TSH Receptor Signaling Research Articles

TSH induces metallothionein 1 in thyrocytes via Gq/11- and PKC-dependent signaling

Metallothioneins (MTs) are cytoprotective proteins acting as scavengers of toxic metal ions or reactive oxygen species. MTs are upregulated in follicular thyroid carcinoma and are regarded as a marker of thyroid stress in Graves' disease. However, the mechanism of MT regulation in thyrocytes is still elusive. In other cellular systems, cAMP-, calcium-, or protein kinase C (PKC)-dependent signaling cascades have been shown to induce MT expression. Of note, all of these three pathways are activated following the stimulation of the TSH receptor (TSHR). Thus, we hypothesized that TSH represents a key regulator of MT expression in thyrocytes. In fact, TSHR stimulation induced expression of MT isoform 1X (MT1X) in human follicular carcinoma cells. In these cells, Induction of MT1X expression critically relied on intact Gq/11 signaling of the TSHR and was blocked by chelation of intracellular calcium and inhibition of PKC. TSHR-independent stimulation of cAMP formation by treating cells with forskolin also led to an upregulation of MT1X, which was completely dependent on PKA. However, inhibition of PKA did not affect the regulation of MT1X by TSH. As in follicular thyroid carcinoma cells, TSH also induced MT1 protein in primary human thyrocytes, which was PKC dependent as well. In summary, these findings indicate that TSH stimulation induces MT1X expression via Gq/11 and PKC, whereas cAMP-PKA signaling does not play a predominant role. To date, little has been known regarding cAMP-independent effects of TSHR signaling. Our findings extend the knowledge about the PKC-mediated functions of the TSHR.

Expression of Thyroid Stimulating Hormone Receptor mRNA in Mouse C2C12 Skeletal Muscle Cells

BackgroundWe analyzed whether thyroid stimulating hormone receptor (TSH-R) is expressed in a skeletal muscle cell line and if TSH has influence on the differentiation of muscle cells or on the determination of muscle fiber types.MethodsTSH-R gene expression was detected with nested real-time polymerase chain reaction (RT-PCR) in C2C12, a mouse skeletal muscle cell line. The effect of TSH on myotube differentiation was assessed by microscopic examination of myotube formation and through the measurement of expression of muscle differentiation markers, i.e., myogenin and myoD, and muscle type-specific genes, i.e., MyHC1, MyHC2a, and MyHC2b, with quantitative RT-PCR before and after incubation of C2C12 myotube with TSH.ResultsTSH-R was expressed in the mouse skeletal muscle cell line. However, treatment with TSH had little effect on the differentiation of muscle cells, although the expression of the muscle differention marker myogenin was significantly increased after TSH treatment. Treatment of TSH did not affect the expression of muscle type-specific genes.ConclusionTSH-R is expressed in a mouse skeletal muscle cell line, but the role of TSH receptor signaling in skeletal muscle needs further investigation.

PDGF Enhances Orbital Fibroblast Responses to TSHR Stimulating Autoantibodies in Graves' Ophthalmopathy Patients

