Identification of a Novel Epitope in the Thyroid-stimulating Hormone Receptor Ectodomain Acting as Intramolecular Signaling Interface

Abstract

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.

FSH and TSH binding to their respective receptors: similarities, differences and implication for glycoprotein hormone specificity

“…Some theoretical models of the TSH-TSHR complex (Kleinau et al 2004, Moyle et al 2004, Nú ñez Miguel et al 2004 were published just before the coordinates of the FSH-FSHR complex became available (Fan & Hendrickson 2005). The structure of the concave surface and b-strands of the TSHR LRD were well defined in the three models (Kleinau et al 2004, Moyle et al 2004, Nú ñez Miguel et al 2004.…”

“…The structure of the concave surface and b-strands of the TSHR LRD were well defined in the three models (Kleinau et al 2004, Moyle et al 2004, Nú ñez Miguel et al 2004. The model of Moyle et al (2004) comprised three domains of the TSHR linked together and the LRD structure was modelled on the structure of ubiquitin ligase.…”

“…The complex shows TSH interacting with the concave surface of the TSHR LRD but in a different position compared with the FSH-FSHR complex. In particular, the hormone lies parallel to the length of the LRD (Kleinau et al 2004). Our earlier model (Nú ñez Miguel et al 2004) consisted of three TSHR domains with the LRD structure based on ribonuclease inhibitor, and showing small helices, instead of small strands, in the convex surface.…”

“…The model shows TSH binding to one of the side surfaces of the LRD (between the concave and convex surfaces) rather than to the concave surface itself. The model of Kleinau et al (2004) consists of parts of the TSHR ectodomain only. The LRD structure was modelled on the structure of the Nogo receptor and showed a good approximation to the structure of the TSHR LRD, which is now known (Sanders et al 2007a).…”

Significance of Ectodomain Cysteine Boxes 2 and 3 for the Activation Mechanism of the Thyroid-stimulating Hormone Receptor

“…It is established that the C-b2 and 3 potentially interact via disulfide bonds (21)(22)(23) and that mutations disrupting the disulfide bridge lead to constitutive activation (24,25). In a previous study we identified new residues within C-b3 that are also involved in receptor stabilization and intramolecular signal transduction based on a suggested homologous molecular model for the C-b2/C-b3 unit (26). By site-directed mutagenesis we defined a region (Asp-403 Glu-404 Asn-406) of the TSHR ectodomain that can act as a switch for constitutive activity.…”