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Molecular Insights into the Transmembrane Domain of the Thyrotropin Receptor.

Chantreau V, Taddese B, Munier M, Gourdin L, Henrion D, Rodien P, Chabbert M - PLoS ONE (2015)

Bottom Line: Depending on the position of the proline substitution, different effects were observed on membrane expression, glycosylation, constitutive cAMP activity and responses to thyrotropin.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.

View Article: PubMed Central - PubMed

Affiliation: UMR CNRS 6214 -INSERM 1083, Laboratory of Integrated Neurovascular and Mitochondrial Biology, University of Angers, Angers, France.

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.

No MeSH data available.


Related in: MedlinePlus

TSH induced Ca2+ response of TSHR mutants.Typical dose-response curves of Ca2+ accumulation induced by TSH on HEK-293 cells transfected with pcDNA3.1 plasmids encoding WT or mutated TSHR. In (a), the cells were transfected with the same amount of TSHR coding plasmids, yielding the membrane expression shown in insert. In (b), the cells were transfected with different amounts of TSHR coding plasmids to obtain similar levels of cell surface expression, shown in insert. The total amount of pcDNA3.1 plasmids was kept constant by addition of empty plasmids. Receptor symbols: closed circles, WT; closed down triangles, L2.58P; open squares, L2.59P; open triangles, I2.60P; closed diamonds, A5.50P; closed squares, L2.58PA5.50P; closed triangles, P2.59PA5.50P; open diamonds, I2.60PA5.50P.
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pone.0142250.g007: TSH induced Ca2+ response of TSHR mutants.Typical dose-response curves of Ca2+ accumulation induced by TSH on HEK-293 cells transfected with pcDNA3.1 plasmids encoding WT or mutated TSHR. In (a), the cells were transfected with the same amount of TSHR coding plasmids, yielding the membrane expression shown in insert. In (b), the cells were transfected with different amounts of TSHR coding plasmids to obtain similar levels of cell surface expression, shown in insert. The total amount of pcDNA3.1 plasmids was kept constant by addition of empty plasmids. Receptor symbols: closed circles, WT; closed down triangles, L2.58P; open squares, L2.59P; open triangles, I2.60P; closed diamonds, A5.50P; closed squares, L2.58PA5.50P; closed triangles, P2.59PA5.50P; open diamonds, I2.60PA5.50P.

Mentions: TSHR activates the Ca2+ pathway in response to TSH [52]. To determine which mutants can activate this pathway, in a first assay, we measured the Ca2+ accumulation in response to TSH for all mutants, in cells transfected with the same maximal amount of pcDNA3.1 plasmid (Fig 7A). Compared to the cAMP pathway with EC50 around 0.2 mUI/ml, the activation of the Ca2+ pathway by the wild type receptor required a five-fold higher concentration of TSH (Fig 7A). Activation to about half the maximum activity of WT was observed only for the highly expressed L2.59 mutant. No significant activation could be detected for the other mutants. Subsequently, in a second assay, we measured the Ca2+ accumulation in response to TSH for the WT control and single mutants (except L2.58P) with comparable cell surface expression (Fig 7B). In these conditions, we did not observe significant Ca2+ accumulation in response to TSH for any of the mutants.


Molecular Insights into the Transmembrane Domain of the Thyrotropin Receptor.

Chantreau V, Taddese B, Munier M, Gourdin L, Henrion D, Rodien P, Chabbert M - PLoS ONE (2015)

TSH induced Ca2+ response of TSHR mutants.Typical dose-response curves of Ca2+ accumulation induced by TSH on HEK-293 cells transfected with pcDNA3.1 plasmids encoding WT or mutated TSHR. In (a), the cells were transfected with the same amount of TSHR coding plasmids, yielding the membrane expression shown in insert. In (b), the cells were transfected with different amounts of TSHR coding plasmids to obtain similar levels of cell surface expression, shown in insert. The total amount of pcDNA3.1 plasmids was kept constant by addition of empty plasmids. Receptor symbols: closed circles, WT; closed down triangles, L2.58P; open squares, L2.59P; open triangles, I2.60P; closed diamonds, A5.50P; closed squares, L2.58PA5.50P; closed triangles, P2.59PA5.50P; open diamonds, I2.60PA5.50P.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4636318&req=5

pone.0142250.g007: TSH induced Ca2+ response of TSHR mutants.Typical dose-response curves of Ca2+ accumulation induced by TSH on HEK-293 cells transfected with pcDNA3.1 plasmids encoding WT or mutated TSHR. In (a), the cells were transfected with the same amount of TSHR coding plasmids, yielding the membrane expression shown in insert. In (b), the cells were transfected with different amounts of TSHR coding plasmids to obtain similar levels of cell surface expression, shown in insert. The total amount of pcDNA3.1 plasmids was kept constant by addition of empty plasmids. Receptor symbols: closed circles, WT; closed down triangles, L2.58P; open squares, L2.59P; open triangles, I2.60P; closed diamonds, A5.50P; closed squares, L2.58PA5.50P; closed triangles, P2.59PA5.50P; open diamonds, I2.60PA5.50P.
Mentions: TSHR activates the Ca2+ pathway in response to TSH [52]. To determine which mutants can activate this pathway, in a first assay, we measured the Ca2+ accumulation in response to TSH for all mutants, in cells transfected with the same maximal amount of pcDNA3.1 plasmid (Fig 7A). Compared to the cAMP pathway with EC50 around 0.2 mUI/ml, the activation of the Ca2+ pathway by the wild type receptor required a five-fold higher concentration of TSH (Fig 7A). Activation to about half the maximum activity of WT was observed only for the highly expressed L2.59 mutant. No significant activation could be detected for the other mutants. Subsequently, in a second assay, we measured the Ca2+ accumulation in response to TSH for the WT control and single mutants (except L2.58P) with comparable cell surface expression (Fig 7B). In these conditions, we did not observe significant Ca2+ accumulation in response to TSH for any of the mutants.

Bottom Line: Depending on the position of the proline substitution, different effects were observed on membrane expression, glycosylation, constitutive cAMP activity and responses to thyrotropin.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.

View Article: PubMed Central - PubMed

Affiliation: UMR CNRS 6214 -INSERM 1083, Laboratory of Integrated Neurovascular and Mitochondrial Biology, University of Angers, Angers, France.

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.

No MeSH data available.


Related in: MedlinePlus