral leukocytes. This demonstrates the somatic nature of the mutations and is consistent with a dominant effect. Activating mutations were dispersed all along the primary structure of the receptor: in the extracellular domain, the extracellular loops, the second, third, fifth, sixth and seventh transmembrane domains and in the third intracytoplasmic loop (Fig. 1). A large proportion of the mutations have been found in the amino-terminal portion of the sixth transmembrane domain, where five contiguous residues (629–633) were mutated in 44% of the nodules in our series (Fig. 1). Whereas other studies failed initially to find a similarly high prevalence of TSHr mutations in autonomous adenomas (Matsuo et al. 1993, Russo et al. 1995, Djuh et al. 1996, Takeshita et al. 1995, Esapa et al. 1997), a consensus is emerging from more recent studies (Porcellini et al. 1994 and 1995, Führer et al. 1997) that, at least in regions with a borderline iodine supply, TSHr mutations are the major cause of the disease. The lower prevalence seen in Japan and in the USA (Matsuo et al. 1993, Russo et al. 1995, Djuh et al. 1996, Takeshita et al. 1995, Esapa et al. 1997), together with the absence of mutations in 12% of our cases, leaves TEM Vol. 9, No. 4, 1998
some room for other pathophysiological mechanisms or gene targets. All mutated receptors identified in these studies were expressed transiently in COS-7 cells and their functional properties analyzed (Parma et al. 1993 and 1997, Porcellini et al. 1995, Paschke et al. 1994, Van Sande et al. 1995). They all displayed an increase in basal stimulation of cAMP production, despite the fact that most of them were expressed at a lower level than the wild-type receptor. With a few notable exceptions, they remained stimulatable by TSH, did not show constitutive activation of the diacylglycerol–inositol phosphate pathway and bound bovine TSH with a better apparent affinity than the wild-type TSHr. Somatic Mutations of the TSHr and the Pathogenesis of Multinodular Goiter We have reported two different activating mutations of the TSHr in a multinodular goiter with two separated autonomous regions (Duprez et al. 1997a), and similar findings have been described independently (Paschke et al. 1996, Tonacchera et al. 1997). Although the exact prevalence of TSHr mutations in multinodular goiter is unknown, these observations demonstrate that the same pathophysiological mechanism may be at work as in solitary adenomas. In areas with mild to moderate iodine deficiency, autonomy is observed frequently on a background of multinodular goiter. In truly endemic regions, iodine supplementation is followed by an increase in non-autoimmune hyperthyroidism (Fradkin and Wolff 1983, Todd et al. 1995, Dremier et al. 1996), which suggests that iodine supplementation could act as a revelator of a proportion of previously asymptomatic autonomies. It will be interesting to look for TSHr mutations in the autonomous portions of these glands. Somatic Mutations of the TSHr and Thyroid Cancer Activating mutations have been found in rare cases of well-differentiated follicular thyroid carcinomas (Suarez et al. 1991, Matsuo et al. 1993, Russo et al. 1995). The rarity of these
observations, and of the malignant transformation of hyperactive thyroid tissue as a whole, illustrates the essentially benign nature of the cAMP mitogenic pathway in thyrocytes. Familial Non-autoimmune Hyperthyroidism: a Germline Counterpart of the Toxic Adenoma Since our original description of two families from northern France (Thomas et al. 1982, Duprez et al. 1994), six additional pedigrees have been identified where hyperthyroidism and thyroid hyperplasia segregate as an autosomal dominant trait in linkage with mutated alleles of the TSHr gene (Tonacchera et al. 1996b, Führer et al. 1996, and our unpublished data) (Fig. 1; Table 1). This new nosological entity, hereditary toxic thyroid hyperplasia (HTTH), is characterized by the following clinical characteristics: autosomal dominant transmission; hyperthyroidism with a variable age of onset (from infancy to adulthood, even within a given family); hyperplastic goiter of variable size, but with a steady growth; and absence of clinical or biological stigmata of autoimmunity. A common observation in the cases described to date is the need for drastic ablative therapy (surgery or radioiodine) to control the disease once the patient has become hyperthyroid. The prevalence of HTTH is difficult to estimate at the present time. It is likely that many cases have been (and still are) mistaken for Graves’ disease, owing to the relative insensitivity and lack of specificity of thyroid-stimulating antibody assays. It is expected that wider knowledge of the existence of the disease will lead to better diagnosis. This is not a purely academic problem, because presymptomatic diagnosis in children of affected families might prevent the development of neuropsychological complications associated with infantile or juvenile hyperthyroidism. Germline Neomutations of the TSHr are Found in Sporadic Congenital Non-autoimmune Hyperthyroidism Neonatal hyperthyroidism is usually caused by transplacental passage 135
Table 1. List of activating mutations of the TSH receptor observed in toxic adenomas, hereditary toxic thyroid hyperplasia and non-immune congenital hyperthyroidism Residue
Somatic
Asp 276 Asn Ser 281 Asn Ser 281 Thr Ser 281 Ile Met 453 Thr Ile 486 Met Ile 486 Phe Ser 505 Arg Ser 505 Asn Val 509 Ala Ile 568 Thr Val 597 Leu Tyr 601 Asn Del 613–621 Asp 619 Gly Ala 623 Ile Ala 623 Val Ala 623 Ser
⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻
Familial
⫻
⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻
of stimulating autoantibodies and is transient. In some cases, an autoimmune origin cannot be found and the hyperthyroidism does not resolve: de novo germline activating mutations of the TSHr have been found in such cases (Kopp et al. 1995, de Roux et al. 1996a, Esapa et al. 1996, Holzapfel et al. 1997). Four cases bearing four different mutations of the TSHr have been described, (Table 1). The four neonates were small for gestational age and presented a goiter with severe hyperthyroidism requiring prompt surgical intervention. The meaning of the pseudo-exophthalmia presented by one of these children remains ambiguous because they all displayed an advanced bone age. Most of the cases with congenital non-autoimmune thyrotoxicosis have mutations also found in toxic adenomas. In contrast, the majority of mutations in HTTH are ‘private’ mutations (Table 1). This is compatible with the hereditary mutations causing a less severely altered phenotype, with a marginal effect on reproductive fitness in the absence of treatment.
