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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

Expression of the TSHR mutants.(a) Cell surface expression and (b) total cell expression of WT and mutated TSHRs, given as the percentage of the wild type control, after transfection of HEK-293 cells by the same amount of TSHR coding pcDNA3.1 plasmid. Receptor expression was determined by flow cytometry measurements of intact (a) or permeabilized cells (b). The histograms represent the mean ± SEM of at least 3 independent experiments, each carried out in triplicates.
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pone.0142250.g003: Expression of the TSHR mutants.(a) Cell surface expression and (b) total cell expression of WT and mutated TSHRs, given as the percentage of the wild type control, after transfection of HEK-293 cells by the same amount of TSHR coding pcDNA3.1 plasmid. Receptor expression was determined by flow cytometry measurements of intact (a) or permeabilized cells (b). The histograms represent the mean ± SEM of at least 3 independent experiments, each carried out in triplicates.

Mentions: HEK-293 cells were transiently transfected with the pcDNA3.1 plasmid containing the sequence of WT or mutated TSH receptors. The receptor expression was measured by flow cytometry on intact and permeabilized cells (Fig 3). The L2.59 mutant had cell membrane expression (130 ± 20%) similar to WT. However the cell membrane expression decreased to about 40% of WT for the I2.60P and A5.50P mutants, to 20% of WT for the L2.58P mutant, and to less than 10% for the double mutants (Fig 3A). In permeabilized cells (Fig 3B), the amount of receptors observed were 60 ± 10% of WT for the L2.59P mutant, about 30% of WT for the L2.58P, I2.60P and A5.50P mutants and about 15% of WT for the double 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)

Expression of the TSHR mutants.(a) Cell surface expression and (b) total cell expression of WT and mutated TSHRs, given as the percentage of the wild type control, after transfection of HEK-293 cells by the same amount of TSHR coding pcDNA3.1 plasmid. Receptor expression was determined by flow cytometry measurements of intact (a) or permeabilized cells (b). The histograms represent the mean ± SEM of at least 3 independent experiments, each carried out in triplicates.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142250.g003: Expression of the TSHR mutants.(a) Cell surface expression and (b) total cell expression of WT and mutated TSHRs, given as the percentage of the wild type control, after transfection of HEK-293 cells by the same amount of TSHR coding pcDNA3.1 plasmid. Receptor expression was determined by flow cytometry measurements of intact (a) or permeabilized cells (b). The histograms represent the mean ± SEM of at least 3 independent experiments, each carried out in triplicates.
Mentions: HEK-293 cells were transiently transfected with the pcDNA3.1 plasmid containing the sequence of WT or mutated TSH receptors. The receptor expression was measured by flow cytometry on intact and permeabilized cells (Fig 3). The L2.59 mutant had cell membrane expression (130 ± 20%) similar to WT. However the cell membrane expression decreased to about 40% of WT for the I2.60P and A5.50P mutants, to 20% of WT for the L2.58P mutant, and to less than 10% for the double mutants (Fig 3A). In permeabilized cells (Fig 3B), the amount of receptors observed were 60 ± 10% of WT for the L2.59P mutant, about 30% of WT for the L2.58P, I2.60P and A5.50P mutants and about 15% of WT for the double 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