&p.1:Abstract Thyrotropin is the primary pituitary hor-mone which stimulates the growth and differentiationof thyroid cells. TSH binds a specific receptor presentin the plasma membrane of thyroid cells and signals theG protein transducers, which activate different effec-tors, mainly adenyl cyclase and phospholipase C. TheTSH receptor belongs to a broad class of receptorsknown as seven-loop receptors because they contain along stretch of amino acids which cross the plasmamembrane seven times. Mutations in the TSH receptorgene have been found in hyperfunctioning thyroid ade-nomas. These mutations are: (a) somatic (present onlyin the tumor), (b) dominant (only one copy of the geneis affected), and (c) lead to the constitutive activation ofthe cAMP signaling cascade. Most mutations whichhave been identified occur in the intracellular loop IIIand in the transmembrane domain VI. Germline muta-tions in the same regions of the receptor have beenfound in congenital nonautoimmune hyperthyroidism.In addition, germ line mutations have been described inthe extracellular domain of the receptor leading to in-creased TSH levels. The clinical implications of thesefindings are discussed.
Abbreviations CREBcAMP-responsive elementbinding protein · PKA Protein kinase A · TSHThyrotropin&bdy:
Introduction
The thyrotropin (TSH) receptor is a specific moleculepresent in the plasma membrane of thyroid cells whichconfers responsiveness to the pituitary hormone TSH.The pituitary gland synthesizes and secretes TSH in re-sponse to the serum levels of thyroid hormones (triiodo-thyronine and thyroxine). Low levels of these hormonesactivate the thyrotropin-releasing hormone, which is the
A. PorcelliniDipartimento di Biologiae Patologia Molecolare e Cellulare “L. Califano,”Centro di Endocrinologia ed Oncologia Sperimentale,Facoltà di Medicina, Università “Federico II,”5 via Sergio Pansini, I-80131 Naples, Italy
G. FenziDipartimento di Endocrinologia, Facoltà di Medicina,Università “Federico II,” 5 via Sergio Pansini, I-80131 Naples, Italy
E.V. Avvedimento (✉)Dipartimento di Medicina Sperimentale,Facoltà di Medicina a Catanzaro, Università di Reggio Calabria,via Tommaso Campanella I-88100, Catanzaro, Italy
&misc:Received: 15 January 1996 / Accepted: 8 March 1996
ENRICO V. AVVEDIMENTOreceived the M.D. degree atthe Medical School, Univer-sity of Napoli “Federico II”(Italy). He is presently Profes-sor of General Pathology atthe Medical School, Catan-zaro, University of Reggio Ca-labria (Italy). He is also asso-ciated to the Dipartimento diBiologia e Patologia Moleco-lare e Cellulare, MedicalSchool, University “FedericoII” Napoli. His major researchinterest is the molecularanalysis of cAMP signalling ingrowth and differentiation
ANTONIO PORCELLINI receivedthe M.D. degree at the MedicalSchool, University of Naples“Federico II” (Italy). Hereceived a Ph.D. degree inOncology at the Dept. ofExperimental Medicine,Medical School in Catanzaro,University of Reggio Calabria.He is presently staff scientistat the National Cancer Insti-tute Fondazione “Pascale”;Napoli. His major researchinterest is TSH receptorsignalling in thyroid cells.
main stimulator of TSH synthesis; conversely, high levelsof thyroxine repress thyrotropin-releasing hormone [1, 2].The selective expression and localization of the TSH re-ceptor in the plasma membrane of thyroid cells makethese cells specifically responsive to the hormone. Recentdata from several laboratories indicate that the expressionof TSH receptor is not restricted to the thyroid; indeed,nonthyroid tissues also express the receptor. The amountsof mature mRNA and protein in these tissues are approxi-mately 50- to 100-fold less than thyroid cells [3–9].These findings might help to explain the occurrence ofextrathyroid symptoms in diseases involving TSH orTSH-like molecules, without affecting our understandingof the biological responses to TSH in the thyroid gland.
TSH and TSH receptor appear early in the developingthyroid gland. The fetal thyroid acquires the capacity toconcentrate and organify iodine at about 10 weeks ofgestation. Both thyroid hormone and TSH are detectablein the blood soon thereafter and increase in concentra-tion during the second trimester [10].
