Ralf Paschke

The Thyrotropin Receptor in Thyroid Diseases

The growth and function of the thyroid are controlled by thyrotropin1 through the activation of its receptor, which belongs to the large family of G protein–coupled receptors. Despite the extreme d...

Current evidence for and against the TSH receptor being the common antigen in Graves' disease and thyroid associated ophthalmopathy

Vassart, 1990). In particular, in Graves’ disease it seems clear that the pathogenesis is adequately explained by the presence of autoantibodies to the TSH receptor (TSHR) which mimic TSH by inhibiting hormone binding (TBII) and/or stimulating CAMP accumulation (TSAB) leading to hyperthyroidism and goitre. The fact that neonates, born to mothers with Graves’ disease, experience transient hyperthyroidism

A Low Molecular Weight Agonist Signals by Binding to the Transmembrane Domain of Thyroid-stimulating Hormone Receptor (TSHR) and Luteinizing Hormone/Chorionic Gonadotropin Receptor (LHCGR)

Many cognate low molecular weight (LMW) agonists bind to seven transmembrane-spanning receptors within their transmembrane helices (TMHs). The thienopyrimidine org41841 was identified previously as an agonist for the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and suggested to bind within its TMHs because it did not compete for LH binding to the LHCGR ectodomain. Because of its high homology with LHCGR, we predicted that thyroid-stimulating hormone receptor (TSHR) might be activated by org41841 also. We show that org41841 is a partial agonist for TSHR but with lower potency than for LHCGR. Analysis of three-dimensional molecular models of TSHR and LHCGR predicted a binding pocket for org41841 in common clefts between TMHs 3, 4, 5, 6, and 7 and extracellular loop 2 in both receptors. Evidence for this binding pocket was obtained in signaling studies with chimeric receptors that exhibited improved responses to org41841. Furthermore, a key receptor-ligand interaction between the highly conserved negatively charged E3.37 and the amino group of org41841 predicted by docking of the ligand into the three-dimensional TSHR model was experimentally confirmed. These findings provide the first evidence that, in contrast to the ectodomain binding of cognate ligands, a LMW agonist can bind to and activate glycoprotein hormone receptors via interaction with their transmembrane domain.

Sorafenib in locally advanced or metastatic patients with radioactive iodine-refractory differentiated thyroid cancer: The phase III DECISION trial.

4 Background: Sorafenib, an orally active inhibitor of VEGFR1-3 and Raf kinases, has shown promising clinical activity in single-arm phase II studies in radioactive iodine (RAI)-refractory differentiated thyroid cancer (DTC). The double-blind, randomized, multicenter phase III DECISION trial examined sorafenib efficacy and safety vs placebo in patients with progressive RAI-refractory DTC. METHODS Patients with locally advanced/metastatic RAI-refractory DTC who progressed in the preceding 14 months were randomized 1:1 to sorafenib 400 mg bid po or placebo. Placebo patients were allowed to receive sorafenib open-label upon progression. The primary endpoint was progression-free survival (PFS) assessed every 8 wks by independent radiologic review using modified RECIST 1.0 and analyzed by stratified log-rank statistics at α = 0.01 (one-sided). Secondary endpoints included overall survival (OS), response rate (RR; complete + partial response [PR]), and safety. RESULTS A total of 417 patients were randomized (207 to sorafenib and 210 to placebo); median age 63 yr, 52% female. Tumor histology by independent assessment was 57% papillary, 25% follicular, and 10% poorly differentiated. 96% of patients had metastatic disease; the most common target lesions were lung (71%), lymph node (40%), and bone (14%). The primary endpoint was met: median PFS 10.8 months (sorafenib) vs 5.8 months (placebo); HR 0.58, 95% CI 0.45-0.75, p<0.0001. Median OS has not been reached in either arm; 70% of placebo patients started open-label sorafenib. RR (all PRs) in the sorafenib vs placebo arms was 12.2% and 0.5% (p<0.0001) and stable disease ≥ 6 months was 42% and 33%, respectively. The most common any-grade treatment-emergent adverse events in the sorafenib arm included hand-foot skin reaction, diarrhea, alopecia, rash/desquamation, fatigue, weight loss and hypertension. One death in each arm was attributed to study drug. CONCLUSIONS Sorafenib significantly improved PFS compared with placebo in patients with progressive RAI-refractory DTC. Tolerability was consistent with the known sorafenib safety profile. CLINICAL TRIAL INFORMATION NCT00895674.