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Sporadic
Residue
Somatic
Familial
Leu 629 Phe Leu 629 Pro Ile 630 Leu Phe 631 Leu Phe 631 Cys Thr 632 Ile Thr 632 Ala Asp 633 Glu Asp 633 Tyr Asp 633 His Asp 633 Ala Met 637 Arg Pro 639 Ser Asn 650 Tyr Val 657 Phe Del 658–661 Asn 670 Ser Cys 672 Tyr
⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻
⫻
⫻
Sporadic
⫻
⫻ ⫻ ⫻
⫻ ⫻
An Emerging Model for Receptor Activation In agreement with current concepts about GPCR activation, the global effect of activating mutations could be the disruption of a structural constraint maintaining the receptor in an inactive state (Kjelsberg et al. 1992). In the case of TSHr, this constraint must be relatively weak because the wildtype receptor displays a significant level of constitutive activation when expressed in COS-7 cells (Duprez et al. 1994, Kosugi and Mori 1995, Van Sande et al. 1995). We have proposed that it is the ‘noisy’ character of the wild-type TSHr that explains the wide spectrum of amino-acid substitutions leading to its activation. As an extension of this concept, it is tempting to speculate that this same high propensity to become activated would explain the activation of the TSHr by autoantibodies in Graves’ disease. The location of some activating amino-acid substitutions in the extracellular loops and the amino-terminal hormone binding segment (Parma et al. 1995, Duprez et al. 1997b, Kopp et al. 1997, Russo et al. 1997) (Fig. 1), together
⫻ ⫻
with the observation that mild proteolytic treatment of the receptor leads to its activation (Van Sande et al. 1996), suggest that an inhibitory interaction might exist between the unliganded amino-terminal segment and the extracellular loops of the serpentine domain. According to this model, the effect of TSH or stimulating autoantibodies would be to release this inhibitory interaction. A Role for TSHr Polymorphisms in Graves’ Autoimmune Hyperthyroidism? Graves’ disease is known to show some familial aggregation but does not constitute a truly hereditary disorder. Instead, it may be understood as a disease of multifactorial origin, where several independently inherited genes, and environmental factors, are involved collectively in pathogenesis. From a theoretical viewpoint, as the receptor is a major autoantigen in Graves’ disease, one of these genes might be that for the TSHr. However, to date, there is no convincing argument in favor of such a hypothesis, and linkage to the TSHr gene has even been excluded in a large study of familial Graves’ disease (de Roux et al. 1996b). TEM Vol. 9, No. 4, 1998
•
Loss-of-function Mutations
Resistance to Thyrotropin Loss-of-function mutations were first described in a family with ‘compensated hypothyroidism’, or TSH resistance, that is, euthyroid status with a normal thyroid gland, normal levels of circulating T3 and T4, but chronically elevated plasma TSH. The trait appeared to be transmitted in the autosomal recessive mode, as it was found in three sisters from normal parents (Sunthornthepvarakul et al. 1995). All three were compound heterozygotes for missense mutations affecting closely located amino acids in the extracellular domain of the receptor (Fig. 2). Consistent with the clinical picture of resistance to TSH as opposed to complete insensitivity, the Pro 162→Ala mutation (inherited from the mother) displayed some residual activity when expressed transiently in COS-7 cells (Sunthornthepvarakul et al. 1995). The paternal mutation, Ile 167→Asn, displayed no detectable residual activity. Thyroid tests were normal in both heterozygous parents, except for a slightly elevated TSH. Thus, loss of function of the TSHr seemed to behave as a truly recessive trait. Additional TSHr mutations were reported in families or apparently sporadic cases of resistance to TSH (de Roux et al. 1996a, Clifton-Bligh et al. 1996) (Fig. 2). One patient was found to be homozygous for the previously described Pro162Ala. Most other patients were compound heterozygotes for a nonsense (null) mutation and a missense mutation, whose location indicates the importance of single residues in hormone binding (Arg 109→Gln: Clifton-Bligh et al. 1996; Cys 390→Trp: de Roux et al. 1996c), in cAMP activation or simply in the ability of the receptor to fold properly. Congenital Hypothyroidism (CH): a Heterogeneous Disorder Recently, a homozygous missense mutation was found in two siblings from consanguineous parents, a boy and a girl, who presented with CH at neonatal screening (Abramowicz et al. 1997). It consisted of the substitution of threonine in place of a highly conserved TEM Vol. 9, No. 4, 1998
Figure 2. Schematic representation of the TSH receptor with an indication of the amino-acid substitutions or mutations responsible for the loss of function seen in resistance to TSH or rare cases of congenital hypothyroidism.
alanine at position 553 (Fig. 2), in the fourth putative transmembrane helix of the serpentine domain. An expression assay in COS-7 cells showed that the mutation resulted in extremely low expression at the cell membrane, in spite of normal intracellular synthesis. The very small amount of receptor that reached the cell membrane seemed, however, to behave normally in terms of hormone binding and cAMP stimulation. However, the in vivo significance of this finding from the overexpressed receptor in COS-7 cells is uncertain. On careful clinical imaging, a very small thyroid gland could be discerned in front of the thyroid cartilage in these children, who had been diagnosed initially with thyroid agenesis. Blood levels of thyroglobulin (Tg) were in the high normal range at the time of diagnosis, an unexpected finding considering the extreme hypoplasia of the gland. This disproportionately high Tg might result from incomplete polarization of
the thyrocytes or from intercellular leakage from dysplastic follicles. The irreversible decrease of Tg seen after T4 therapy suggests that full maturation of the thyroid might depend on autostimulation by thyroid hormones. Again, thyroid tests were essentially normal in both parents, as well as in asymptomatic heterozygous siblings, demonstrating the autosomal recessive inheritance of the defect. This observation, together with two other cases described recently (Bieberman et al. 1997, Gagné et al., in press) (Fig. 2) and a mouse model (Beamer et al. 1981, Stein et al. 1994), indicate that severe loss of function of the TSHr, while compatible with the development of a thyroid anlage and its migration in front of the trachea, results in profound hypoplasia and congenital hypothyroidism. These data indicate that TSHr defects are responsible for a subset of CH cases that might be difficult to distinguish clinically from true thyroid 137
agenesis, except when blood Tg levels are measured and found to be elevated. However, a TSHr defect is probably an uncommon cause of autosomal recessive CH, as demonstrated by a recent linkage study (Ahlbom et al. 1997). The cause(s) of sporadic thyroid dysgenesis must certainly be sought elsewhere, and discussion of this topic is outside the scope of this review. Suffice it to say that mutations in the thyroid- and kidney-specific transcription factor Pax-8 have recently been identified in a minority of such cases (Macchia et al. 1997). •
Conclusion
The analysis of spontaneous mutations of the TSHr gene has been a particularly rewarding experience. The phenotypic selection made by the clinicians has sifted the significant events out of the thousands of possible mutations one could envisage to test by site-directed mutagenesis. It is our hope that further studies, building on these observations, will contribute to our understanding of the mechanisms of activation of the receptor, both by its normal ligand, and by the autoantibodies of Graves’ disease patients. •
Acknowledgements
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Paschke R, Tonacchera M, Van Sande J, Parma J, Vassart G: 1994. Identification and functional characterization of two new somatic mutations causing constitutive activation of the thyrotropin receptor in hyperfunctioning autonomous adenomas of the thyroid. J Clin Endocrinol Metab 79:1785–1789.
