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ABSTRACT: 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 Gαs and the Gαq pathway. We observed a synergistic increase for basal activity of the Gαs 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 Gαs-mediated signaling. No single CAM revealed constitutive activity for the Gαq pathway. The double mutations with CAMs from TMH1, 2, 3 and 6 also exhibited increase for basal Gαq signaling. Our results suggest that TMH2, 6, 7 show selective preferences towards Gαs signaling, and TMH1, 2, 3, 6 for Gαq signaling.
Keywords.G protein-coupled receptors-constitutively activating mutations-synergism-G protein selectivity-thyroid-stimulating hormone receptor
Cellular and Molecular Life Sciences CMLS 04/2012; 65(24):4028-4038. · 6.57 Impact Factor
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[show abstract]
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ABSTRACT: 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.
Cellular and Molecular Life Sciences CMLS 12/2008; 65(24):4028-38. · 6.57 Impact Factor
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ABSTRACT: Several mutations that decrease the basal signaling activity of G-protein coupled receptors (GPCRs) with pathogenic implications are known. Here we study the molecular mechanisms responsible for this phenotype and investigate how basal and further activated receptor conformations are interrelated. In the basally active thyroid stimulating hormone receptor (TSHR) we combined spatially-distant mutations with opposing effects on basal activity in double-mutations and characterized mutant basal and TSH induced signaling. Mutations lowering basal activity always have a suppressive influence on TSH induced signaling and on constitutively activating mutations (CAMs). Our results suggest that the conformation of a basally 'silenced' GPCR might impair its intrinsic capacity for signaling compared to the wild-type. Striking differences in conformation and intramolecular interactions between TSHR models built using the crystal structures of inactive rhodopsin and partially active opsin help illuminate the molecular details underlying mutations decreasing basal activity.
Cellular and Molecular Life Sciences CMLS 11/2008; 65(22):3664-76. · 6.57 Impact Factor
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ABSTRACT: The majority of constitutively activating human TSH receptor (hTSHR) mutations are located in the transmembrane helices as well as in the extracellular (ECLs) and intracellular loops. S(281) is one of two positions in the ectodomain in which activating hTSHR mutations have been identified in vivo (S(281)T, I, and N). To investigate the functional properties of this key residue in more detail, S(281) was replaced by each of the other 19 amino acids. Many substitutions led to constitutive receptor activation, suggesting that S(281) plays a pivotal role in maintaining the receptor in its inactive state. Strikingly, all substitutions with aromatic residues (S(281)W, F, Y, and H) show expression similar to that of wild-type hTSHR and are tolerated at this position because they maintain basal activity or express only slight constitutive activity. Three-dimensional modeling of the hTSHR suggested that S(281) and surrounding residues are in close proximity to ECL1. To investigate the possible importance of an aromatic environment between the ectodomain in the vicinity of S(281) and ECL1, aromatic residues Y(279), Y(476), H(478), Y(481), Y(482), and H(484) were replaced by alanine. Functional characterization showed impaired cell surface expression and signaling for Y(279)A and Y(481)A, in contrast to the other alanine mutants. However, substitutions of Y(279) and Y(481) with other aromatic residues exhibited surface expression and signaling comparable to wild-type hTSHR. Our results suggest that Y(279) in the extracellular domain and probably Y(481) in the ECL1 also are involved in an aromatic environment around S(281) in the hTSHR, which is important for functional receptor conformation and intramolecular receptor signaling.
Endocrinology 05/2006; 147(4):1753-60. · 4.46 Impact Factor