It is still unclear whether TSH is essential for com-plete development of the thyroid. TSH deficiency duringembryogenesis can be complemented by other glycopro-tein hormones such as human chorionic gonadotropin,which has been shown to bind and stimulate the TSH re-ceptor [11, 12]. TSH appears to be necessary both in vivoand in vitro for the induction and maintenance of thyroidhormone biosynthesis and expression of thyroid differen-tiation genes. In the absence of TSH stimulation, thyro-globulin, thyroperoxidase, and iodine uptake are down-regulated [13–15]. These features constitute the pheno-type of secondary hypothyroidism caused by the absenceof TSH either congenital or after hypophysectomy [1, 2].
Structure of TSH receptor
TSH belongs to a restricted family of hormones, knownas glycoprotein hormones, which includes luteinizinghormone, follicle-stimulating hormone, and human chori-onic gonadotropin. These hormones have a common α
subunit and homologous but distinct β subunits. The res-olution of the structure of human chorionic gonadotropinat 3 Å has provided important insights into the α/β het-erodimer alignments in all members of the glycoproteinhormone family and might help to understand how thesehormones interact with their cognate receptors. Thesehormones contain an unusual structure, similar to a mole-cular seatbelt, which assists a common subunit to com-bine with four different subunits to form the four hor-mones of the family. The intertwined heterodimers have alarge surface-to-volume ratio, resulting in a single-layerstructure, which contact the large extracellular domain ofthe receptor [16]. It is likely that all the glycoprotein hor-mones interact with their receptors in a similar fashion.
The TSH receptor belongs to a family of receptorswhich signal to G proteins. These receptors, known as Gprotein coupled receptors, possess common structuralmotifs [17–19]. The general architecture of these recep-tors is shown in Fig. 1. The features of this family of re-ceptors are: (a) an extracellular segment which binds thehormone, (b) a membrane-spanning domain, and (c) anintracellular segment.
The second segment of G-coupled receptors showssome distinctive features and appears to be the most pre-served motif in this family of receptors. It is formed by along stretch of amino acids crossing the plasma mem-brane seven times. There are seven membrane-spanningsegments interconnected with three extracellular andthree intracellular (cytoplasmic side) loops. The architec-ture of this motif is invariant, and receptors as differentas those of rodopsin and TSH are almost structurallyidentical in the dimension and disposition of these do-mains [20].
The third segment at the COOH terminus of this fami-ly of receptors contains a segment of variable lengthwhich lies on the cytoplasmic side of the membrane.
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Fig. 1 A schematic diagram of a G protein coupled receptor. Atthe NH terminus is shown the extracellular segment which can beof variable length. The receptor is inserted in the plasma mem-brane by seven transmembrane α-helices, each 19–24 amino acidslong. These domains are separated by extracellular and intracellu-lar loops. The intracellular segment at the COOH terminus can bepalmitoylated at a cystein residue, as in the β-adrenergic receptor&/fig.c:
Fig. 2 Structure of TSH receptor. Note the large extracellular seg-ment, which is characteristic of the receptors for glycoprotein hor-mones. C, Cysteine residues. The arrangements of the transmem-brane domains have been deduced from rodopsin receptor [16]&/fig.c:
In summary, the TSH receptor contains (a) a large ex-tracellular domain which represents the binding site forthe hormone, (b) seven transmembrane loops, and (c) arelatively short cytoplasmic tail in the C terminus(Fig. 2).