Thyroid-stimulating hormone receptor (TSHR) stimulating autoantibodies are associated with Graves' ophthalmopathy (GO), the orbital manifestation of Graves' disease (GD). TSHR autoantibody levels and orbital TSHR expression levels correlate positively with GO disease activity. Platelet-derived growth factors (PDGF) are increased in GO and potently activate orbital fibroblast effector functions. We investigated the possible relationship between PDGF and TSHR expression on orbital fibroblasts and how that influences the immunopathological effects of TSHR autoantibodies on orbital fibroblast activity. Orbital fibroblasts were stimulated with PDGF-AA, PDGF-AB, and PDGF-BB, and TSHR expression was determined by flow cytometry. Stimulatory effects of bovine TSH and GD immunoglobulins on orbital fibroblasts (with or without PDGF-BB preincubation) were determined by IL-6, IL-8, chemokine (C-C motif) ligand (CCL)-2, CCL5, CCL7, and hyaluronan ELISA. The TSHR blocking antibody K1-70 and the cAMP inhibitor H89 were used to determine involvement of TSHR signaling. PDGF-AB and PDGF-BB stimulation increased TSHR expression on orbital fibroblasts, whereas PDGF-AA did not. Furthermore, stimulation with bovine TSH and immunoglobulins from GD patients induced IL-6, IL-8, CCL2, and hyaluronan production by orbital fibroblasts, and PDGF-BB preincubation enhanced this response of orbital fibroblasts. Blocking studies with a TSHR blocking antibody and a cAMP inhibitor inhibited these effects, indicating the involvement of TSHR signaling and thus of TSHR stimulating autoantibodies herein. These findings indicate that PDGF-B containing PDGF isoforms amplify the immunopathological effects of TSHR-stimulating autoantibodies in GO patients by stimulating TSHR expression on orbital fibroblasts.

Monogenic Thyroid Disorder

Monogenic disorder is a single gene disorder resulted of a single mutated gene. Monogenic disorder has benefits in early diagnosis and precious prediction of disease course. Furthermore, monogenic disorder could provide an informative knowledge to the understanding of related pathophysiology. Thyroid monogenic disorder could occur in various steps, such as thyroid development, hormonogenesis, TSH-receptor signaling, thyroid hormone transport and end organ response. Here, we reviewed of congenital hypothyroidism, congenital hyperthyroidism and thyroid hormone resistance syndrome.

New Pathogenic Thyrotropin Receptor Mutations Decipher Differentiated Activity Switching at a Conserved Helix 6 Motif of Family A GPCR

In this paper we report two new TSH receptor (TSHR) mutations. One mutation (Pro639(6.50)Leu) was identified in two siblings with congenital hypothyroidism, and a second mutation (Cys636(6.47)Arg) was found in a patient suffering from nonautoimmune hyperthyroidism. Both mutations are located in transmembrane helix (TMH) 6 at the conserved Cys(6.47)-Trp(Met)(6.48)-Leu(Ala)(6.49)-Pro(6.50) motif of family A G protein-coupled receptors (GPCR). To study the pathogenic mechanisms, we tested patients' mutations and further side chain variations regarding their effects on TSHR signaling. Substitution Pro639Leu fully inactivates the promiscuous TSHR for cAMP (Gs) and IP (Gq) signaling. In contrast, Cys636Arg leads to constitutive activation of Gs. Organization of TSHR in oligomers was not modified by mutations at position 636. Interestingly, it is known from crystal structures of GPCR that Pro(6.50) is located at a TMH6 kink-distortion, which is a pivot during activation-related helical movements. However, the cell surface expressions of all mutants at position 639 were comparable to wild type, indicating a helical conformation like wild type. Until now, only naturally occurring constitutively activating mutations in TSHR TMH6 have been reported, but here we present the first pathogenic inactivating mutation (Pro639Leu). Our data are indicative of differentiated regulation of Gs and Gq signaling at particular TMH6 positions, but without any effects on TSHR oligomer constellation. Details of signaling modulation by each mutant at positions 636(6.47) and 639(6.50) help us to understand high conservation of these amino acids in family A GPCR. Described molecular (pathogenic) mechanisms are likely not unique for TSHR.

Comparative proteomic analysis to dissect differences in signal transduction in activating TSH receptor mutations in the thyroid