Contacts between extracellular loop two and transmembrane helix six determine basal activity of the thyroid-stimulating hormone receptor

A number of alanine mutations in extracellular loop two (ECL2) of the thyroid-stimulating hormone receptor (TSHR) were found to increase or decrease basal activity when compared with the wild type receptor. K565A was identified as a mutant with decreased basal activity, and strongly impaired hormone induced signaling activity. To gain insights into how ECL2 mutants affect basal activity, we focused on constitutively activating pathogenic mutant I568V in ECL2, which exhibits elevated basal activity. Because our molecular model suggests that Ile-568 is embedded in an environment of hydrophobic residues provided by transmembrane helix bundle, we tested mutants in this region to identify potential interaction partner(s) for Ile-568. Indeed, the double mutant I568V/I640L (ECL2/TMH6) suppresses the increased basal activity exhibited by I568V alone. We suggest a spatial and functional relationship between ECL2 and TMH6 in which side chain interaction between Ile-568 and Ile-640 constrains the receptor in a conformation with low basal activity. Although the single mutant I640L exhibits basal activity lower than wild type, its differently branched and bulkier side chain complements the reduced side chain bulk in I568V, restoring wild type basal activity to the double mutant. This scenario is confirmed by the reciprocal double mutant I640V/I568L. The combination of basally increased activity of I640V and basally decreased activity of mutant I568L also restores basal activity of wild type TSHR. These and other mutant phenotypes reported here support a dynamic interface between TMH6 and ECL2. Disruption of this critical interface for signaling by introduction of mutations in TSHR can either increase or decrease basal activity.

Extended hormone binding site of the human thyroid stimulating hormone receptor: distinctive acidic residues in the hinge region are involved in bovine thyroid stimulating hormone binding and receptor activation.

The human thyroid stimulating hormone receptor (hTSHR) belongs to the glycoprotein hormone receptors that bind the hormones at their large extracellular domain. The extracellular hinge region of the TSHR connects the N-terminal leucine-rich repeat domain with the membrane-spanning serpentine domain. From previous studies we reasoned that apart from hormone binding at the leucine-rich repeat domain, additional multiple hormone contacts might exist at the hinge region of the TSHR by complementary charge-charge recognition. Here we investigated highly conserved charged residues in the hinge region of the TSHR by site-directed mutagenesis to identify amino acids interacting with bovine TSH (bTSH). Indeed, the residues Glu-297, Glu-303, and Asp-382 in the TSHR hinge region are essential for bTSH binding and partially for signal transduction. Side chain substitutions showed that the negative charge of Glu-297 and Asp-382 is necessary for recognition of bTSH by the hTSHR. Multiple combinations of alanine mutants of the identified positions revealed an increased negative effect on hormone binding. An assembled model suggests that the deciphered acidic residues form negatively charged patches at the hinge region resulting in an extended binding mode for bTSH on the hTSHR. Our data indicate that certain positively charged residues of bTSH might be involved in interaction with the identified negatively charged amino acids of the hTSHR hinge region. We demonstrate that the hinge region represents an extracellular intermediate connector for both hormone binding and signal transduction of the hTSHR.

Gene expression analysis reveals evidence for inactivation of the TGF-β signaling cascade in autonomously functioning thyroid nodules

Molecular eventsthat lead to the development of autonomously functioning thyroid nodules (AFTNs) are somatic mutations of the thyrotropin receptor (TSHR) in approximately 60% of the nodules and less frequently, somatic mutations in the Gsα protein. However, AFTNs without known mutations indicate that other causes remain to be identified. Moreover, the impact of constitutively activating TSHR mutations on the signal transduction network of the thyroid epithelial cell is unknown. We therefore investigated gene expression in 15 AFTNs and their surrounding tissue using Affymetrix GeneChips. Most prominently, data analysis revealed a changed pattern of gene expression in the TGF-β signaling cascade and 25 differentially regulated genes in AFTNs, including thyroid peroxidase, type I iodothyronine deiodinase and sialyltransferase (SIAT) 1. Strikingly coexpression of SIAT 1 and TSHR in COS-7 cells increased TSH binding and cell surface expression of the TSHR. Moreover, differences in gene expression patterns for AFTNs with and without TSHR mutations indicate specific alterations of signal transduction in AFTNs without TSHR mutations. These results suggest that AFTNs with TSHR mutations harbor further mechanisms of forward stimulation. Furthermore, they give important leads to elucidate the molecular etiology of AFTNs without TSHR mutations.

Detection of PAX8/PPARG and RET/PTC rearrangements is feasible in routine air-dried fine needle aspiration smears.

