Signaling‐sensitive amino acids surround the allosteric ligand binding site of the thyrotropin receptor

Abstract

The thyrotropin receptor [thyroid-stimulating hormone receptor (TSHR)], a G-protein-coupled receptor (GPCR), is endogenously activated by thyrotropin, which binds to the extracellular region of the receptor. We previously identified a low-molecular-weight (LMW) agonist of the TSHR and predicted its allosteric binding pocket within the receptor's transmembrane domain. Because binding of the LMW agonist probably disrupts interactions or leads to formation of new interactions among amino acid residues surrounding the pocket, we tested whether mutation of residues at these positions would lead to constitutive signaling activity. Guided by molecular modeling, we performed site-directed mutagenesis of 24 amino acids in this spatial region, followed by functional characterization of the mutant receptors in terms of expression and signaling, measured as cAMP accumulation. We found that mutations V421I, Y466A, T501A, L587V, M637C, M637W, S641A, Y643F, L645V, and Y667A located in several helices exhibit constitutive activity. Of note is mutation M637W at position 6.48 in transmembrane helix 6, which has a significant effect on the interaction of the receptor with the LMW agonist. In summary, we found that a high proportion of residues in several helices surrounding the allosteric binding site of LMW ligands in the TSHR when mutated lead to constitutively active receptors. Our findings of signaling-sensitive residues in this region of the transmembrane bundle may be of general importance as this domain appears to be evolutionarily retained among GPCRs.

From Molecular Details of the Interplay between Transmembrane Helices of the Thyrotropin Receptor to General Aspects of Signal Transduction in Family A G-protein-coupled Receptors (GPCRs)

“…L587V mutation data of constitutive activation (Ref. 46 and Fig. 5) are consistent with the ␣-helix conformation in the second model.…”

New Small Molecule Agonists to the Thyrotropin Receptor

“…This allosteric mode of action may result from the inherent property of small molecules, including their small size and hydrophobic nature. However, it has been shown that the TSHR contains clusters of residues with allosteric potential encompassing signaling-sensitive amino acids within the helices of the TMD (21). To determine if our lead molecules were allosteric modulators, as expected, we tested their receptorstimulating activity on a chimeric receptor in which the major part of its ectodomain consists of the LHR sequence fused to the native human TSHR TMD.…”

“…SMLs by virtue of their small size can permeate the cell and dock with polar and nonpolar residues in the TMD region and exert their effects in an allosteric manner (21). In the case of the TSHR, the different transmembrane helices and intracellular and extracellular loops connecting these helices have been studied extensively for activating and inactivating mutations (22), which are the ''hot-spots'' in the receptor, having been found in human pathological conditions (21).…”

“…SMLs by virtue of their small size can permeate the cell and dock with polar and nonpolar residues in the TMD region and exert their effects in an allosteric manner (21). In the case of the TSHR, the different transmembrane helices and intracellular and extracellular loops connecting these helices have been studied extensively for activating and inactivating mutations (22), which are the ''hot-spots'' in the receptor, having been found in human pathological conditions (21). Based on such modeling and mutational analysis of TSHR ''hot-spots,'' there appear to be two clusters of residues spanning the TM helices that are preferred mutation sites, as well as potential sites for docking of selected small molecules (20,23).…”

Intracellular iodinated compounds affect sodium iodide symporter expression through TSH-mediated signaling pathways

“…TSHr is a G protein-coupled receptor that transfers extracellular signals mainly by the TSH/TSHr-caMP-PKa and TSH/TSHr-Plc-iP3 pathways (18,19). The mechanism by which TSH regulates the expression and activity of niS through the cAMP-PKA pathway has been partially clarified by several studies.…”