In the thyroid, cAMP controls both thyroid growth and function. Gain-of-function mutations in the thyroid-stimulating hormone receptor (TSHR) lead to constitutive cAMP formation and are a major cause of autonomous thyroid adenomas. The impact of activating TSHR mutations on the signal transduction network of the thyrocyte is not fully understood. To gain more insights into constitutive TSHR signaling, rat thyrocytes (FRTL-5 cells) with stable expression of three activating TSHR mutants (mutTSHR: A623I, L629F and Del613-621), which differ in their functional characteristics in vitro, were analyzed by a quantitative proteomic approach and compared to the wild-type TSHR (WT-TSHR). This study revealed (1) differences in the expression of Rab proteins suggesting an increased TSHR internalization in mutTSHR but not in the WT-TSHR; (2) differential stimulation of PI3K/Akt signaling in mutTSHR vs. WT-TSHR cells, (3) activation of Epac, impairing short-time Akt phosphorylation in both, mutTSHR and WT-TSHR cells. Based on the analysis of global changes in protein expression patterns, our findings underline the complexity of gain-of-function TSHR signaling in thyrocytes, which extends beyond pure cAMP and/or IP formation. Moreover, evidence for augmented endocytosis in the mutTSHR, adds to a new concept of TSHR signaling in thyroid autonomy. Further studies are required to clarify whether the observed differences in Rab, PI3K and Epac signaling may contribute to differences in the phenotypic presentation, i.e. stimulation of function and growth of thyroid autonomy in vivo.

Small-Molecule Thyrotropin Receptor Agonist Activates Naturally Occurring Thyrotropin-Insensitive Mutants and Reveals Their Distinct Cyclic Adenosine Monophosphate Signal Persistence

Subclinical hypothyroidism (SHT), characterized by normal thyroid hormone levels maintained by elevated thyrotropin (TSH), predisposes patients to health problems as they age. Some cases arise from mutations of the TSH receptor (TSHR) that confer TSH resistance. This resistance might be circumvented by TSHR agonists with different modes of binding compared with TSH. We hypothesized that the recently discovered small-molecule TSHR agonist C2, with its unique mode of receptor binding, would activate mutant TSHRs associated with SHT, facilitating their study. HEK-EM293 cells transiently expressing TSHR variants-wild-type TSHR or mutants C41S, L252P, L467P, or C600R-were analyzed for TSH or C2-induced cyclic adenosine monophosphate (cAMP) signaling to establish C2 as a mutant TSHR agonist. These cells were also pretreated with TSH or C2 to characterize each mutant receptor's ability to maintain and desensitize cAMP signaling. We showed that C2 could activate the TSH-unresponsive TSHR ectodomain mutants C41S and L252P but had no effect on the serpentine mutant L467P. We found that TSH and C2 could acutely activate the serpentine mutant C600R. Preincubation with C2 caused persistent cAMP signaling and receptor desensitization in wild-type TSHR and cAMP signal persistence with no detectable desensitization in the cases of C41S and L252P. The small-molecule agonist C2 is a useful pharmacological tool for the study of mutant TSHRs. It revealed that some naturally occurring TSH-insensitive mutants can mediate induction of cAMP elevation upon stimulation with C2 and that this signal is differentially maintained within cells.

TSH Receptor Function Is Required for Normal Thyroid Differentiation in Zebrafish

TSH is the primary physiological regulator of thyroid gland function. The effects of TSH on thyroid cells are mediated via activation of its membrane receptor [TSH receptor (TSHR)]. In this study, we examined functional thyroid differentiation in zebrafish and characterized the role of TSHR signaling during thyroid organogenesis. Cloning of a cDNA encoding zebrafish Tshr showed conservation of primary structure and functional properties between zebrafish and mammalian TSHR. In situ hybridization confirmed that the thyroid is the major site of tshr expression during zebrafish development. In addition, we identified tpo, iyd, duox, and duoxa as novel thyroid differentiation markers in zebrafish. Temporal analyses of differentiation marker expression demonstrated the induction of an early thyroid differentiation program along with thyroid budding, followed by a delayed onset of duox and duoxa expression coincident with thyroid hormone synthesis. Furthermore, comparative analyses in mouse and zebrafish revealed for the first time a thyroid-enriched expression of cell death regulators of the B-cell lymphoma 2 family during early thyroid morphogenesis. Knockdown of tshr function by morpholino microinjection into embryos did not affect early thyroid morphogenesis but caused defects in later functional differentiation. The thyroid phenotype observed in tshr morphants at later stages comprised a reduction in number and size of functional follicles, down-regulation of differentiation markers, as well as reduced thyroid transcription factor expression. A comparison of our results with phenotypes observed in mouse models of defective TSHR and cAMP signaling highlights the value of zebrafish as a model to enhance the understanding of functional differentiation in the vertebrate thyroid.