Macchia PE, Lapi P, Chiovati L, Busslinger M, Fenzi GF, Di Lauro R: 1997. Identification of a mutation in the PAX-8 gene in a patient with thyroid ectopy [abst]. J Endocrinol Invest 20 (Supp):84.
Paschke R, Fuehrer D, Holzapfel HP: 1996. Identification of different somatic thyrotropin receptor mutations in toxic multinodular goiter [abst]. J Endocrinol Invest 6 (Supp):28.
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Patten JL, Johns DR, Valle D, et al.: 1990. Mutation in the gene encoding the stimulatory G protein of adenylate cyclase in Albright’s hereditary osteodystrophy. N Engl J Med 322:1412–1419. Porcellini A, Ciullo I, Laviola L, Amabile G, Fenzi G, Avvedimento VE: 1994. Novel mutations of thyrotropin receptor gene in thyroid hyperfunctioning adenomas. Rapid identification by fine needle aspiration biopsy. J Clin Endocrinol Metab 79:657–661. Porcellini A, Ciullo I, Pannain S, Fenzi G, Avvedimento VE: 1995. Somatic mutations in the VI transmembrane segment of the thyrotropin receptor constitutively activate cAMP signalling in thyroid hyperfunctioning adenomas. Oncogene 11: 1089–1093. Robbins LS, Nadeau JH, Johnson KR, et al.: 1993. Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function. Cell 72:827–834. Robinson PR, Cohen GB, Zhukovsky EA, Oprian DD: 1992. Constitutively active mutants of Rhodopsin. Neuron 9:719–725. Roger P, Reuse S, Maenhaut C, Dumont JE: 1995. Multiple facets of the modulation of growth by cAMP. Vitam Horm 51:59–191. Russo D, Arturi F, Schlumberger M, et al.: 1995. Activating mutations of the TSH receptor in differentiated thyroid carcinomas. Oncogene 11:1907–1911. Russo D, Arturi F, Chiefari E, Filetti S: 1997. Molecular insights into TSH receptor abnormality and thyroid disease. J Endocrinol Invest 20:36–47. Stein SA, Oates EL, Hall CR, et al.: 1994. Identification of a point mutation in the thyrotropin receptor of the hyL/hyL hypothyroid mouse. Mol Endocrinol 8:129–138. Strader CD, Fong TM, Tota MR, Underwood D: 1994. Structure and function of G protein-coupled receptors. Ann Rev Biochem 63:101–132. Suarez HG, du Villard JA, Caillou B, Schlumberger M, Parmentier C, Monier R: 1991. gsp mutations in human thyroid tumours. Oncogene 6:677–679. Sunthornthepvarakul T, Gottschalk M, Hayashi Y, Refetoff S: 1995. Resistance to thyrotropin caused by mutations in the thyrotropin receptor gene. N Engl J Med 323:155–160. Takeshita A, Nagayama Y, Yokoyama N, et al.: 1995. Rarity of oncogenic mutations in the thyrotropin receptor of autonomously functioning thyroid nodules in Japan. J Clin Endocrinol Metab 80:2607–2611.
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Thomas JL, Leclère J, Hartemann P, et al.: 1982. Familial hyperthyroidism without evidence of autoimmunity. Acta Endocrinol 100:512–518. Todd CH, Allain T, Gomo ZA, Hasler JA, Ndiweni M, Oken E: 1995. Increase in thyrotoxicosis associated with iodine supplements in Zimbabwe. Lancet 346:1563–1564. Tonacchera M, Van Sande J, Parma J, et al.: 1996a. TSH receptor and disease. Clin Endocrinol 44:621–633.
Tonacchera M, Van Sande J, Cetani F, et al.: 1996b. Functional characteristics of three new germline mutations of the thyrotropin gene causing autosomal dominant toxic thyroid hyperplasia. J Clin Endocrinol Metab 8:547–554. Tonacchera M, Chiovato L, Fiore E, et al.: 1997. Elevated incidence of TSH-R gene mutations in toxic adenoma and in hot nodules of toxic multinodular goiters [abst]. J Endocrinol Invest 20 (Supp):4. Van Sande J, Massart C, Costagliola S, et al.:
Molecular Properties of the CRF Receptor Joachim Spiess, Frank M. Dautzenberg, Sabine Sydow, Richard L. Hauger, Andreas Rühmann, Thomas Blank and Jelena Radulovic
Research into the biology of corticotropin-releasing factor (CRF) has been intensified significantly by the structural characterization of the CRF receptor (CRF-R). Two receptor subtypes, CRF-R1 and CRF-R2, and three functional splice variants of CRF-R2 have been discovered. It appears that ligand binding requires interaction of the N-terminal domain with one or two other extracellular domains of the CRF-R. In contrast to the mammalian CRF-R1, the frog CRF-R1 discriminates between naturally occurring CRF-like peptides. Corticotropin-releasing factor (CRF or CRH), a 41 amino acid residue polypeptide (Spiess et al. 1981), which stimulates hypophyseal corticotropin (ACTH) secretion (Vale et al. 1981), has been recognized as an early chemical signal triggering many responses to stress. Initially, the activation of the hypothalamic hypophyseal adrenal axis was at the center of the research interest. It has been demonstrated that this axis is initiated by the release J. Spiess, F.M. Dautzenberg, S. Sydow, A. Rühmann, T. Blank and J. Radulovic are at the Max-Planck Institute for Experimental Medicine, Department of Molecular Neuroendocrinology, Hermann-Rein-Strasse 3, 37075 Goettingen, Germany; and R.L. Hauger is at the University of California San Diego, Department of Psychiatry, 9500 Gilman Drive, La Jolla, CA 92093-0603, USA.
