Molecular Insights into the Transmembrane Domain of the Thyrotropin Receptor

Abstract

The thyrotropin receptor (TSHR) is a G protein-coupled receptor (GPCR) that is member of the leucine-rich repeat subfamily (LGR). In the absence of crystal structure, the success of rational design of ligands targeting the receptor internal cavity depends on the quality of the TSHR models built. In this subfamily, transmembrane helices (TM) 2 and 5 are characterized by the absence of proline compared to most receptors, raising the question of the structural conformation of these helices. To gain insight into the structural properties of these helices, we carried out bioinformatics and experimental studies. Evolutionary analysis of the LGR family revealed a deletion in TM5 but provided no information on TM2. Wild type residues at positions 2.58, 2.59 or 2.60 in TM2 and/or at position 5.50 in TM5 were substituted to proline. Depending on the position of the proline substitution, different effects were observed on membrane expression, glycosylation, constitutive cAMP activity and responses to thyrotropin. Only proline substitution at position 2.59 maintained complex glycosylation and high membrane expression, supporting occurrence of a bulged TM2. The TSHR transmembrane domain was modeled by homology with the orexin 2 receptor, using a protocol that forced the deletion of one residue in the TM5 bulge of the template. The stability of the model was assessed by molecular dynamics simulations. TM5 straightened during the equilibration phase and was stable for the remainder of the simulations. Our data support a structural model of the TSHR transmembrane domain with a bulged TM2 and a straight TM5 that is specific of glycoprotein hormone receptors.

Structure and activation of the TSH receptor transmembrane domain

“…GPHRs have an alanine at position 5.50 (Ballesteros–Weinstein numbering [27]) of TM5 in contrast to other GPCRs that have a conserved Pro in this position and consequently a kink in the structure of TM5. Therefore, the models of the structures of TM5 of GPHRs show a regular α helix without a kink, as predicted previously by Kleinau et al [15] and Chantreau et al [11]. TSHR mutation A593 5.50 P is likely to induce a kink in the TM5 structure that would probably affect folding of the mutated receptor.…”

“…1), but not in the structure of TM5, as previously predicted by Chantreau et al [11]. GPHRs have an alanine at position 5.50 (Ballesteros–Weinstein numbering [27]) of TM5 in contrast to other GPCRs that have a conserved Pro in this position and consequently a kink in the structure of TM5.…”

“…To date, a comparison of the inactive and active conformations of the GPHRs to infer an activation mechanism of the TMD has not been carried out. The majority of previously published models of the active state of GPHRs were based only on one template [11–13, 15–18]. In one study, a model of the TSHR TMD was produced based on 15 GPCRs in their inactive conformations, 3 GPCRs in partially active conformations and 1 in fully active conformation [14].…”

Comparative Study of the Steroidogenic Effects of Human Chorionic Gonadotropin and Thieno[2,3-D]pyrimidine-Based Allosteric Agonist of Luteinizing Hormone Receptor in Young Adult, Aging and Diabetic Male Rats

“…It should be noted that, in a large number of GPCRs, a high-affinity orthosteric site is located within the transmembrane domain. It can be assumed that in evolutionary precursors of the LHCGR and other receptors of pituitary glycoprotein hormones, the transmembrane orthosteric site, due to structural changes in the transmembrane domain, including the replacement of the proline residue in the fifth transmembrane region, which is highly conserved in Class A GPCRs [ 46 , 47 , 48 ], has lost the ability to bind hormonal regulators and, as a result, transformed into an allosteric site. It can be assumed that in the course of evolution, in LHCGR and structurally related TSHR and follicle-stimulating hormone receptor, the transmembrane orthosteric site has lost the ability to bind hormones and converted to an allosteric site.…”

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

“…This structural prediction was later confirmed in crystal structures of receptors that do not have a proline at position 5×50 and which do indeed have a regular alpha-helical TMH5 such as the Sphingosine 1-phosphate receptor 1 [alanine in position 5×50; PDB entry 3V2W (127)], the P2Y12 receptor [asparagine in position 5×50; PDB entry 4NTJ (128)], and the lysophosphatidic acid receptor 1 [threonine in position 5×50, LPAR1, PDB entry 4Z34 (129)]. These structural implications for Ala593 in TMH5 of TSHR (126) were recently confirmed by others (104). …”