A New Small-Molecule Antagonist Inhibits Graves' Disease Antibody Activation of the TSH Receptor

Graves' disease (GD) is caused by persistent, unregulated stimulation of thyrocytes by thyroid-stimulating antibodies (TSAbs) that activate the TSH receptor (TSHR). We previously reported the first small-molecule antagonist of human TSHR and showed that it inhibited receptor signaling stimulated by sera from four patients with GD. Our objective was to develop a better TSHR antagonist and use it to determine whether inhibition of TSAb activation of TSHR is a general phenomenon. We aimed to chemically modify a previously reported small-molecule TSHR ligand to develop a better antagonist and determine whether it inhibits TSHR signaling by 30 GD sera. TSHR signaling was measured in two in vitro systems: model HEK-EM293 cells stably overexpressing human TSHRs and primary cultures of human thyrocytes. TSHR signaling was measured as cAMP production and by effects on thyroid peroxidase mRNA. We tested analogs of a previously reported small-molecule TSHR inverse agonist and selected the best NCGC00229600 for further study. In the model system, NCGC00229600 inhibited basal and TSH-stimulated cAMP production. NCGC00229600 inhibition of TSH signaling was competitive even though it did not compete for TSH binding; that is, NCGC00229600 is an allosteric inverse agonist. NCGC00229600 inhibited cAMP production by 39 ± 2.6% by all 30 GD sera tested. In primary cultures of human thyrocytes, NCGC00229600 inhibited TSHR-mediated basal and GD sera up-regulation of thyroperoxidase mRNA levels by 65 ± 2.0%. NCGC00229600, a small-molecule allosteric inverse agonist of TSHR, is a general antagonist of TSH receptor activation by TSAbs in GD patient sera.

Subunit Interactions Influence TSHR Multimerization

The TSH receptor (TSHR) is the key molecule influencing thyroid growth and development and is an antigenic target in autoimmune thyroid disease. The TSHR exists in monomeric and multimeric forms, and it has been shown previously that multimeric complexes of the TSHR preferentially localize in lipid rafts. However, unlike other glycoprotein hormone receptors, the TSHR exists in several forms on the cell membrane due to intramolecular cleavage of its ectodomain, which causes the production of α- and β-subunits of various lengths. After cleavage and reduction of disulfide bonds, α-subunits consisting of the receptor ectodomain may be lost from the cell surface by receptor shedding, leading to accumulation of excess β-subunits within the membrane. Because cell surface expression of these various forms of the TSHR is critical to receptor signaling and autoimmune responses, we set out to model the influence of β-subunits on full-length TSHRs. To study this interaction, we generated three truncated ectodomain β-subunits linked to green fluorescent protein (named β-316, -366, and -409) as examples of native cleaved forms of the TSHR. These constructs were transfected into human embryonic kidney 293 cells in the presence and absence of the full-length receptor. Whereas the β-316 and β-366 forms showed cell surface expression, the expression of β-409 was primarily intracellular. Cotransfection of the β-subunits with a full-length hemagglutinin-tagged wild-type (WT) receptor (HT-WT-TSHR) in both transient and stable systems caused a significant decrease in surface expression of the full-length WT receptors. This decrease was not seen with control plasmid consisting of a plasma membrane-targeted protein tagged to red fluorescent protein. To ascertain if this response was due to homointeraction of the truncated β-constructs with the WT-TSHRs, we immunoprecipitated membranes prepared from the cotransfected cells using antihemagglutinin and then probed with anti-green fluorescent protein. These studies confirmed dimerization of the β-subunits with the WT full-length receptor, and this interaction was further observed in vivo by fluorescence resonance energy transfer. We then studied the functional consequences of this interaction on TSHR signaling by examining Gαs-mediated signals. The well-expressed truncated constructs, when coexpressed with full-length TSHR, did not alter constitutive cAMP levels, but there was a significant decrease in TSH-induced cAMP generation. Furthermore, we observed that truncated β-316 and β-366 had faster internalization rate, which may lead to a significant decrease in the expression of the full-length receptor on the cell surface, thus contributing to the decreased signaling response. However, the decrease in surface receptors may also be due to inhibition of newly formed receptors reaching the surface as result of receptor-receptor interaction. It is well known that under normal physiological conditions both cleaved and uncleaved TSHR forms coexist on the cell surface of normal thyrocytes. Our studies allow us to conclude, therefore, that multimerization of cleaved/ truncated forms of the β-subunits with the full-length TSHR has a profound influence on TSHR internalization and signaling. Hence, the degree of intramolecular cleavage must also modulate TSHR signaling.