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of hypothalamic CRF into the hypophyseal portal system; the CRF signal is then amplified by hypophyseal corticotropin secretion and finally by secretion of cortisol from the adrenal cortex [for review, see Owens and Nemeroff (1991)]. Research on CRF has been enhanced to a significant extent by pathophysiological considerations. A role for CRF has been proposed in endocrine diseases such as Cushing’s disease (Jones 1990), as well as inflammation and cognitive diseases such as Alzheimer’s disease [reviewed by De Souza (1995)]. •
Multiplicity of Receptor Forms
With the structural characterization of the CRF receptor (CRF-R) and its subtypes (Chang et al. 1993, Chen et al. 1993, Vita et al. 1993; Lovenberg et al.
1996. Specific activation of the thyrotropin receptor by trypsin. Mol Cell Endocrinol 119:161–168. Van Sande J, Parma J, Tonacchera M, Swillens S, Dumont J, Vassart G: 1995. Somatic and germline mutations of the TSH receptor gene in thyroid diseases. J Clin Endocrinol Metab 80:2577–2585. Vassart G, Dumont JE: 1992. The thyrotropin receptor and the regulation of thyrocyte function and growth. Endocr Rev 13:596–611.
1995, Kishimoto et al. 1995, Perrin et al. 1995, Stenzel et al. 1995, Liaw et al. 1996, Yu et al. 1996, Dautzenberg et al. 1997), research into the action of the CRF was enhanced significantly. Cloning of cDNAs from different species and tissues has revealed that all species investigated so far produce two receptor subtypes, subtypes 1 and 2 (CRF-R1 and CRF-R2). CRF-R1 is a 415 amino acid protein that is expressed mainly in the brain and pituitary (Chen et al. 1993). CRF-R2 is expressed in the form of three functional splice variants, α (411–413 amino acids), β (431–438 amino acids) and γ (397 amino acids) (Kishimoto et al. 1995, Lovenberg et al. 1995, Stenzel et al. 1995, Sperle et al. 1997). In rodents, CRF-R2 is mainly a peripheral receptor produced in the heart and in blood vessels, whereas CRF-R2α has been found only in the central nervous system (CNS). In contrast to rodents, both human CRF-R2α and CRF-R2β are coexpressed in peripheral organs and the CNS (Valdenaire et al. 1997), whereas CRF-R2γ, which has been isolated only from humans, was found only in the brain (Sperle et al. 1997). Interestingly, only CRF-R2α was isolated from amphibian species (Dautzenberg et al. 1997). The evolutionary divergence of the human and rodent forms of the CRF-R must have occurred relatively late (Fig. 1). It is unclear at this time whether the observed differences in tissue distribution and variety of forms between humans and rodents affect the pattern of physiological events after CRF release. This question needs to be addressed to determine the limitations of rodent models in this context.
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Mutations of the thyrotropin receptor (TSHr) can be loss of function or gain of function. Loss-of-function mutations can affect a variety of loci in the TSHr gene. Their most common manifestation is resistance to TSH; they may also be the cause of a subset of cases of congenital hypothyroidism. Gain-of-function mutations are of greater theoretical interest. Somatic mutations constitutively activating the TSHr are the major cause of benign toxic thyroid adenomas, and of some cases of multinodular goiters. They underlie hereditary toxic thyroid hyperplasia, and have been found in cases of sporadic congenital non-autoimmune hyperthyroidism. A role for TSHr polymorphisms in Graves’ disease has not been documented.
Over the past few years, mutations in the thyrotropin receptor (TSHr) gene have been identified as a cause of acquired, hereditary or congenital thyroid diseases (Tonacchera et al. 1996a). Depending on their nature, the mutations cause either loss of function or gain of function, leading to hypoor hyperthyroidism, respectively. The loss-of-function mutations are relatively trivial events interfering with normal receptor function – there are hundreds of different ways to destroy the precise functional architecture of L. Duprez, J. Van Sande, P. Rodien and J.E. Dumont are at the Institut de Recherche Interdisciplinaire, Faculty of Medicine, University of Brussels, Belgium; J. Parma is at the Service de Génétique Médicale, Hôpital Erasme, University of Brussels, Belgium; and G. Vassart and M. Abramowicz are at the Institut de Recherche Interdisciplinaire and the Service de Génétique Médicale.
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a protein. However, the identification of a large series of gain-of-function mutations in the TSHr gene was one of the first examples of a new pathophysiological mechanism, in which diseases are caused by mutations that increase the basal activity of a receptor and make it constitutively active, in the absence of its normal agonist. In addition, the study of the structure– function relationships of these constitutively active mutants has led to a better understanding of the mechanisms implicated in the normal activation of the receptor by TSH, and it provides a rationale for the understanding of its activation by autoantibodies in Graves’ disease. The aim of this short review is to summarize what has been learnt over recent years by the identification and functional analyses of the different types of TSHr mutations.