Coupling of the receptorto the heterotrimeric G proteins
Once activated by the binding of hormone, the TSH re-ceptor contacts and activates the G protein complex. TheG protein is a trimeric complex containing three differ-ent subunits, Gα, Gβ, and Gγ, only the first of which ac-tually binds and hydrolyzes GTP. All G proteins share acommon mechanism by which the information is trans-duced from the receptor through a GTP-activated α sub-unit or a free β/γ heterodimer to specific downstream ef-fectors (in the case of TSH, to adenylyl cyclase via aGαs). GTP acts as molecular switch that transfers infor-mation in the form of conformational change. Binding ofGTP causes the α subunit to dissociate from the dimerβ/γ and from the receptor and to associate with the effec-tor, thus resulting in activation. Free α subunits were ini-tially shown to activate two effectors (adenylyl cyclase
and cGMP phosphodiesterase). The β/γ dimers werethought to reverse the action of α by reforming the inac-tive (GDP-bound) heterotrimer and guiding Gα back tothe receptor for reactivation (G proteins amplify the sig-nal from the receptor, since they can transduce the signalmany times from the same receptor molecule). Now it isquite clear that β/γ dimers play a discrete role in signaltransmission by activating effectors, independently of α(Fig. 3) [21]. To date there is no evidence indicating thatTSH receptor activates effector(s) via β/γ dimers. Gαappears to be the main target for the activated TSH re-ceptor. The crystal structure of transducin α-GTP at 2 Åresolution has revealed the detailed anatomy of the ac-tive subunit. The Gα subunit is composed of two do-mains, one structurally homologous to small GTPasesand another unique to G proteins. Bound GTP is deeplyburied in a cleft between the two domains [22]. An acti-vated receptor turns on a G protein by turning away theG-specific (helical) domain of α from the GTPase do-main. The helical domain restricts movement out of andinto the guanine binding pocket of the GTPase domainsuch as a “teapot lid” (Fig. 4). To promote replacementof GDP by GTP, the activated receptor pushes the lidaway from the “pot.” It does so by loosening a coopera-tive net of hydrogen bonds which connects residues inboth domains with one another and the guanine ring ofthe bound nucleotide. The Gα helical domain works asan intrinsic guanine-nucleotide-dissociation inhibitorthat is positively regulated by hormone receptors andpresumably inhibits the exit of bound guanine nucleo-tide. The Gα switch-off reaction, i.e., GTP hydrolysis,also depends on the helical domain. The guanidiumgroup of an arginine (Arg-174 located in the linker se-quence between the helical and GTPase domains of Gαtransducin) is positioned close to the γ-phosphate ofbound GTP, where it stabilizes a transition state of theGTPase reaction (Fig. 4) [23]. This structural motif ex-plains why cholera toxin stimulates Gα and increasescAMP. Cholera toxin ADP-ribosylates the arginine 174,inhibits GTP hydrolysis and keeps Gα in active confor-mation [24]. Mutations of Gα subunit leading to the sub-stitution of this arginine and to suppression of GTPaseactivity have been found in several endocrine tumors[25–27].
569
Fig. 3 Signaling of TSH receptor to adenylyl cyclase via G pro-tein αs. General view of the complex TSH receptor G protein. Theinactive heterotrimeric G protein complex is shown. α subunit dis-sociates from β/γ upon binding of the TSH to the receptor. αs-GTPactivates adenylyl cyclase&/fig.c:
Fig. 4 Activation of Gα by thereceptor. Magnification of Gprotein complex in inactive state(GDP bound, left) or active state(GTP bound, right). The criticalarginine in position 174 (fromtransducin α), which stimulatesthe GTPase reaction, is shown(empty circle). The active recep-tor contacts Gα and stimulatesthe GDP-GTP exchange reac-tion. The segment of the receptorshown (embedded in the lipidbilayer) is the III intracytoplas-mic loop. Black circles, thehydrogen bonds loosened by thecontact with the active receptor &/fig.c:
TSH activation of cAMP signaling
Multiple types of evidence indicate that the predominantsignalling pathway induced by TSH in the thyroid is me-diated by Gαs coupled to the activated receptor. Recentlyit has been reported that TSH stimulates the members ofall four G protein families (Gs, Gq/11, Gi, and G12) [28].At present, however, the functional implications of mul-tiple signalling pathways elicited by the activated TSHreceptor are not clear. cAMP, which is generated by ade-nylyl cyclase following the interaction with Gαs, appar-ently mimics most of the biological effects of TSH withrespect to growth and differentiation of thyroid cells.cAMP binds and activates the tetrameric enzyme cAMP-dependent protein kinase A (PKA). This enzyme isformed by two dimerized regulatory (R) and two catalyt-ic subunits (C). In eukaryotic cells multiple forms of reg-ulatory and catalytic subunits assemble together to gen-erate several PKA holoenzymes [29, 30]. The specificfunctions of these different types of PKA still remainelusive. There are two main forms of PKA. The twotypes of holoenzyme differ in the structure of the R sub-unit incorporated (RI or RII), but the C subunits are ei-ther identical or very similar. Type I is essentially a solu-
ble cytosolic enzyme, while type IIα is located mainly inthe cytoskeleton, and type IIβ is found in the Golgi, cen-trosome, and perinuclear area [31]. Type II PKA is an-chored to the membranes by binding of the regulatorysubunits to cellular proteins localized in different com-partments [32, 33]. Regulatory subunits I and II also dif-fer with respect to binding and dissociation affinities tocAMP and turnover. RI is very sensitive to cAMP (Kd1 mM, t1/2 31 h), while RII is less responsive to cAMP(Kd 10 mM, t1/2 125 h) [34]. These findings suggest thatPKAI and PKAII decode cAMP signals that differ intheir duration and intended target. It is likely that PKAIis transiently activated by weak cAMP signals whereasPKAII responds to high and persistent cAMP levels.