Persistent cAMP signaling by thyrotropin (TSH) receptors is not dependent on internalization

Evidence was presented that thyrotropin [thyroid-stimulating hormone (TSH)]-stimulated persistent cAMP signaling is dependent on receptor (with G-protein α subunits and adenylyl cyclase) internalization. Because it is not clear whether G proteins and adenylyl cyclase internalize with receptors, we tested whether persistent cAMP signaling by TSH receptor (TSHR) is dependent on internalization. We measured persistent TSHR signaling as an accumulation of cAMP in HEK-EM293 cells permanently expressing human TSHRs incubated with isobutylmethylxanthine for 30 min after washing the cells to remove unbound TSH, and TSHR internalization by fluorescence microscopy using Alexa-tagged TSH and binding assays using (125)I-TSH. TSHRs, but not the closely related lutropin or follitropin receptors, exhibit persistent cAMP signaling. TSHRs were not internalized by 30 min incubation with unlabeled TSH; however, expression of β-arrestin-2 promoted TSHR internalization that was inhibited by dynasore, a dynamin inhibitor. Expression of β-arrestin-2 had no effect on TSHR cAMP signaling, dynasore inhibited TSHR cAMP signaling in the absence or presence of TSHR internalization, and expression of a dominant-negative mutant dynamin, which inhibited internalization, had no effect on persistent cAMP signaling. Persistent cAMP signaling was specifically inhibited by a small molecule TSHR antagonist. We conclude that TSHRs do not have to be internalized to exhibit persistent cAMP signaling.

Thyrotropin (TSH) Receptor Residue E251 in the Extracellular Leucine-Rich Repeat Domain Is Critical for Linking TSH Binding to Receptor Activation

The TSH receptor (TSHR) ectodomain comprises a tubular leucine-rich repeat domain (LRD) and a hinge [or signaling specificity domain (SSD)]. TSH binds to both the LRD and SSD, leading to signal transduction by the transmembrane domain. The SSD structure and spatial orientation to the other components are unknown. We exploited a fortuitous observation to obtain mechanistic insight into the relationship between TSH binding and signal transduction. A mouse TSHR cDNA generated by PCR was found to express a receptor with poor TSH-induced cAMP generation despite normal TSH binding. Progressive reversion to wild-type of six mutations revealed E251K in the LRD to be critical for reduced signal transduction in both mouse and human TSHR. An I286F substitution in the SSD had a much weaker effect and was additive with E251K. To our knowledge, there are no previous examples of specific amino acid mutations in the TSHR LRD that dissociate TSH binding from TSHR signal transduction. To prevent flailing of the TSHR LRD, its position vis-à-vis the SSD must be stabilized by multiple amino acid interactions. The present data suggest that TSHR residue E251 is one of these residues involved in stabilizing the LRD relative to the SSD, thereby enabling ligand binding to transduce a signal by the latter. That the E251K mutation can reduce signal transduction despite high-affinity TSH binding comparable with the wild-type TSHR provides mechanistic insight into the coupling between ligand binding and receptor activation.