•
Background
The TSHr belongs to the superfamily of seven transmembrane domain receptors coupled to G proteins (G-protein-coupled receptors; GPCRs) (Nagayama and Rapoport 1992, Vassart and Dumont 1992). More precisely, it is a member of a subfamily whose prototypic members are rhodopsin and the adrenergic receptors (Helmreich and Hofmann 1996). A common characteristic of GPCRs is the presence of seven transmembrane segments, subdividing the molecules into an extracellular domain (aminoterminal segment plus three extracellular loops), a transmembrane barrel (seven α helices) and an intracytoplasmic domain (three intracytoplasmic loops plus the carboxyl-terminal segment). In the prototypic GPCRs, the ligand binds to structures within the pocket between transmembrane helices (e.g. biogenic amine receptors) and/or to residues of the extracellular domain (neuropeptide receptors) (Strader et al. 1994). Together with the other glycoprotein hormone receptors [luteinizing hormone/chorionic gonadotropin receptor (LH/CGr) and follicle-stimulating hormone receptor (FSHr)], the TSHr is characterized by a particularly long amino-terminal segment (398 residues), which is responsible for high-affinity binding of TSH (Parmentier et al. 1989). This portion of the receptor is glycosylated and essentially made of ‘leucine repeat’ motifs. The availability of the tridimensional structure of one prototypic protein made of leucine repeats (the ribonuclease inhibitor) has allowed the building of models of the amino-terminal hormone binding segment of glycoprotein receptors (Kobe and Deisenhofer 1993 and 1995, Kajava et al. 1995). Whereas the amino-terminal domain of the LH/CG receptors is capable of high-affinity
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by its normal agonist or autoantibodies, or secondary to an activating mutation, is expected to cause concomitant hyperplasia and hyperfunction (Roger et al. 1995). If the activation is taking place in a single cell, it is expected to promote clonal expansion of hyperfunctional thyrocytes. The structural and functional dichotomy of the TSHr poses the problem of understanding its activation mechanism in the light of what is known of other GPCRs. Clearly, the bulky TSH cannot fit within the transmembrane pocket of the receptor; how then does binding of the hormone to the extracellular amino terminus result in activation of the serpentine portion? As will become apparent, the distribution of activating mutations along the receptor’s primary sructure provides clues for the elaboration of a model of receptor activation. • Figure 1. Schematic representation of the TSH receptor with an indication of the amino-acid substitutions or deletions responsible for its constitutive activation.
hormone binding when expressed alone, no convincing data of this sort have been obtained for the TSHr. Also, the TSHr is unique because of its dimeric structure: a proportion of the molecules, synthesized as a single polypeptidic chain, undergoes a still poorly defined maturation step, leading to a cleavage near the border between the amino terminus and the first transmembrane helix (Couet et al. 1996a, Chazenbalk et al. 1997). As suggested initially (Vassart and Dumont 1992), a 50 amino-acid residue segment located in this region and with no counterpart in the LH/CG or FSH receptors, probably plays a key role in this maturation step (Chazenbalk et al. 1997). After cleavage, the amino terminus remains mostly attached to the ‘serpentine’ portion by disulfide bond(s), but there is evidence of continuous shedding of the amino terminus by a mechanism involving protein disulfide isomerase (PDI) (Couet et al. 1996b). Similar to the protein, the TSHr gene (located on chromosome 14q31) can be subdivided into two segments with
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different functional significance: a large exon encodes the carboxy-terminal portion of the ectodomain, the serpentine and intracellular domains (residues 294–764), while a series of nine exons code for the remainder of the ectodomain (Gross et al. 1991). The intracytoplasmic domain of GPCRs contains the site(s) of interaction with G proteins (O’Dowd et al. 1988), the transducers between activated receptors and the various effectors (Neer 1995). The human TSHr interacts functionally with both the Gs and Gq proteins, the former interaction mediating the effects of TSH on tissue growth and hormone secretion, the latter being responsible for stimulation of hormone synthesis (Dumont et al. 1992). Contrary to many GPCRs, including the LH/CG receptor, the TSHr exhibits very limited homologous desensitization. This should be remembered when interpreting the effects of activating mutations and the stimulatory immunoglobulins of Graves’ disease. The continuous stimulation of the receptor, whether
Gain-of-function Mutations
From our knowledge of thyroid cell physiology, constitutive activation of the cAMP-dependent regulatory cascade in human thyrocytes is expected to cause unregulated growth of autonomous hyperfunctioning tissue (Dumont et al. 1989). Transgenic mouse models of this situation have been constructed (Ledent et al. 1992, Michiels et al. 1994). Up until now, mutations in two molecules of the cAMP regulatory cascade have been associated with thyroid hyperfunction: G␣s and the TSH receptor. Mutations in G␣s resulting in decrease of GTPase activity have been identified in a proportion of somatotropic adenoma (Lyons et al. 1990) and in McCune– Albright syndrome (Patten et al. 1990). The possibility that spontaneous mutations activating the TSHr exist was predicted by earlier studies with the adrenergic and melanocortin receptors, as well as with rhodopsin (Cotecchia et al. 1992, Robinson et al. 1992, Robbins et al. 1993). Somatic Mutations Activating the TSHr Constitutively are the Major Cause of Benign Toxic Thyroid Adenomas Solitary autonomous adenoma and multinodular goiter are the two major TEM Vol. 9, No. 4, 1998
