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Differences in Signal Activation by LH and hCG are Mediated by the LH/CG Receptor's Extracellular Hinge Region.

Grzesik P, Kreuchwig A, Rutz C, Furkert J, Wiesner B, Schuelein R, Kleinau G, Gromoll J, Krause G - Front Endocrinol (Lausanne) (2015)

Bottom Line: These helix preserving modifications showed no effect on hormone-induced signaling.This opposite effect on signaling by hLH and hCG can be explained by distinct sites of hormone interaction in the hinge region.In conclusion, our analysis provides details of the differences between hLH- and hCG-induced signaling that are mainly determined in the L2-beta loop of the hormones and in the hinge region of the receptor.

View Article: PubMed Central - PubMed

Affiliation: Leibniz Institut für Molekulare Pharmakologie (FMP) , Berlin , Germany.

ABSTRACT
The human lutropin (hLH)/choriogonadotropin (hCG) receptor (LHCGR) can be activated by binding two slightly different gonadotropic glycoprotein hormones, choriogonadotropin (CG) - secreted by the placenta, and lutropin (LH) - produced by the pituitary. They induce different signaling profiles at the LHCGR. This cannot be explained by binding to the receptor's leucine-rich-repeat domain (LRRD), as this binding is similar for the two hormones. We therefore speculate that there are previously unknown differences in the hormone/receptor interaction at the extracellular hinge region, which might help to understand functional differences between the two hormones. We have therefore performed a detailed study of the binding and action of LH and CG at the LHCGR hinge region. We focused on a primate-specific additional exon in the hinge region, which is located between LRRD and the serpentine domain. The segment of the hinge region encoded by exon10 was previously reported to be only relevant to hLH signaling, as the exon10-deletion receptor exhibits decreased hLH signaling, but unchanged hCG signaling. We designed an advanced homology model of the hormone/LHCGR complex, followed by experimental characterization of relevant fragments in the hinge region. In addition, we examined predictions of a helical exon10-encoded conformation by block-wise polyalanine (helix supporting) mutations. These helix preserving modifications showed no effect on hormone-induced signaling. However, introduction of a structure-disturbing double-proline mutant LHCGR-Q303P/E305P within the exon10-helix has, in contrast to exon10-deletion, no impact on hLH, but only on hCG signaling. This opposite effect on signaling by hLH and hCG can be explained by distinct sites of hormone interaction in the hinge region. In conclusion, our analysis provides details of the differences between hLH- and hCG-induced signaling that are mainly determined in the L2-beta loop of the hormones and in the hinge region of the receptor.

No MeSH data available.


Mutations introduced in the middle of the LGCGR hinge region. (A) Sequence of LHCGR constructs of helix supporting block-wise polyalanine and directed helix-disturbing proline mutations within the exon10-helix and adjacent helix. (B) Localization of the GFP fluorescence signals of the LHCGR constructs in transiently transfected HEK 293 cells by confocal LSM. The GFP-signals of the fusions (left panel, green) and the Trypan blue signals of the membranes of the same cells (central panels, red) were computer-overlayed (right panels, yellow). GFP fluorescence is detectable only for transfected cells, whereas all cells show cell surface trypan blue fluorescence. All constructs are expressed on the plasma membrane surface.
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Figure 5: Mutations introduced in the middle of the LGCGR hinge region. (A) Sequence of LHCGR constructs of helix supporting block-wise polyalanine and directed helix-disturbing proline mutations within the exon10-helix and adjacent helix. (B) Localization of the GFP fluorescence signals of the LHCGR constructs in transiently transfected HEK 293 cells by confocal LSM. The GFP-signals of the fusions (left panel, green) and the Trypan blue signals of the membranes of the same cells (central panels, red) were computer-overlayed (right panels, yellow). GFP fluorescence is detectable only for transfected cells, whereas all cells show cell surface trypan blue fluorescence. All constructs are expressed on the plasma membrane surface.

Mentions: To reveal the structure–function properties of the exon10-encoded part of the LHCGR hinge region, an alanine-block scan was performed. Since polyalanine constructs are prone to form helix structures and helix formation was predicted, the 27 exon10 amino acids (Q290-L316) were systematically substituted by five (LHCGR-Ala1–LHCGR-Ala3) and six (LHCGR-Ala4 and LHCGR-ALa5) alanines in a row. The predicted adjacent helix (construct LHCGR-Ala6) contains an alanine-block substitution of the amino acids 318–324 (Figure 5A). This region has been described as signaling sensitive (23). Since alanine blocks are capable of mimicking a helical structure (38), we introduced proline mutations which might disturb potential helical portions, such as the double proline mutant at the position Q303P/E305P. Additionally, in the wild-type LHCGR, position M320 was mutated to proline.