Nuclear responses to cAMP are dependent on specifictranscription factors activated by PKA. For example, fol-lowing phosphorylation of specific serine residues(mainly serine at position 133) the transcription factorCREB (cAMP-responsive element binding protein), be-comes active and stimulates the transcription of cAMP-induced promoters [35, 36]. cAMP, generated by TSHsignaling, dissociates the PKA holoenzyme. Regulatorysubunits release free C subunit, which migrates from thecytoplasm to the nucleus (Fig. 5) [37 38, 39], where itphosphorylates the transcription factor CREB. Phosphor-ylated CREB binds a nuclear adapter CREB-bindingprotein or a similar 300-kDa protein (p300) which bridg-es CREB to TFIIB and TBP, essential components of theRNA polymerase initiation transcription complex(Fig. 5) [40, 41]. The ultimate result is the stimulation oftranscription of cAMP-responsive promoters.
TSH also stimulates the expression of thyroid specificgenes: thyroglobulin, thyroperoxidase, and iodine carrier[13–15]. The molecular mechanism of the induction ofthese genes is not yet well established, but there are indi-cations that some of these effects are mediated by the ac-tivation of thyroid-transacting factors TTF1 and PAX8[42].
The mechanism by which TSH regulates the synthesisof its own receptor is of particular interest. TSH receptor
570
Fig. 5 cAMP signaling from the membrane to the nucleus in thy-roid follicular cells. This schematic diagram illustrates the trans-mission of cAMP stimulus from the membrane (TSH receptor ad-enylyl cyclase) to the nucleus. cAMP binds a regulatory subunit ofPKA (RIIβ), which is localized on the membranes of the Golgi ap-paratus and centrosome via an anchor protein (A). The enzymedissociates and free catalytic subunit (C) enters the nucleus, whereit phosphorylates the transcription factor CREB, which binds anadapter p300. p300 bridges CREB to the RNA polymerase initia-tion complex (TFIIB). TTF1, a specific thyroid transacting factor,is indicated in the phosphorylated form. It is not yet known wheth-er PKA directly phosphorylates TTF1 (-?-). p27 is a specific cy-clin kinase inhibitor which has been shown in macrophages to beinduced by cAMP [66]. Tg, thyroglobulin, which represents a pro-totype of cAMP-induced gene. The precise molecular mechanismgoverning Tg induction by cAMP is not yet known&/fig.c:
gene is activated by low cAMP levels [43] and TTF1[44]. High cAMP concentrations inhibit and downregu-late the accumulation of TSH receptor mRNA in rat thy-roid cells [43]. This effect probably involves a short-liferepressor activated by cAMP PKA. Downregulation ofTSH receptor by cAMP may desensitize the TSH path-way in the presence of high cAMP levels.
Recent evidence obtained from primary cultures ofdog thyroid cells indicates that TSH enhances the surviv-al of thyrocytes by preventing apoptosis [45]. It is un-clear whether this finding reflects a specific role of TSHin vivo or is due to the adjustment of the primary cells tothe in vitro conditions.
Somatic mutations of TSH receptor genein thyroid hyperfunctioning adenomas
Thyroid hyperfunctioning adenomas are clonal neo-plasms of thyroid gland characterized by autonomousclonal growth (TSH-independent), hypersecretion of thy-roid hormones, and TSH suppression [46]. This effect iscaused by the high serum levels of thyroid hormoneswhich inhibit TSH secretion. These adenomas constitute
a common cause of hyperthyroidism and are more fre-quent in endemic goiter areas [47]. On thyroid scansthese lesions appear as single hyperfunctioning (hot)nodules because they concentrate the radioactive elementto a greater extent than the resting paranodular and con-tralateral tissue deprived of its normal tonic TSH stimu-lation. These nodules are formed by preferential replica-tion of a single follicular cell [1, 2].
Mutations in the TSH receptor gene have been foundin thyroid-hyperfunctioning adenomas. The general fea-tures of these mutations are: (a) somatic, i.e., present on-ly in the tumor tissue and not in the surrounding normalgland, (b) dominant, i.e., only one copy of the gene is af-fected, and (c) constitutive activation of the cAMP sig-naling cascade. This has been shown by expressing themutated versions of the receptor in nonthyroid cells(COS7 or NIH3T3).