clinical presentations of thyroid autonomy in countries with low or borderline iodine supply. Thyroid adenomas are encapsulated benign tumors, characterized mainly by autonomy, a variable degree of hyperfunction and usually a slow growth. This definition can be extended to at least some adenomatous nodules lacking a capsule, provided their autonomy is documented. Eight years after the identification of the first mutation in Gs (Lyons et al. 1990), and five years after the demonstration of TSHr receptor mutations in autonomous adenomas (Parma et al. 1993), the situation can be summarized as follows. In our series of 33 adenomatous nodules, 82% were found to harbor a somatic mutation in the TSHr gene and 6% in the G␣s gene (Parma et al. 1997). All these mutations were found in the heterozygous state in the nodular tissue and not in the adjacent tissue or the peripheral leukocytes. This demonstrates the somatic nature of the mutations and is consistent with a dominant effect. Activating mutations were dispersed all along the primary structure of the receptor: in the extracellular domain, the extracellular loops, the second, third, fifth, sixth and seventh transmembrane domains and in the third intracytoplasmic loop (Fig. 1). A large proportion of the mutations have been found in the amino-terminal portion of the sixth transmembrane domain, where five contiguous residues (629–633) were mutated in 44% of the nodules in our series (Fig. 1). Whereas other studies failed initially to find a similarly high prevalence of TSHr mutations in autonomous adenomas (Matsuo et al. 1993, Russo et al. 1995, Djuh et al. 1996, Takeshita et al. 1995, Esapa et al. 1997), a consensus is emerging from more recent studies (Porcellini et al. 1994 and 1995, Führer et al. 1997) that, at least in regions with a borderline iodine supply, TSHr mutations are the major cause of the disease. The lower prevalence seen in Japan and in the USA (Matsuo et al. 1993, Russo et al. 1995, Djuh et al. 1996, Takeshita et al. 1995, Esapa et al. 1997), together with the absence of mutations in 12% of our cases, leaves TEM Vol. 9, No. 4, 1998
some room for other pathophysiological mechanisms or gene targets. All mutated receptors identified in these studies were expressed transiently in COS-7 cells and their functional properties analyzed (Parma et al. 1993 and 1997, Porcellini et al. 1995, Paschke et al. 1994, Van Sande et al. 1995). They all displayed an increase in basal stimulation of cAMP production, despite the fact that most of them were expressed at a lower level than the wild-type receptor. With a few notable exceptions, they remained stimulatable by TSH, did not show constitutive activation of the diacylglycerol–inositol phosphate pathway and bound bovine TSH with a better apparent affinity than the wild-type TSHr. Somatic Mutations of the TSHr and the Pathogenesis of Multinodular Goiter We have reported two different activating mutations of the TSHr in a multinodular goiter with two separated autonomous regions (Duprez et al. 1997a), and similar findings have been described independently (Paschke et al. 1996, Tonacchera et al. 1997). Although the exact prevalence of TSHr mutations in multinodular goiter is unknown, these observations demonstrate that the same pathophysiological mechanism may be at work as in solitary adenomas. In areas with mild to moderate iodine deficiency, autonomy is observed frequently on a background of multinodular goiter. In truly endemic regions, iodine supplementation is followed by an increase in non-autoimmune hyperthyroidism (Fradkin and Wolff 1983, Todd et al. 1995, Dremier et al. 1996), which suggests that iodine supplementation could act as a revelator of a proportion of previously asymptomatic autonomies. It will be interesting to look for TSHr mutations in the autonomous portions of these glands. Somatic Mutations of the TSHr and Thyroid Cancer Activating mutations have been found in rare cases of well-differentiated follicular thyroid carcinomas (Suarez et al. 1991, Matsuo et al. 1993, Russo et al. 1995). The rarity of these
observations, and of the malignant transformation of hyperactive thyroid tissue as a whole, illustrates the essentially benign nature of the cAMP mitogenic pathway in thyrocytes. Familial Non-autoimmune Hyperthyroidism: a Germline Counterpart of the Toxic Adenoma Since our original description of two families from northern France (Thomas et al. 1982, Duprez et al. 1994), six additional pedigrees have been identified where hyperthyroidism and thyroid hyperplasia segregate as an autosomal dominant trait in linkage with mutated alleles of the TSHr gene (Tonacchera et al. 1996b, Führer et al. 1996, and our unpublished data) (Fig. 1; Table 1). This new nosological entity, hereditary toxic thyroid hyperplasia (HTTH), is characterized by the following clinical characteristics: autosomal dominant transmission; hyperthyroidism with a variable age of onset (from infancy to adulthood, even within a given family); hyperplastic goiter of variable size, but with a steady growth; and absence of clinical or biological stigmata of autoimmunity. A common observation in the cases described to date is the need for drastic ablative therapy (surgery or radioiodine) to control the disease once the patient has become hyperthyroid. The prevalence of HTTH is difficult to estimate at the present time. It is likely that many cases have been (and still are) mistaken for Graves’ disease, owing to the relative insensitivity and lack of specificity of thyroid-stimulating antibody assays. It is expected that wider knowledge of the existence of the disease will lead to better diagnosis. This is not a purely academic problem, because presymptomatic diagnosis in children of affected families might prevent the development of neuropsychological complications associated with infantile or juvenile hyperthyroidism. Germline Neomutations of the TSHr are Found in Sporadic Congenital Non-autoimmune Hyperthyroidism Neonatal hyperthyroidism is usually caused by transplacental passage 135
Table 1. List of activating mutations of the TSH receptor observed in toxic adenomas, hereditary toxic thyroid hyperplasia and non-immune congenital hyperthyroidism Residue
Somatic
Asp 276 Asn Ser 281 Asn Ser 281 Thr Ser 281 Ile Met 453 Thr Ile 486 Met Ile 486 Phe Ser 505 Arg Ser 505 Asn Val 509 Ala Ile 568 Thr Val 597 Leu Tyr 601 Asn Del 613–621 Asp 619 Gly Ala 623 Ile Ala 623 Val Ala 623 Ser
⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻
Familial
⫻
⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻
of stimulating autoantibodies and is transient. In some cases, an autoimmune origin cannot be found and the hyperthyroidism does not resolve: de novo germline activating mutations of the TSHr have been found in such cases (Kopp et al. 1995, de Roux et al. 1996a, Esapa et al. 1996, Holzapfel et al. 1997). Four cases bearing four different mutations of the TSHr have been described, (Table 1). The four neonates were small for gestational age and presented a goiter with severe hyperthyroidism requiring prompt surgical intervention. The meaning of the pseudo-exophthalmia presented by one of these children remains ambiguous because they all displayed an advanced bone age. Most of the cases with congenital non-autoimmune thyrotoxicosis have mutations also found in toxic adenomas. In contrast, the majority of mutations in HTTH are ‘private’ mutations (Table 1). This is compatible with the hereditary mutations causing a less severely altered phenotype, with a marginal effect on reproductive fitness in the absence of treatment.