Activation of Dual Oxidases Duox1 and Duox2

Dual oxidases were initially identified as NADPH oxidases producing H(2)O(2) necessary for thyroid hormone biosynthesis. The crucial role of Duox2 has been demonstrated in patients suffering from partial iodide organification defect caused by bi-allelic mutations in the DUOX2 gene. However, the Duox1 function in thyroid remains elusive. We optimized a functional assay by co-expressing Duox1 or Duox2 with their respective maturation factors, DuoxA1 and DuoxA2, to compare their intrinsic enzymatic activities under stimulation of the major signaling pathways active in the thyroid in relation to their membrane expression. We showed that basal activity of both Duox isoenzymes depends on calcium and functional EF-hand motifs. However, the two oxidases are differentially regulated by activation of intracellular signaling cascades. Duox1 but not Duox2 activity is stimulated by forskolin (EC(50) = 0.1 microm) via protein kinase A-mediated Duox1 phosphorylation on serine 955. In contrast, phorbol esters induce Duox2 phosphorylation via protein kinase C activation associated with high H(2)O(2) generation (phorbol 12-myristate 13-acetate EC(50) = 0.8 nm). These results were confirmed in human thyroid cells, suggesting that Duox1 is also involved in thyroid hormonogenesis. Our data provide, for the first time, detailed insights into the mechanisms controlling the activation of Duox1-2 proteins and reveal additional phosphorylation-mediated regulation.

Antibody-induced modulation of TSH receptor post-translational processing

The post-translational processing of the TSH receptor (TSHR) includes intra-molecular cleavage with the loss of a 50 amino acid ectodomain region and the formation of two subunits (alpha and beta), followed by likely alpha subunit shedding. TSHR antibodies (TSHR-Abs), which are directed at the ectodomain, may influence thyroid function by stimulating or inhibiting TSHR signaling or may bind without any such influence (the neutral group of antibodies). When we examined the characteristics of a series of monoclonal TSHR-Abs, we found that many were able to inhibit receptor cleavage and enhance TSHR expression. This was especially apparent with the neutral type of TSHR-Abs directed to the cleaved region of the ectodomain (aa 316-366). Indeed, such inhibition appeared to be epitope dependent with TSHR-Abs directed to regions after residues 335-354 showing no such activity. We propose that this aberrant process, whereby TSHR-Abs influence antigen processing, is a novel mechanism for the maintenance and exacerbation of autoimmune thyroid disease.

Lipid Rafts Are Triage Centers for Multimeric and Monomeric Thyrotropin Receptor Regulation

The TSH receptor (TSHR), a heptahelical G protein-coupled receptor on the surface of thyrocytes, is a major autoantigen and physiological regulator of the thyroid gland. Unlike other G protein-coupled receptors, the TSHR undergoes posttranslational cleavage of its ectodomain, leading to the existence of several forms of the receptor on the plasma membrane. We previously hypothesized that to achieve high fidelity and specificity of TSH ligand or TSHR autoantibody signaling, the TSHR may compartmentalize into microdomains within the plasma membrane. In support of this hypothesis we have shown previously that TSHRs reside in GM1 ganglioside-enriched lipid rafts in the plasma membrane of TSHR-expressing cells. In this study, we further explored the different forms of TSHRs that reside in lipid rafts. We studied both TSHR-transfected cells and rat thyrocytes, using both nondetergent biochemical analyses and receptor-lipid raft colocalization. Using the biochemical approach, we observed that monomeric receptors existed in both raft and nonraft fractions of the cell surface in the steady state. We also demonstrated that the multimeric forms of the receptor were preferentially partitioned into the lipid microdomains. Different TSHR forms, including multimers, were dynamically regulated both by receptor-specific and postreceptor-specific modulators. TSH ligand and TSHR antibody of the stimulating variety induced a decrease of multimeric forms in the raft fractions. In addition, multimeric and monomeric forms of the receptor were both associated with Gsalpha within and without the rafts. Although failure to achieve total lipid raft disruption prevented a conclusion regarding the relative power of TSHR signaling within and without the raft domains, these data showed clearly that not only were a significant proportion of TSHRs residing within lipid microdomains but that constitutive multimerization of TSHRs was actually regulated within the lipid rafts.

