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.
Glycoprotein hormone receptors (GPHRs) differ from the other seven transmembrane receptors mainly through a complex activation mechanism that requires the binding of a large hormone toward a large N-terminal ectodomain. The intramolecular mechanism of the signal transduction to the serpentine domain upon hormone binding at the ectodomain is not understood. To identify determinants at the GPHR ectodomain that may be involved in signal transduction, we first searched for homologous structural features. Based on high sequence similarity to the determined structures of the Nogo-receptor ectodomain and the intermolecular complex of the Interleukin-8 ligand (IL8) and the Nterminal peptide of the IL8 receptor (IL8RA), the hypothesis was developed that portions of the intramolecular components, Cysteine-box-2 and Cysteine-box-3, of the GPHR ectodomain interact and localize at the interface between ectodomain and serpentine domain. Indeed, point mutations within the D 403 EFN 406 motif at Cysteine-box-3 of the thyrotropin receptor resulted in increased basal cAMP levels, suggesting that this motif may be important for transduction of the signal from the ectodomain to the transmembrane domain. New indications are provided about the tight spatial cooperation and relative location of the new epitope and other determinants at the thyrotropin receptor ectodomain, such as the leucine-rich repeat motif Ser 281 and the cysteine boxes. According to the high sequence conservation, the results are of general relevance for the signal transduction mechanism of other glycoprotein hormone receptors such as choriogonadotrophic/luteinizing hormone receptor and follicle-stimulating hormone receptor.
In 27 families with familial non-autoimmune hyperthyroidism (FNAH) reported up to date, the onset of hyperthyroidism varies from 18 months to 60 years. Also the manifestation of goitres is variable in these families. A 74-year-old woman first presented at the age of 69 years with tachyarrhythmia and hypertension. After initial treatment of her hypertension and oral anticoagulation for her intermittent atrial fibrillation, a thyroid workup revealed a suppressed TSH and normal fT3 and fT4. TPO, TSH receptor (TSHR), and thyroglobulin antibodies were negative. Thyroid ultrasound revealed a thyroid volume of 102 ml with several nodules with diameters of up to 2.6 cm right and up to 1.8 cm left. Scintigraphy showed a homogeneous Technetium-99 m ((99 m)Tc) uptake of 1.27%. She was subsequently treated with 1 GBq radioiodine ((131)I). At the age of 74, her thyroid function was normal and her thyroid volume decreased to 90 ml. Because of the diffuse (99 m)Tc uptake and the negative TPO, TSHR, and thyroglobulin antibodies, genetic analysis of her TSHR gene was performed, in spite of her negative family history for hyperthyroidism. Sequencing revealed a N670S TSHR germline mutation. Previous in vitro characterisation of this TSHR mutation suggests a weak constitutive activity, yet the experimental data are ambiguous. This case illustrates the necessity to analyse patients with hyperthyroidism accompanied by diffuse (99 m)Tc uptake and negative TPO, TSHR, and thyroglobulin antibodies for TSHR germline mutations. Moreover, it demonstrates that TSHR germline mutations may first lead to longstanding nodular goitrogenesis before the late manifestation of subclinical hyperthyroidism.
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