BACKGROUND The diagnostic limitations of fine needle aspiration (FNA), like the indeterminate category, can be partially overcome by molecular analysis. As PAX8/PPARG and RET/PTC rearrangements have been detected in follicular thyroid carcinomas (FTCs) and papillary thyroid carcinomas (PTCs), their detection in FNA smears could improve the FNA diagnosis. To date, these rearrangements have never been analyzed in routine air-dried FNA smears, but only in frozen tissue, formalin-fixed paraffin-embedded (FFPE) tissue, and in fresh FNA material. Fixed routine air-dried FNA samples have hitherto been judged as generally not suitable for testing these rearrangements in a clinical setting. Therefore, the objective of the present study was to investigate the feasibility of extracting RNA from routine air-dried FNA smears for the detection of these rearrangements with real-time polymerase chain reaction (RT-PCR). METHODS A new method for RNA extraction from routine air-dried FNA smears was established, which allowed analysis for the presence of four variants of PAX8/PPARG and RET/PTC 1 and RET/PTC 3, which were analyzed in 106 routine FNA smears and the corresponding surgically obtained FFPE tissues using real-time quantitative PCR (RT-qPCR). To assess RNA quality, an intron-spanning PAX8 cDNA was amplified. RESULTS Acceptable RNA quality was obtained from 95% of the FNA samples and 92% of the FFPE samples. PAX8/PPARG was detected in 4 of 96 FFPEs and in 6 of 96 FNAs. PAX8/PPARG was present in 4 of 10 FTCs and in 3 of 42 follicular adenomas (FAs). Similarly, RET/PTC was found in 3 of 96 FFPEs and in 4 of 96 FNAs. Two of 21 PTC samples and 3 of 42 FA samples carried this rearrangement. CONCLUSION These data are the first to show the feasibility of extracting RNA from routine air-dried FNA smears for the detection of PAX8/PPARG and RET/PTC rearrangements with RT-qPCR. These promising methodological advances, if confirmed in larger series of FNA and FFPE samples, may lead to the introduction of molecular analysis of routine air-dried FNA smears in everyday practice.

Evidence for cooperative signal triggering at the extracellular loops of the TSH receptor.

The mechanisms governing transition of the thyroid stimulating hormone (TSH) receptor (TSHR) from basal to active conformations are poorly understood. Considering that constitutively activating mutations (CAMs) and inactivating mutations in each of the extracellular loops (ECLs) trigger only partial TSHR activation or inactivation, respectively, we hypothesized that full signaling occurs via multiple extracellular signal propagation events. Therefore, individual CAMs in the extracellular region were combined to create double and triple mutants. In support of our hypothesis, combinations of mutants in the ECLs are in some cases additive, while in others they are even synergistic, with triple mutant I486A/I568V/V656F exhibiting a 70‐fold increase in TSH‐independent signaling. The proximity but likely different spatial orientation of the residues of activating and inactivating mutations in each ECL supports a dual functionality to facilitate signal induction and conduction, respectively. This is the first report for G‐protein coupled receptors, suggesting that multiple and cooperative signal propagating events at all three ECLs are required for full receptor activation. Our findings provide new insights concerning molecular signal transmission from extracellular domains toward the transmembrane helix bundle of the glycoprotein hormone receptors.—Kleinau, G., Jaeschke, H., Mueller, S., Raaka, B. M., Neumann, S., Paschke, R., Krause, G. Evidence for cooperative signal triggering at the extracellular loops of the TSH receptor. FASEB J. 22, 2798–2808 (2008)

RET germline mutation in codon 791 in a family representing 3 generations from age 5 to age 70 years: should thyroidectomy be performed?

OBJECTIVE To describe a kindred with a rare RET germline mutation in codon 791 and discuss potential management strategies. METHODS We present clinical and biochemical data as well as results of mutation analysis in our study subjects and provide an overview of related published reports. RESULTS Multiple endocrine neoplasia type 2 (MEN 2) is a familial cancer syndrome characterized by the development of medullary thyroid carcinoma (MTC), pheochromocytoma, and parathyroid hyperplasia or adenoma. Germline mutations in RET are responsible for this autosomal dominant syndrome. Familial MTC is a variant of MEN 2A and can be caused by RET mutations in codon 791. Deaths from gene carriers with mutations in these codons have not yet been reported. In general, gene carriers with these RET mutations have late-onset MTC. Because only a few kindreds with this specific mutation have been identified and no long-term follow-up data are available, management of these patients can be a challenge. We illustrate the difficulties with decisions about not only when to perform thyroidectomy in these patients but also whether thyroidectomy should even be considered in such gene carriers with a benign course. Our reported kindred included four carriers with a codon 791 RET germline mutation, one of whom had the rare concomitant occurrence of acromegaly and MEN 2A. The 70-year-old mother had acromegaly and hyperparathyroidism but normal serum calcitonin levels and normal findings on thyroid ultrasound examination. She refused pentagastrin testing and any surgical intervention. The 37-year-old daughter had hypothyroidism, a small thyroid gland, and negative results of pentagastrin stimulation testing of calcitonin. The 18-year-old grandson also had a negative pentagastrin test result and normal thyroid ultrasound findings. The 5-year-old granddaughter had normal results of thyroid ultrasonography. In all patients, we recommended thyroidectomy. CONCLUSION Prospective studies are needed to clarify which patients with codon 791 RET germline mutation should undergo thyroidectomy.