Structural determinants for G-protein activation and specificity in the third intracellular loop of the thyroid-stimulating hormone receptor

The selectivity of G-protein recognition is determined by the intracellular loops (ICLs) of seven-transmembrane-spanning receptors. In a previous study, we have shown that the N-terminal and central portions of ICL2 from F525 to D530 participate in dual Galphas-/Galphaq-protein activation by the thyroid-stimulating hormone receptor (TSHR). ICL3 is another major determinant for G-protein activation. Therefore, the aim of our study was to identify important amino acids within ICL3 of the TSHR to gain insight in more detail about its specific function for Galphas- and Galphaq-protein activation and selectivity. Single-alanine substitutions of residues in the N-terminal, middle, and C-terminal region of ICL3 were generated. N-terminal residues Y605 and V608 and C-terminal positions K618, K621, and I622 were identified as selectively important for Galphaq activation, whereas mutations in the center of ICL3 had no effect on TSHR signaling. Our findings provide evidence for an amino acid pattern in the N- and C-terminal part of ICL3, which is involved in Galphaq-mediated signaling. Furthermore, molecular modeling of interaction of TSHR ICL2 and 3 with Galphaq suggests three potential contact sites: TSHR C-terminal ICL3 with beta5-6 loop of Galphaq, TSHR ICL2 residues I523-R531 with beta2-3 loop and N-terminal helix of Galphaq, and TSHR ICL2/transmembrane helix (TMH) 3+ICL3/TMH6 with C-terminal tail of Galphaq.

A hydrophobic cluster in the center of the third extracellular loop is important for TSH receptor signaling

Objectives: Previous reports on the follicle stimulating hormone receptor (FSHR) and choriogonadotrophic/luteinizing hormone receptor (CG/LHR), which belong to the glycoproteinhormone receptor (GPHR) family, suggest that the extracellular loop (ECL) 3 could be a key domain for ligand binding and intramolecular receptor signaling. In contrast to ECLs 1 and 2 of GPHRs, the ECL3 displays high sequence homology, particularly in the central portion of the loop. Therefore, we opted to identify amino acids with functional importance within ECL3 of the thyroid stimulating hormone receptor (TSHR).

A Hydrophobic Cluster in the Center of the Third Extracellular Loop Is Important for Thyrotropin Receptor Signaling

Previous reports on the follicle-stimulating hormone receptor and choriogonadotropic/LH receptor, which belong to the glycoprotein hormone receptor family, suggest that the extracellular loop (ECL) 3 could be a key domain for ligand binding and intramolecular receptor signaling. In contrast to ECLs 1 and 2 of glycoprotein hormone receptors, the ECL3 displays high sequence homology, particularly in the central portion of the loop. Therefore, we opted to identify amino acids with functional importance within ECL3 of the TSH receptor (TSHR). Single alanine substitutions of all residues in ECL3 were generated. Functional characterization revealed the importance of five amino acids in the central portion of ECL3 and K660 at the ECL3/transmembrane helix (TMH) 7 junction for TSHR signaling. Decrease of G(s) activation and loss of G(q) activation by substitutions of K660 demonstrates a role for this position for TSHR conformation and signal transduction. By molecular modeling, we predicted potential interaction partners of K660:E409 and D410 in the N terminus of TMH1 and D573 in the ECL2. Complementary double mutants did not reconstitute G(s)/G(q)-mediated signaling, suggesting that K660 is not directly involved in a structural unit between ECL3 and the N terminus of TMH1. These results support a TSHR model in which the side chain of K660 is orientated toward the backbone of ECL2. Moreover, our findings provide evidence that a hydrophobic cluster, comprising residues 652-656 of ECL3, strongly influences intramolecular signal transduction and G protein activation of the TSHR.