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Sporadic
Residue
Somatic
Familial
Leu 629 Phe Leu 629 Pro Ile 630 Leu Phe 631 Leu Phe 631 Cys Thr 632 Ile Thr 632 Ala Asp 633 Glu Asp 633 Tyr Asp 633 His Asp 633 Ala Met 637 Arg Pro 639 Ser Asn 650 Tyr Val 657 Phe Del 658–661 Asn 670 Ser Cys 672 Tyr
⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻ ⫻
⫻
⫻
Sporadic
⫻
⫻ ⫻ ⫻
⫻ ⫻
An Emerging Model for Receptor Activation In agreement with current concepts about GPCR activation, the global effect of activating mutations could be the disruption of a structural constraint maintaining the receptor in an inactive state (Kjelsberg et al. 1992). In the case of TSHr, this constraint must be relatively weak because the wildtype receptor displays a significant level of constitutive activation when expressed in COS-7 cells (Duprez et al. 1994, Kosugi and Mori 1995, Van Sande et al. 1995). We have proposed that it is the ‘noisy’ character of the wild-type TSHr that explains the wide spectrum of amino-acid substitutions leading to its activation. As an extension of this concept, it is tempting to speculate that this same high propensity to become activated would explain the activation of the TSHr by autoantibodies in Graves’ disease. The location of some activating amino-acid substitutions in the extracellular loops and the amino-terminal hormone binding segment (Parma et al. 1995, Duprez et al. 1997b, Kopp et al. 1997, Russo et al. 1997) (Fig. 1), together
⫻ ⫻
with the observation that mild proteolytic treatment of the receptor leads to its activation (Van Sande et al. 1996), suggest that an inhibitory interaction might exist between the unliganded amino-terminal segment and the extracellular loops of the serpentine domain. According to this model, the effect of TSH or stimulating autoantibodies would be to release this inhibitory interaction. A Role for TSHr Polymorphisms in Graves’ Autoimmune Hyperthyroidism? Graves’ disease is known to show some familial aggregation but does not constitute a truly hereditary disorder. Instead, it may be understood as a disease of multifactorial origin, where several independently inherited genes, and environmental factors, are involved collectively in pathogenesis. From a theoretical viewpoint, as the receptor is a major autoantigen in Graves’ disease, one of these genes might be that for the TSHr. However, to date, there is no convincing argument in favor of such a hypothesis, and linkage to the TSHr gene has even been excluded in a large study of familial Graves’ disease (de Roux et al. 1996b). TEM Vol. 9, No. 4, 1998
•
Loss-of-function Mutations
Resistance to Thyrotropin Loss-of-function mutations were first described in a family with ‘compensated hypothyroidism’, or TSH resistance, that is, euthyroid status with a normal thyroid gland, normal levels of circulating T3 and T4, but chronically elevated plasma TSH. The trait appeared to be transmitted in the autosomal recessive mode, as it was found in three sisters from normal parents (Sunthornthepvarakul et al. 1995). All three were compound heterozygotes for missense mutations affecting closely located amino acids in the extracellular domain of the receptor (Fig. 2). Consistent with the clinical picture of resistance to TSH as opposed to complete insensitivity, the Pro 162→Ala mutation (inherited from the mother) displayed some residual activity when expressed transiently in COS-7 cells (Sunthornthepvarakul et al. 1995). The paternal mutation, Ile 167→Asn, displayed no detectable residual activity. Thyroid tests were normal in both heterozygous parents, except for a slightly elevated TSH. Thus, loss of function of the TSHr seemed to behave as a truly recessive trait. Additional TSHr mutations were reported in families or apparently sporadic cases of resistance to TSH (de Roux et al. 1996a, Clifton-Bligh et al. 1996) (Fig. 2). One patient was found to be homozygous for the previously described Pro162Ala. Most other patients were compound heterozygotes for a nonsense (null) mutation and a missense mutation, whose location indicates the importance of single residues in hormone binding (Arg 109→Gln: Clifton-Bligh et al. 1996; Cys 390→Trp: de Roux et al. 1996c), in cAMP activation or simply in the ability of the receptor to fold properly. Congenital Hypothyroidism (CH): a Heterogeneous Disorder Recently, a homozygous missense mutation was found in two siblings from consanguineous parents, a boy and a girl, who presented with CH at neonatal screening (Abramowicz et al. 1997). It consisted of the substitution of threonine in place of a highly conserved TEM Vol. 9, No. 4, 1998
Figure 2. Schematic representation of the TSH receptor with an indication of the amino-acid substitutions or mutations responsible for the loss of function seen in resistance to TSH or rare cases of congenital hypothyroidism.
alanine at position 553 (Fig. 2), in the fourth putative transmembrane helix of the serpentine domain. An expression assay in COS-7 cells showed that the mutation resulted in extremely low expression at the cell membrane, in spite of normal intracellular synthesis. The very small amount of receptor that reached the cell membrane seemed, however, to behave normally in terms of hormone binding and cAMP stimulation. However, the in vivo significance of this finding from the overexpressed receptor in COS-7 cells is uncertain. On careful clinical imaging, a very small thyroid gland could be discerned in front of the thyroid cartilage in these children, who had been diagnosed initially with thyroid agenesis. Blood levels of thyroglobulin (Tg) were in the high normal range at the time of diagnosis, an unexpected finding considering the extreme hypoplasia of the gland. This disproportionately high Tg might result from incomplete polarization of
the thyrocytes or from intercellular leakage from dysplastic follicles. The irreversible decrease of Tg seen after T4 therapy suggests that full maturation of the thyroid might depend on autostimulation by thyroid hormones. Again, thyroid tests were essentially normal in both parents, as well as in asymptomatic heterozygous siblings, demonstrating the autosomal recessive inheritance of the defect. This observation, together with two other cases described recently (Bieberman et al. 1997, Gagné et al., in press) (Fig. 2) and a mouse model (Beamer et al. 1981, Stein et al. 1994), indicate that severe loss of function of the TSHr, while compatible with the development of a thyroid anlage and its migration in front of the trachea, results in profound hypoplasia and congenital hypothyroidism. These data indicate that TSHr defects are responsible for a subset of CH cases that might be difficult to distinguish clinically from true thyroid 137
agenesis, except when blood Tg levels are measured and found to be elevated. However, a TSHr defect is probably an uncommon cause of autosomal recessive CH, as demonstrated by a recent linkage study (Ahlbom et al. 1997). The cause(s) of sporadic thyroid dysgenesis must certainly be sought elsewhere, and discussion of this topic is outside the scope of this review. Suffice it to say that mutations in the thyroid- and kidney-specific transcription factor Pax-8 have recently been identified in a minority of such cases (Macchia et al. 1997). •
Conclusion
The analysis of spontaneous mutations of the TSHr gene has been a particularly rewarding experience. The phenotypic selection made by the clinicians has sifted the significant events out of the thousands of possible mutations one could envisage to test by site-directed mutagenesis. It is our hope that further studies, building on these observations, will contribute to our understanding of the mechanisms of activation of the receptor, both by its normal ligand, and by the autoantibodies of Graves’ disease patients. •
Acknowledgements
This study was supported by the Belgian Programme on University Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Service for Sciences, Technology and Culture. It was also supported by grants from the Fonds de la Recherche Scientifique Médicale, the FNRS, Télévie, the European Union (Biomed), Association Belge de Recherche contre le Cancer and Association de Recherche Biomédicale et de Diagnostic. L.D. is Aspirant of the Fonds National de la Recherche Scientifique. References Abramowicz MJ, Duprez L, Parma J, Vassart G, Heinrichs C: 1997. Familial congenital hypothyroidism due to inactivating mutation of the thyrotropin receptor causing profound hypoplasia of the thyroid gland. J Clin Invest 99:3018–3024.
