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. 2015 Feb;156(2):488-98.
doi: 10.1210/en.2014-1509. Epub 2014 Nov 19.

Transmembrane domains of attraction on the TSH receptor

Rauf Latif  1 , M Rejwan Ali, Mihaly Mezei, Terry F Davies
Affiliations

Transmembrane domains of attraction on the TSH receptor

Rauf Latif et al. Endocrinology. 2015 Feb.

Abstract

The TSH receptor (TSHR) has the propensity to form dimers and oligomers. Our data using ectodomain-truncated TSHRs indicated that the predominant interfaces for oligomerization reside in the transmembrane (TM) domain. To map the potentially interacting residues, we first performed in silico studies of the TSHR transmembrane domain using a homology model and using Brownian dynamics (BD). The cluster of dimer conformations obtained from BD analysis indicated that TM1 made contact with TM4 and two residues in TM2 made contact with TM5. To confirm the proximity of these contact residues, we then generated cysteine mutants at all six contact residues predicted by the BD analysis and performed cysteine cross-linking studies. These results showed that the predicted helices in the protomer were indeed involved in proximity interactions. Furthermore, an alternative experimental approach, receptor truncation experiments and LH receptor sequence substitution experiments, identified TM1 harboring a major region involved in TSHR oligomerization, in agreement with the conclusion from the cross-linking studies. Point mutations of the predicted interacting residues did not yield a substantial decrease in oligomerization, unlike the truncation of the TM1, so we concluded that constitutive oligomerization must involve interfaces forming domains of attraction in a cooperative manner that is not dominated by interactions between specific residues.