Thyroid‐stimulating hormone receptor levels and binding affinity in the thyroid gland of growth‐retarded mice

Growth-retarded (grt/grt) mice are congenitally primary hypothyroid. Our previous study indicated that thyroid-stimulating hormone (TSH) responsiveness was defective in the grt/grt thyroid gland. We now report additional studies of impaired grt/grt thyroid function. Semiquantitative RT-PCR confirmed that TSH receptor (TSHR) mRNA expression in the grt/grt thyroid was significantly decreased compared with +/+ thyroids. Scatchard analysis revealed that grt/grt and +/+ mice have only one type of TSH binding site. grt/grt thyroids had fewer TSH binding sites, although this did not apparently affect the affinity of TSH for its receptor. The present data suggest that reduced TSHR levels or defects in TSHR signaling could be one of the possible defective sites in the grt/grt thyroid gland.

Thyrotropin (TSH)-Induced Production of Vascular Endothelial Growth Factor in Thyroid Cancer Cellsin Vitro: Evaluation of TSH Signal Transduction and of Angiogenesis-Stimulating Growth Factors

Thyroid tumor growth requires angiogenesis, and vascular endothelial growth factor (VEGF) has been shown to be the most important endothelial mitogen. TSH is the major thyrotropic hormone, but its impact to modulate VEGF production has not yet been studied. Several other growth factors have also been shown to affect thyroid cancer cell growth and function in vitro. Therefore, the aim of the current study was to 1) establish the effect of TSH on VEGF as well as 2) evaluate the TSH signal transduction of this effect, and 3) screen other growth factors for the ability to modulate VEGF in thyroid cancer cell lines. HTC, a follicular cancer cell line lacking endogenous TSH receptor (TSHr), its receptor positive variant (HTC TSHr), and a cell line of Huerthle cell origin (XTC) were used. After stimulation with growth factors in vitro [TSH; epidermal growth factor (EGF), IGF, placenta growth factor, TGF-alpha, TGF-beta1, fibroblast growth factor, platelet-derived growth factor, and hepatocyte growth factor] cells were analyzed for VEGF gene expression by Northern blotting and for VEGF protein by enzyme immunoassay. TSHr signal transduction was evaluated by analyzing the effect of stimulators (cholera toxin, 8-bromo-cAMP, forskolin, and 12-O-tetradecanoyl-phorbol-13-acetate) and inhibitors (2',5'-dideoxyadenosine and staurosporine) on VEGF protein levels under basal and TSH-stimulated conditions. TSH increased VEGF mRNA and protein in a dose-dependent manner in HTC TSHr and XTC cells by up to 40%. The effects of TSH were mediated by protein kinase C (PKC), rather than protein kinase A (PKA), stimulation, because inhibition of PKC by staurosporine resulted in a decrease in VEGF production of up to 65%, whereas inhibition of the PKA signal transduction pathway (2',5'-dideoxyadenosine) resulted in only a minor decrease. TSH was not the most powerful stimulator of VEGF production. TGF-beta1 and EGF were 1.5- to 2-fold more potent. Placenta growth factor and TGF-alpha did not induce VEGF production in TSHr-positive HTC cells, whereas they did induce VEGF production in TSHr-negative HTC cells. In thyroid cancer cell lines, TSH induces VEGF production involving the PKC, rather than the PKA, pathway. However, EGF and TGF-beta increase the capacity of thyroid cancer cells to provide VEGF more effectively than TSH. In the absence of a functioning TSHr, additional growth factors, such as TGF-alpha, increase capacity for VEGF stimulation.