All identified mutations have been mapped to exon 10of the thyroid receptor gene. Exon 10 encodes the seven-loop-spanning segment and the COOH terminus of thereceptor. A complete catalogue of the mutations pub-lished to date is reported in Fig. 6.
The segments of the receptor affected are: (a) I and IIextracellular loops (residues 486 and 568) [48], (b) IIIintracellular loop (residues 619 and 623) [49], (c) VI in-tramembrane-spanning segment (residues 631, 632, and633) [50, 51]. Mutations in the I and II extracellularloops increase the affinity of TSH to the receptor, sug-gesting that the binding of TSH stabilizes an active con-formation of the receptor. Mutations in the III intracellu-lar loop and in the VI membrane-spanning segment donot affect the binding affinity of TSH [49 50, 51, 52]. Inthese cases the constitutive activation of the receptormay depend on conformational changes induced by the
571
Fig. 6 Localization of activating mutations in the TSH receptorgene. Black circles, the wild-type codons mutated in thyroid hy-perfunctioning adenomas; arrows, the substituted residues (whitecircles). These mutations are somatic and dominant (see text).Black triangles, the codons found mutated in rare cases of congen-ital hyperthyroidism; white triangles, the substituted amino acids.These mutations are constitutional and dominant. Note that thewhite circle inscribed in the white trianglerepresents the samemutation found in one hyperfunctioning adenoma and also in con-genital hyperthyroidism [51, 57]&/fig.c:
mutated residues. In vitro mutagenesis of receptors ofthe same family has revealed that the residues in the IIIintracellular loop and VI membrane-spanning segmentare uniquely suited to keep the receptor in an inactivestate [53]. Any change of the residues in these regionsresults in activation of the receptor [54]. This is also sug-gested by the analysis of the mutated residues in 631,632, and 633 positions in the TSH receptor [49–52].
These mutant forms of the receptor, expressed bytransfection in nonthyroid cells, constitutively activateadenylyl cyclase. The basal concentration of cAMP (inthe absence of the hormone) is higher in cells expressingall the mutants described above. Addition of TSH result-ed in a significant increase in cAMP levels. Differencesin the response to TSH may depend on the individual ex-perimental conditions. The general picture emergingfrom these studies is that these mutations constitutivelyactivate cAMP signaling (adenylyl cyclase and transcrip-tion of cAMP-induced genes), and do not change the af-finity of the receptor for the TSH.
TSH is also known to activate inositol phosphate hy-drolysis via phospholipase C [55]. The mutants in thesegments b and c fail to increase basal or induced inosi-tol phosphate hydrolysis [49, 52]. Mutants in the seg-ment a, however, have been shown to also activate theinositol phosphate-Ca2+ cascade [49] (see Fig. 6). Thesedata suggest that the normal configuration of the I and IIextracellular loop contributes to the silencing of the emp-ty TSH receptor and point to possible phenotypic differ-ences between mutants b and c versus the mutants insegment a.
Recently we have expressed the type c mutated recep-tors in thyroid cells; interestingly, some mutations result-ed in TSH-independent growth and some others did notchange the pattern of the TSH-dependent proliferation(A.P, in preparation).
Germ-line mutations in the TSH receptor gene
Constitutive activation
TSH receptor mutations have been found in cases of con-genital nonautoimmune hyperthyroidism. The mutationsare constitutional and are present in only one allele [56].The segments of the receptor affected are all located inthe membrane-spanning domains: the III [56]; VI [57]and VII segments [56] (see Fig. 6). Note that somaticmutations in thyroid-hyperfunctioning adenomas alsofrequently map in the VI transmembrane domain (com-pare black and white circles in Fig. 6). Cloning and ex-pression of these mutants in nonthyroid cells resulted inhigher constitutive activation of adenylyl cyclase thanthe wild-type receptor. Inositol metabolism was not af-fected. TSH affinity to the receptor was the same in themutants and the wild-type receptors [56]. The biochemi-cal properties of these mutants are indistinguishablefrom those of somatic mutants found in hyperfunctioningthyroid adenomas. It is worth noting that the age of diag-
nosis and the severity of the symptoms are highly vari-able [56, 57]. Although these mutations are inherited in adominant fashion, the genetic background and environ-mental factors may influence the severity of the clinicalphenotype.
