Preferences of transmembrane helices for cooperative amplification of Gαs and Gαq signaling of the thyrotropin receptor

III. Medical Department, University of Leipzig, Germany.
Cellular and Molecular Life Sciences CMLS (Impact Factor: 5.81). 12/2008; 65(24):4028-38. DOI: 10.1007/s00018-008-8530-3
Source: PubMed


The majority of constitutively activating mutations (CAMs) of the thyroid-stimulating hormone receptor display a partially activated receptor. Thus, full receptor activation requires a multiplex activation process. To define impacts of different transmembrane helices (TMHs) on cooperative signal transduction, we combined single CAMs in particular TMHs to double mutations and measured second messenger accumulation of the G(alpha)s and the G(alpha)q pathway. We observed a synergistic increase for basal activity of the G(alpha)s pathway, for all characterized double mutants except for two combinations. Each double mutation, containing CAMs in TMH2, 6 and 7 showed the highest constitutive activities, suggesting that these helices contribute most to G(alpha)s-mediated signaling. No single CAM revealed constitutive activity for the G(alpha)q pathway. The double mutations with CAMs from TMH1, 2, 3 and 6 also exhibited increase for basal G(alpha)q signaling. Our results suggest that TMH2, 6, 7 show selective preferences towards G(alpha)s signaling, and TMH1, 2, 3, 6 for G(alpha)q signaling.

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    • "Several mutations identified by mutagenesis studies (L417V, TMH1 [64]; S562A, ECL2 [64]; Y605A, TMH5 [41]; N658A, ECL3 [65]) of the TSHR are characterized by the same functional finding. By simultaneous combination of CAMs in the TSHR it was recently shown that the transmembrane helices are characterized by different preferences for cooperative amplification of Gs and Gq mediated signaling pathways [66]. These examples indicate that for full and multiple GPHR activation in terms of dual Gs and Gq coupling, highly specific structural conformations of the intracellular region must be induced by the entire receptor protein. "
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    ABSTRACT: In this study we wanted to gain insights into selectivity mechanisms between G-protein-coupled receptors (GPCR) and different subtypes of G-proteins. The thyrotropin receptor (TSHR) binds G-proteins promiscuously and activates both Gs (cAMP) and Gq (IP). Our goal was to dissect selectivity patterns for both pathways in the intracellular region of this receptor. We were particularly interested in the participation of poorly investigated receptor parts. We systematically investigated the amino acids of intracellular loop (ICL) 1 and helix 8 using site-directed mutagenesis alongside characterization of cAMP and IP accumulation. This approach was guided by a homology model of activated TSHR in complex with heterotrimeric Gq, using the X-ray structure of opsin with a bound G-protein peptide as a structural template. We provide evidence that ICL1 is significantly involved in G-protein activation and our model suggests potential interactions with subunits Gα as well as Gβγ. Several amino acid substitutions impaired both IP and cAMP accumulation. Moreover, we found a few residues in ICL1 (L440, T441, H443) and helix 8 (R687) that are sensitive for Gq but not for Gs activation. Conversely, not even one residue was found that selectively affects cAMP accumulation only. Together with our previous mutagenesis data on ICL2 and ICL3 we provide here the first systematically completed map of potential interfaces between TSHR and heterotrimeric G-protein. The TSHR/Gq-heterotrimer complex is characterized by more selective interactions than the TSHR/Gs complex. In fact the receptor interface for binding Gs is a subset of that for Gq and we postulate that this may be true for other GPCRs coupling these G-proteins. Our findings support that G-protein coupling and preference is dominated by specific structural features at the intracellular region of the activated GPCR but is completed by additional complementary recognition patterns between receptor and G-protein subtypes.
    PLoS ONE 03/2010; 5(3):e9745. DOI:10.1371/journal.pone.0009745 · 3.23 Impact Factor
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    ABSTRACT: Up to date, 14 patients with sporadic non-autoimmune hyperthyroidism (SNAH) caused by sporadic germline mutations in the TSH receptor (TSHR) gene have been reported. Despite considerable differences in the activity of hyperthyroidism, all SNAH case reports concluded that the demonstrated constitutive activity explains the phenotype. Recently, linear regression analysis (LRA) of constitutive activity as a function of TSHR expression determined by 125I-bTSH binding or fluorescence activated cell sorting analysis was described as a more reliable way of characterizing the in vitro activity (IVA) of a constitutively activating TSHR mutation. Therefore, we analyzed a possible genotype-phenotype correlation in a systematic review of the case reports and investigated the TSHR mutation's LRA in selected cases. We determined the LRA for all sporadic germline mutations which had not previously been reported. Moreover, we systematically evaluated all case reports of SNAH for evidence of an association of the clinical course (CC) with the IVA of the mutated TSHR. The LRA determined were: M453T (5.2+/-0.8), L512Q (4.5+/-0.7), I568T (25.6+/-6.3), F631L (45.9+/-9.4), T632I (14.5+/-2.7), D633Y (16.4+/-6.4). None of the 10 examined clinical signs showed a significant association with the LRA. Moreover, the comparison of the CC of patients harboring the same mutation (S281N, M453T, I568T, S505N) also showed no relation of the clinical activity with a high LRA. Considering the different diagnostic circumstances, therapeutic strategies and the limitations of a systematic analysis of case reports due to the restricted number of case reports and limited follow-up we found no consistent relation of the TSHR mutation's IVA determined by LRA with the CC of patients with SNAH. This may also be due to the action of genetic, epigenetic, and environmental modifiers.
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    ABSTRACT: The resistance to thyrotropin (TSH) action is the disease associated with molecular defects hampering the adequate transmission of TSH stimulatory signal into thyroid cells. The defect may in principle affect every step along the cascade of events following the binding of TSH to its receptor (TSHR) on thyroid cell membranes. After the description of the first family affected with loss-of-function (LOF) TSHR mutations in 1995, there is now evidence that TSH resistance is a disease with a broad range of expressivity going from severe congenital hypothyroidism (CH) with thyroid hypoplasia to mild hyperthyrotropinemia (hyperTSH) associated with an apparent euthyroid state. More severe forms occur in patients with disrupting biallelic TSHR mutations and follow a recessive pattern of inheritance. Differential diagnosis in these cases includes the exclusion of other causes of thyroid dysgenesis, such as mutations in thyroid transcription factors. More mild forms may instead occur in patients with monoallelic TSHR defects following a dominant mode of inheritance. In these cases we described the dominant negative effect exerted by some LOF mutants on the activity of the wild-type TSHR. Differential diagnosis involves the exclusion of mild hypothyroidism in autoimmune thyroid disease or pseudohypoparathyroidism associated with genetic or epigenetic defects at the GNAS locus. This review will focus on the prevalence of TSHR mutations, on the molecular mechanisms leading to TSH resistance and on the variable clinical expression of this disease.
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