Differences in Signal Activation by LH and hCG are Mediated by the LH/CG Receptor's Extracellular Hinge Region.

Grzesik P, Kreuchwig A, Rutz C, Furkert J, Wiesner B, Schuelein R, Kleinau G, Gromoll J, Krause G - Front Endocrinol (Lausanne) (2015)

Mutations introduced in the middle of the LGCGR hinge region. (A) Sequence of LHCGR constructs of helix supporting block-wise polyalanine and directed helix-disturbing proline mutations within the exon10-helix and adjacent helix. (B) Localization of the GFP fluorescence signals of the LHCGR constructs in transiently transfected HEK 293 cells by confocal LSM. The GFP-signals of the fusions (left panel, green) and the Trypan blue signals of the membranes of the same cells (central panels, red) were computer-overlayed (right panels, yellow). GFP fluorescence is detectable only for transfected cells, whereas all cells show cell surface trypan blue fluorescence. All constructs are expressed on the plasma membrane surface.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585211&req=5

Figure 5: Mutations introduced in the middle of the LGCGR hinge region. (A) Sequence of LHCGR constructs of helix supporting block-wise polyalanine and directed helix-disturbing proline mutations within the exon10-helix and adjacent helix. (B) Localization of the GFP fluorescence signals of the LHCGR constructs in transiently transfected HEK 293 cells by confocal LSM. The GFP-signals of the fusions (left panel, green) and the Trypan blue signals of the membranes of the same cells (central panels, red) were computer-overlayed (right panels, yellow). GFP fluorescence is detectable only for transfected cells, whereas all cells show cell surface trypan blue fluorescence. All constructs are expressed on the plasma membrane surface.
Mentions: To reveal the structure–function properties of the exon10-encoded part of the LHCGR hinge region, an alanine-block scan was performed. Since polyalanine constructs are prone to form helix structures and helix formation was predicted, the 27 exon10 amino acids (Q290-L316) were systematically substituted by five (LHCGR-Ala1–LHCGR-Ala3) and six (LHCGR-Ala4 and LHCGR-ALa5) alanines in a row. The predicted adjacent helix (construct LHCGR-Ala6) contains an alanine-block substitution of the amino acids 318–324 (Figure 5A). This region has been described as signaling sensitive (23). Since alanine blocks are capable of mimicking a helical structure (38), we introduced proline mutations which might disturb potential helical portions, such as the double proline mutant at the position Q303P/E305P. Additionally, in the wild-type LHCGR, position M320 was mutated to proline.

Bottom Line: These helix preserving modifications showed no effect on hormone-induced signaling.This opposite effect on signaling by hLH and hCG can be explained by distinct sites of hormone interaction in the hinge region.In conclusion, our analysis provides details of the differences between hLH- and hCG-induced signaling that are mainly determined in the L2-beta loop of the hormones and in the hinge region of the receptor.

View Article: PubMed Central - PubMed

Affiliation: Leibniz Institut für Molekulare Pharmakologie (FMP) , Berlin , Germany.

ABSTRACT
The human lutropin (hLH)/choriogonadotropin (hCG) receptor (LHCGR) can be activated by binding two slightly different gonadotropic glycoprotein hormones, choriogonadotropin (CG) - secreted by the placenta, and lutropin (LH) - produced by the pituitary. They induce different signaling profiles at the LHCGR. This cannot be explained by binding to the receptor's leucine-rich-repeat domain (LRRD), as this binding is similar for the two hormones. We therefore speculate that there are previously unknown differences in the hormone/receptor interaction at the extracellular hinge region, which might help to understand functional differences between the two hormones. We have therefore performed a detailed study of the binding and action of LH and CG at the LHCGR hinge region. We focused on a primate-specific additional exon in the hinge region, which is located between LRRD and the serpentine domain. The segment of the hinge region encoded by exon10 was previously reported to be only relevant to hLH signaling, as the exon10-deletion receptor exhibits decreased hLH signaling, but unchanged hCG signaling. We designed an advanced homology model of the hormone/LHCGR complex, followed by experimental characterization of relevant fragments in the hinge region. In addition, we examined predictions of a helical exon10-encoded conformation by block-wise polyalanine (helix supporting) mutations. These helix preserving modifications showed no effect on hormone-induced signaling. However, introduction of a structure-disturbing double-proline mutant LHCGR-Q303P/E305P within the exon10-helix has, in contrast to exon10-deletion, no impact on hLH, but only on hCG signaling. This opposite effect on signaling by hLH and hCG can be explained by distinct sites of hormone interaction in the hinge region. In conclusion, our analysis provides details of the differences between hLH- and hCG-induced signaling that are mainly determined in the L2-beta loop of the hormones and in the hinge region of the receptor.

No MeSH data available.