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Figures

Figure 1.
Figure 1.
A, TSHR β-subunit dimerization. Western blotting of a truncated TSHR (β-366), partially devoid of the ectodomain, continued to demonstrate oligomerization with dimeric forms and oligomeric forms outnumbering the monomers (∼70 kDa). WT-type monomeric form 100- to 120-kDa and oligomeric forms (>220 kDa) are shown in the left lane. TMD appeared to be the primary site of the oligomer interface. B, Dimer poses of TSHR-TMD protomers. The individual centers of the second protomer (yellow ball) of the TSHR in the membrane plane are shown for the configurations in which the BD was successfully able to form a dimer. The likely contacts are restricted to essentially opposite sides of the central seven-helix bundle (shown as orange clusters).
Figure 2.
Figure 2.
A, Structure of a TSHR dimer. This is a representation of the dimer structure at the center of the largest cluster viewed from the side. The residues that make contact in this helical association are represented by black and steel gray ball structures. The residues involved were as follows: TM1 (V433) made contact with TM4 (L551); TM2 (L467) made contact with TM5 (T588); and TM2 (A470) made contact with TM5 (N590). Inset, A close-up top view of the interacting residues in the corresponding helices. TM5 and TM4 are in blue and TM1 and TM2 are in orange. B, Cross-linking of cysteine-mutated TSHRs. To confirm the proximity of the BD predicted residues, we generated a full-length TSHR construct having all six predicted residues (three pairs) mutated to cysteine and then transfected this construct into CHO cells to obtain a stable line (Mut6). The percent expression of surface receptors in WT TSHR- and Mut6-transfected cells was 83.25 ± 2.89 and 47.35 ± 16.78, respectively. Total membrane lysates prepared from the Mut6 and WT cells were then cross-linked using CuSO4 and phenanthroline as described in Materials and Methods. The right lane in the Mut6 immunoblot panel shows increased oligomeric forms (≥220 kDa) in the presence of the cross-linker when compared with the vehicle-treated lysate (left lane). There was also a marked corresponding decrease in the monomeric forms (100–120 kDa) in the treated lane. The right WT immunoblot panel shows that the WT receptor lysate treated with or without (−/+) the cross-linker in the same way did not reveal an enhanced oligomer formation or any increased higher cross-linked bands than the constitutive oligomeric forms. Bottom graph, Densitometry of monomer to dimer ratio of the respective immunoblots.
Figure 3.
Figure 3.
A–D, Truncations of the TSHR-TMD. The eight helices of the TSHR transmembrane domain are depicted by cylindrical structures connected to each other by their extra- and intracellular loops. The WT TSHR is shown in panel A, whereas panels B–D are structures with the paired truncations used in subsequent studies. The dotted line shows the actual connection of the ECLs and ICLs in the various truncated constructs. ECL, extracellular loop. E, Expression of pair-wise deleted mutants. WT TSHR and the truncated and paired TSHR mutants (TM1+TM2; TM3+TM4; and TM5+TM6) had a YFP tag at their carboxyl tail and were transfected into HEK293 cells and checked for total expression of YFP by flow cytometry in unfixed cells 48 hours after the transfection. The y-axis indicates the percentage of YFP-positive cells. Significant expression (from 50% to 90%) of truncated receptors was observed in these transiently transfected HEK293 cells. UT, untruncated. F, Western blotting of double TM truncated TSHRs. This figure illustrates the TSHR species identified in a nonreducing immunoblot of total membranes (100 μg/lane) prepared from the cells described in panel E after probing with the TSHR antibody (M1 at 2 μg/mL). There was a substantial decrease in TSHR oligomers (bands of ≥ to 220 kDa) with the TM1+TM2 deletion construct compared with WT TSHRs or other truncated pairs. Monomeric receptors of the expected 100–120 kDa were seen with the WT construct.
Figure 4.
Figure 4.
A, Individual TM1 and TM2 truncated TSHRs. Total membranes prepared from constructs transfected into HEK293 cells with either TM1 deleted or TM2 deleted were immunoblotted using a TSHR antibody. There was a marked decrease in oligomers seen with the TM1 deleted mutant compared with WT TSHR and the TM2 deleted receptors. B, TSHR monomer to oligomer ratios. Densitometry of panel A yielded monomer to oligomer ratios of 1.0 compared with 1.4 for WT and thus emphasized the decreased formation of oligomers with the TM1 mutant compared with WT receptors. C, FRET analysis of the TM1 truncated construct. As detailed in Materials and Methods, FRET studies were carried out using CFP-tagged and YFP-tagged WT and TM1-deleted TSHR constructs after cotransfecting these tagged receptors into HEK293 cells. As controls we used CTV and C5V constructs tagged with CFP and Venus reporter genes. CTV is a construct in which the CFP (donor) is tagged to Venus (reporter) by a fixed set of amino acids that gives a positive FRET, whereas C5V is a truncated version of this arrangement in which the FRET efficiency is very low (56). FRET efficiency was measured for these different constructs after donor spectral bleed-through and acceptor spectral bleed-through subtraction using the pFRET software as described. The data indicate a significant decrease (75%) in FRET efficiency with the TM1-deleted receptors over that of WT TSHRs, illustrating decreased dimer or oligomer formation in TM1 truncated receptors. D, FRET images. WT (left panel) and TM1 and TM2 truncated (right panel) TSHR FRET images, after spectral bleed-through correction, showing the different fluorescent intensity obtained in the FRET analyses. The graphs below show the pixels measured across the images with their intensity (gray values) in the y-axis. Note the different scales needed to illustrate the decreased intensity obtained with the TM1 truncated receptor compared with that of the WT TSHR.
Figure 5.
Figure 5.
A, Aligned protein sequence of LHR and TSHR TM1 region. The protein sequences of the TSHR and LHR TM1 region were aligned to show the 54% conservation. The residues marked in red are the nonconserved substitutions found only in the LH receptor TM1 region. The residues of interest in TM1 for oligomer formation is marked in blue and underlined. LHR, LH receptor. B, Surface expression of the LH receptor substituted construct. Fluorescence-activated cell sorter analyses were performed with cells expressing WT-TSHR (upper right box) and TSHRs substituted with the LH receptor TM1 sequence (LHRsb) (lower right box) and TSHR truncated for TM1 (lower left box). These constructs had YFP on their carboxyl end and the unfixed cells were stained for surface TSHR expression with anti-TSHR (M1), which was subsequently detected with an antimouse PE as indicated in the x-axis. The upper left box shows the background binding with the second antibody alone. Some (29.2%) of the LH receptor TM1 substituted cells had surface expression of this chimeric receptor. C, The LH receptor TM1 restores TSHR TM1δ oligomerization. The densitometry of the Western blotting of lysates were prepared from WT-, LHRsb-, and TM1δ TSHR-expressing cells and probed for receptor forms with anti-TSHR (M1). As shown here, the TSHR TM1δ construct showed reduced monomer to oligomer ratio, but the substitution of LH receptor residues into the TSHR TM1δ truncated construct restored the oligomeric forms almost to the original levels seen with WT TSHR (gray filled bar). PE, Phycoerythrin.
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