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Ahlbom BE, Yaqoob M, Larsson A, Ilicki A, Annerén G, Wadelius C: 1997. Genetic and linkage analysis of familial congenital hypothyroidism: exclusion of linkage to the TSH receptor gene. Hum Genet 99:186–190.
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Molecular Properties of the CRF Receptor Joachim Spiess, Frank M. Dautzenberg, Sabine Sydow, Richard L. Hauger, Andreas Rühmann, Thomas Blank and Jelena Radulovic
Research into the biology of corticotropin-releasing factor (CRF) has been intensified significantly by the structural characterization of the CRF receptor (CRF-R). Two receptor subtypes, CRF-R1 and CRF-R2, and three functional splice variants of CRF-R2 have been discovered. It appears that ligand binding requires interaction of the N-terminal domain with one or two other extracellular domains of the CRF-R. In contrast to the mammalian CRF-R1, the frog CRF-R1 discriminates between naturally occurring CRF-like peptides. Corticotropin-releasing factor (CRF or CRH), a 41 amino acid residue polypeptide (Spiess et al. 1981), which stimulates hypophyseal corticotropin (ACTH) secretion (Vale et al. 1981), has been recognized as an early chemical signal triggering many responses to stress. Initially, the activation of the hypothalamic hypophyseal adrenal axis was at the center of the research interest. It has been demonstrated that this axis is initiated by the release J. Spiess, F.M. Dautzenberg, S. Sydow, A. Rühmann, T. Blank and J. Radulovic are at the Max-Planck Institute for Experimental Medicine, Department of Molecular Neuroendocrinology, Hermann-Rein-Strasse 3, 37075 Goettingen, Germany; and R.L. Hauger is at the University of California San Diego, Department of Psychiatry, 9500 Gilman Drive, La Jolla, CA 92093-0603, USA.
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of hypothalamic CRF into the hypophyseal portal system; the CRF signal is then amplified by hypophyseal corticotropin secretion and finally by secretion of cortisol from the adrenal cortex [for review, see Owens and Nemeroff (1991)]. Research on CRF has been enhanced to a significant extent by pathophysiological considerations. A role for CRF has been proposed in endocrine diseases such as Cushing’s disease (Jones 1990), as well as inflammation and cognitive diseases such as Alzheimer’s disease [reviewed by De Souza (1995)]. •
Multiplicity of Receptor Forms
With the structural characterization of the CRF receptor (CRF-R) and its subtypes (Chang et al. 1993, Chen et al. 1993, Vita et al. 1993; Lovenberg et al.
1996. Specific activation of the thyrotropin receptor by trypsin. Mol Cell Endocrinol 119:161–168. Van Sande J, Parma J, Tonacchera M, Swillens S, Dumont J, Vassart G: 1995. Somatic and germline mutations of the TSH receptor gene in thyroid diseases. J Clin Endocrinol Metab 80:2577–2585. Vassart G, Dumont JE: 1992. The thyrotropin receptor and the regulation of thyrocyte function and growth. Endocr Rev 13:596–611.
1995, Kishimoto et al. 1995, Perrin et al. 1995, Stenzel et al. 1995, Liaw et al. 1996, Yu et al. 1996, Dautzenberg et al. 1997), research into the action of the CRF was enhanced significantly. Cloning of cDNAs from different species and tissues has revealed that all species investigated so far produce two receptor subtypes, subtypes 1 and 2 (CRF-R1 and CRF-R2). CRF-R1 is a 415 amino acid protein that is expressed mainly in the brain and pituitary (Chen et al. 1993). CRF-R2 is expressed in the form of three functional splice variants, α (411–413 amino acids), β (431–438 amino acids) and γ (397 amino acids) (Kishimoto et al. 1995, Lovenberg et al. 1995, Stenzel et al. 1995, Sperle et al. 1997). In rodents, CRF-R2 is mainly a peripheral receptor produced in the heart and in blood vessels, whereas CRF-R2α has been found only in the central nervous system (CNS). In contrast to rodents, both human CRF-R2α and CRF-R2β are coexpressed in peripheral organs and the CNS (Valdenaire et al. 1997), whereas CRF-R2γ, which has been isolated only from humans, was found only in the brain (Sperle et al. 1997). Interestingly, only CRF-R2α was isolated from amphibian species (Dautzenberg et al. 1997). The evolutionary divergence of the human and rodent forms of the CRF-R must have occurred relatively late (Fig. 1). It is unclear at this time whether the observed differences in tissue distribution and variety of forms between humans and rodents affect the pattern of physiological events after CRF release. This question needs to be addressed to determine the limitations of rodent models in this context.
© 1998, Elsevier Science Ltd, 1043-2760/98/$19.00. PII: S1043-2760(98)00037-X
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