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

• DOI: 10.1124/mol.119.116947
• Publication Date: 2019-08-10T06:35:12Z
• Authors: Patrick Marcinkowski; Annika Kreuchwig; Sandro Mendieta; Inna Hoyer; Franziska Witte; Jens Furkert; Claudia Rutz; Dieter Lentz; Gerd Krause; Ralf Schülein
• Keywords: Signaling proteins; Interface (matter)

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