Rearrangement of the Extracellular Domain/Extracellular Loop 1 Interface Is Critical for Thyrotropin Receptor Activation

Abstract

The thyroid stimulating hormone receptor (TSHR) is a G protein-coupled receptor (GPCR) with a characteristic large extracellular domain (ECD). TSHR activation is initiated by binding of the hormone ligand TSH to the ECD. How the extracellular binding event triggers the conformational changes in the transmembrane domain (TMD) necessary for intracellular G protein activation is poorly understood. To gain insight in this process, the knowledge on the relative positioning of ECD and TMD and the conformation of the linker region at the interface of ECD and TMD are of particular importance. To generate a structural model for the TSHR we applied an integrated structural biology approach combining computational techniques with experimental data. Chemical cross-linking followed by mass spectrometry yielded 17 unique distance restraints within the ECD of the TSHR, its ligand TSH, and the hormone-receptor complex. These structural restraints generally confirm the expected binding mode of TSH to the ECD as well as the general fold of the domains and were used to guide homology modeling of the ECD. Functional characterization of TSHR mutants confirms the previously suggested close proximity of Ser-281 and Ile-486 within the TSHR. Rigidifying this contact permanently with a disulfide bridge disrupts ligand-induced receptor activation and indicates that rearrangement of the ECD/extracellular loop 1 (ECL1) interface is a critical step in receptor activation. The experimentally verified contact of Ser-281 (ECD) and Ile-486 (TMD) was subsequently utilized in docking homology models of the ECD and the TMD to create a full-length model of a glycoprotein hormone receptor. Glycoprotein hormones (GPHs)3 normally regulate crucial processes in metabolism and reproduction by activating GPHRs. This is especially true for TSHR, which can cause several clinically relevant conditions like hypo-and hyperthyroidism when it malfunctions. Yet the mechanism of how extracellular ligand binding induces the structural changes required for intracellular G protein activation is unknown. We pursued an integrated structural biology approach using modeling guided by experimental data to generate experimentally supported full-length TSHR models. It is expected that some insights gleaned from a TSHR model can be generalized to other GPHRs. These models in turn create testable hypotheses on the mechanism of GPHR activation and can promote drug development to treat GPHR-associated diseases.GPHs bind to the ECD of their respective receptors (Fig. 1A) and consequently initiate activation, which is presumably propagated by induction of conformational changes within the ECD's hinge region (HR) (1-4). Interestingly, GPHRs still possess a binding site within the TMD not associated with physiological receptor activation but accessible to low molecular weight agonists and allosteric modulators (5-7). Another important aspect of GPHR function and physiology is posttranslational modification, including disulfide bond formation, glycosylation, tyrosine sulfation, and p...

Intervention Strategies into Glycoprotein Hormone Receptors for Modulating (Mal–)function, with Special Emphasis on the TSH Receptor

“…Concerning the transmembrane domain GPHR share high sequence similarity and overlapping features within class A GPCRs, which are visible in determined GPCR structures [49,50]. Therefore, so far for larger GPHR structures only homology molecular models assembled by variant fragments revealed insights into structure-function relationships of ligand-GPHR complexes (e. g., [29,[51][52][53][54][55]) such as the here suggested LHR full length model as monomer (also representative for FSHR and TSHR) in ▶ Fig. 2.…”

Structure and activation of the TSH receptor transmembrane domain

“…However, a receptor model obtained in this way would not show either inactive or active conformation. In addition, Schaarschmidt et al [10] have obtained an active conformation of the TSHR TMD based on five partially active and two fully active GPCR template structures. A model of the active TSHR TMD, obtained in this way, is likely to represent the inactive conformation in its cytoplasmic region rather than the active conformation.…”

“…Also, the crystal structure of the LRD and the ECD of the human FSH receptor (FSHR) bound to hFSH has been determined [8, 9]. No experimental structures of the TMD of the TSHR are available, although several models of the structure of the TSHR TMD have been published [10–18]. …”

Structural–Functional Features of the Thyrotropin Receptor: A Class A G-Protein-Coupled Receptor at Work

“…Functionally supportive data for assembling the SD and the extracellular region are rather rare (135, 189). More detailed methods for building these models are described in our own previous publications on the TSHR or other GPCRs [e.g., Ref.…”