Thyrotropin Receptor: Allosteric Modulators Illuminate Intramolecular Signaling Mechanisms at the Interface of Ecto- and Transmembrane Domain

Abstract

The large TSH-bound ectodomain of the thyrotropin receptor (TSHR) activates the transmembrane domain (TMD) indirectly via an internal agonist (IA). The ectodomain/TMD interface consists of a converging helix, a Cys-Cys-bridge-linked IA, and extracellular loops (ECL). To investigate the intramolecular course of molecular activation, especially details of the indirect activation, we narrowed down allosteric inhibition sites of negative allosteric modulator (NAM) by mutagenesis, homology modeling, and competition studies with positive allosteric modulator (PAM). From the inhibitory effects of NAM S37a on: 1) chimeras with swapped ectodomain, 2) stepwise N-terminal truncations, 3) distinct constitutively active mutations distributed across the hinge region and ECL, but not across the TMD, we conclude that S37a binds at the ectodomain/TMD interface, between the converging helix, ECL1, and the IA. This is also supported by the noncompetitive inhibition of PAM-C2-activation by S37a in the TSHR-TMD construct lacking the ectodomain. Mutagenesis studies on the IA and ECL were guided by our refined model of the ectodomain/TMD interface and indicate an interaction with the TSHR-specific residues E404 (preceding IA) and H478 (ECL1). At this new allosteric interaction site, NAM S37a blocks both TSH-and PAM-induced activation of the TSHR. Our refined models, mutations, and new allosteric binding pocket helped us to gain more detailed insights into the intramolecular course of TSHR activation at the ectodomain/TMD interface, including the delocalization of the converging helix and rearrangement of the conformation of IA. These changes are embedded between the ECL and cooperatively trigger active conformations of TMD. SIGNIFICANCE STATEMENT The intramolecular activation mechanisms of the TSHR appear to be distinct from those of other G protein-coupled receptors, as the TSHR has a uniquely large N-terminal ectodomain that includes the hormone binding site and an internal agonist sequence. We present new molecular and structural insights into the interface between ectodomain and transmembrane domain in the TSHR, as well as the transfer of activation to the transmembrane domain. This knowledge is critical for understanding activation or inhibition of the receptor by allosteric ligands. We have identified a new allosteric antagonist binding pocket that is located exactly at this interface and possesses specific features that may allow the generation of potent highly TSHR-selective drugs, of potential value for the treatment of Graves' orbitopathy.

The intramolecular agonist is obligate for activation of glycoprotein hormone receptors

“…Substitution of E1.27 with Asp, Asn, and Gln did not compensate for function, indicating that the Glu side‐chain participates in a structurally well‐defined interaction(s). As also recently proposed, 44 our structural homology model (Figure 2) suggests a potential interaction of E409 with K660 in TM7, which is also known to have an essential functional role in GPHR to achieve an active state 48‐50 . At this position, a Lys is obligatory and cannot be substituted by any other amino acid without a loss of functionality.…”

Modulating TSH Receptor Signaling for Therapeutic Benefit

“…4). The new allosteric binding site is distant from both the orthosteric TSH binding site and the known allosteric binding site within the TMD [73].…”

“…Stereoselective synthesis and enantiomer separation resulted recently in the enantiopure molecule S37a -a micro-molar and, above all, highly TSHR-specif-ic antagonist [72]. S37a forms a bent and rigid structure [73] (Fig. 4b) and inhibits in vitro the TSHR activation by the monoclonal TSAb M22 [23] and the allosteric small molecule agonist compound 2 (C2) [74].…”

New Therapeutic Horizons for Graves’ Hyperthyroidism

“…VA-K-14 gives minor antagonism of FSH signaling in vitro, whereas S37a appears to be more specific. Elegant structural studies have shown that S37a binds TSHR in the juxta-membrane region between the C-terminal end of the extracellular domain, which has an internal agonist function, and the first extracellular loop ( 81 ). VA-K-14 and ANTAG3 most likely fit within a hydrophobic pocket that more directly stabilizes the transmembrane domains in an “off” conformation ( 17 , 19 ).…”