Inactivation
TSH receptor mutations leading to the loss of its biologi-cal activity are inherited as recessive traits. Three siblingshave been described who presented normal serum con-centrations of thyroid hormones and high concentrationsof TSH (20-fold). They had mutations in both alleles ofthe receptor gene, one inherited from each parent. Themutant gene from the father had no activity while the ma-ternal gene had reduced biological activity. These muta-tions have been mapped in the extracellular domain of thereceptor (Fig. 7). Definite evidence on the molecularmechanism leading to the loss of biological activity ofthese receptors has not been documented yet [58]. Cellsexpressing the maternal and paternal mutated receptorsrequired approximately 20 times more TSH to producethe level of activity observed in cells transfected withwild-type receptor [58]. One possible mechanism to ex-plain the high TSH serum levels found in these patients isthe reduced affinity of these receptors for TSH. A definiteanswer to this question will be produced by the directmeasurement of TSH affinity to the mutated receptor.
572
Fig. 7 Mutations in the TSH receptor gene leading to inactivationof the receptor. Localization of two constitutional mutations lead-ing to partial TSH resistance (see text). Black circles, the wild-type residues with the corresponding substitutions (arrows)
A recessive mutation in the TSH receptor gene hasbeen described in hypothyroid mice (hyt). The mutationis localized in the IV transmembrane domain and inhibitshormone binding [59]. This finding suggests that somesegments of the transmembrane domains control thebinding of TSH to the receptor. It is necessary thereforeto carefully reanalyze TSH binding affinity of the mutat-ed a, b, and c receptors (see Fig. 6).
Clinical implications
The discovery of TSH receptor mutations will changeour understanding of the pathogenesis of several thyroiddiseases. For example, other somatic mutations affectingthe extracellular, TSH-binding region of the receptormight have a role in common causes of thyroid dysfunc-tion, such as chronic autoimmune thyroiditis and Graves’disease. The TSH receptor is a well-recognized antigenthat interacts with stimulatory (Graves’ disease) andblocking (autoimmune hypothyroidism) autoantibodies[1, 2, 60]. A somatic mutation in the receptor gene in athyroid follicular cell could make the receptor antigenic,leading to the production of different antibodies whichcan block or stimulate the mutated and normal receptor.Recently a substitution of threonine for proline at posi-tion 52 has been found in a patient with autoimmune hy-perthyroidism [61]. The same mutant receptor has beenshown to increase TSH-stimulated cAMP accumulationwhen expressed in nonthyroid cells [62]. However, thebiological significance of these findings is unclear, andevidence for the involvement of the TSH receptor muta-tions in Graves’ disease is equivocal [63, 64].
The most frequent mutations described in thyroid-hy-perfunctioning adenomas have been detected in the RNAextracted from fine needle aspiration biopsy of the nod-ules [50]. Since these changes in the nucleotide sequenceof the gene lead to the loss or a gain of a restriction site,they can be easily screened and detected by a restrictionanalysis using specific enzymes. The possibility to rec-ognize the various TSH receptor mutants by restrictionenzyme analysis of FNAB specimens may ultimatelylead to the recognition of new mutations and to the earlydiagnosis of thyroid-hyperfunctioning adenomas.
An important question still unanswered concerns thecause(s) of these somatic mutations. A marked increasein autonomous hyperfunctioning nodules has beenreported in human iodine-deficient goitres, which isassociated with initial high TSH plasma levels [47]. It isalso known that in the larger nodules there is a higherhormone production leading to progressive and severehyperthyroidism [65]. These clinical observations sug-gest that TSH chronic stimulation might positively selectcells with activating mutations of the receptor. Thesecells are extremely sensitive to TSH and have a specificgrowth advantage. Other growth factors (insulin-likegrowth factor I and epidermal growth factor) can also in-duce growth of thyroid cells and may contribute to theinitial polyclonal expansion of follicular cells. We sug-
gest that the growth stimulus elicited by TSH amplifiesand facilitates clonal expansion of cells bearing activat-ing mutations of the TSH receptor. A recent finding fromour laboratory that 90% of the 632 mutant originates inmultinodular goiters is consistent with this idea (A.P. etal., in preparation).
We anticipate that more mutations of the TSH recep-tor gene will be found in the near future, providing great-er insights into how TSH works and explaining the causeof various thyroid diseases.
&p.2:AcknowledgementsThe author’s own research reported here hasbeen supported by grants from EC, AIRC (Associazione ItalianaRicerca Cancro), C.N.R., Progetti Finalizzati “Ingegneria Genet-ica,” and “ACRO.” We apologize for omitting relevant primary ref-erences due to space constraints.
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