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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.


There are differences in the L2-beta loop for hLH and hCG. This gives rise to tighter binding for hLH than for hCG at sTyr331, the second binding site at LHCGR. (A) Homology model details of superimposed hormones hLH and hCG extracellularly bound to LRRD and hinge region of LHCGR based on the crystal structure of FSH/FSHR [4AY9, Ref. (9)]. (B) Sequence differences in L2-beta loop of these glycoprotein hormones (cjCG: New world monkey Callithrix jacchus). Gray: positions representing interacting elements of the sTyr moiety of the LHCGR hinge region. White on black background: differences in prolines between FSH and hLH, hCG, and even between hLH (red boxed) and hCG. (C) hLH was modeled based on the hCG structure (PDB: 1HRP, light green). Close up view: the L1-alpha loop is similar for hLH (dark blue) and hCG (cyan). The differing conformations of the L2-beta loop for hLH is based on a fragment in the crystal structure of Insulysin (3TUV) with identical sequence PPLPQ. The resulting backbone conformation of hLH (red) clearly differs from that in hCG structure (light green). The additional proline in hLH (see red box in B) restricts the conformational degree of freedom of βR63 (red) located in the N-terminal flank of the L2-beta loop. (D) Detailed view for hLH. The resulting binding cavity for sTyr331 formed by the L1-alpha loop (dark blue surface) and L2-beta loop (red surface) is more surrounded in hLH, and is flanked by the βR63 (red) and thus provides an additional H-bond donor for the interaction with the sTyr331 (yellow) of LHCGR compared to (E) detailed view for hCG (light green), where βR63 does not interact with sTyr331 and the binding pocket is much wider.
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Figure 3: There are differences in the L2-beta loop for hLH and hCG. This gives rise to tighter binding for hLH than for hCG at sTyr331, the second binding site at LHCGR. (A) Homology model details of superimposed hormones hLH and hCG extracellularly bound to LRRD and hinge region of LHCGR based on the crystal structure of FSH/FSHR [4AY9, Ref. (9)]. (B) Sequence differences in L2-beta loop of these glycoprotein hormones (cjCG: New world monkey Callithrix jacchus). Gray: positions representing interacting elements of the sTyr moiety of the LHCGR hinge region. White on black background: differences in prolines between FSH and hLH, hCG, and even between hLH (red boxed) and hCG. (C) hLH was modeled based on the hCG structure (PDB: 1HRP, light green). Close up view: the L1-alpha loop is similar for hLH (dark blue) and hCG (cyan). The differing conformations of the L2-beta loop for hLH is based on a fragment in the crystal structure of Insulysin (3TUV) with identical sequence PPLPQ. The resulting backbone conformation of hLH (red) clearly differs from that in hCG structure (light green). The additional proline in hLH (see red box in B) restricts the conformational degree of freedom of βR63 (red) located in the N-terminal flank of the L2-beta loop. (D) Detailed view for hLH. The resulting binding cavity for sTyr331 formed by the L1-alpha loop (dark blue surface) and L2-beta loop (red surface) is more surrounded in hLH, and is flanked by the βR63 (red) and thus provides an additional H-bond donor for the interaction with the sTyr331 (yellow) of LHCGR compared to (E) detailed view for hCG (light green), where βR63 does not interact with sTyr331 and the binding pocket is much wider.

Mentions: Structural homology models for the extracellular domains of LHCGR were built on the basis of the available FSHR crystal structures (27). The procedure for the homologous hTSHR modeling has been already described in detail (28). Disulfide bridges between cysteines of cysteine boxes Cb-2 and Cb-3 were built as suggested by the FSHR crystal structure. The sulfated sTyr335 of hFSHR was already known to be mandatory for hormone binding and signaling and interacts tightly with amino acids of the hormone subunits between the L2-beta loop and the L1-alpha loop (27). Both interaction models, hLHCGR/hLH and hLHCGR/hCG, were built (Figure 3). For the complex with bound hCG, hFSH in the template structure of the hFSH–hFSHR complex was substituted with the crystal structure of hCG [PDB-code: 1HRP, Ref. (29)]. In the hLH model, the alpha and beta-subunit of this hormone structure were used as template. Except for sequence 70PPLPQ74 that is different in the L2 loop of the beta-subunit, the corresponding fragment of another template 3TUV from PDB was used. On the basis of the N- and C-terminal fragments of the L2-beta loop of the crystal structures of CG (1QFW, 1HRP) and by overlapping superimposition with the elongated conformation of the 70PPLPQ74 fragment, the homology model of L2-beta loop was changed for hLH. Initially, side chains of the homology models were subjected to conjugate gradient minimizations [until they converged at a termination gradient of 0.05 kcal/(mol*Å)]. The AMBER F99 force field was used. Finally, the models were minimized without constraints. Structural modifications and homology modeling procedures were performed with Sybyl X2.0 (Certara, Inc., St. Louis, MI, USA).


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)

There are differences in the L2-beta loop for hLH and hCG. This gives rise to tighter binding for hLH than for hCG at sTyr331, the second binding site at LHCGR. (A) Homology model details of superimposed hormones hLH and hCG extracellularly bound to LRRD and hinge region of LHCGR based on the crystal structure of FSH/FSHR [4AY9, Ref. (9)]. (B) Sequence differences in L2-beta loop of these glycoprotein hormones (cjCG: New world monkey Callithrix jacchus). Gray: positions representing interacting elements of the sTyr moiety of the LHCGR hinge region. White on black background: differences in prolines between FSH and hLH, hCG, and even between hLH (red boxed) and hCG. (C) hLH was modeled based on the hCG structure (PDB: 1HRP, light green). Close up view: the L1-alpha loop is similar for hLH (dark blue) and hCG (cyan). The differing conformations of the L2-beta loop for hLH is based on a fragment in the crystal structure of Insulysin (3TUV) with identical sequence PPLPQ. The resulting backbone conformation of hLH (red) clearly differs from that in hCG structure (light green). The additional proline in hLH (see red box in B) restricts the conformational degree of freedom of βR63 (red) located in the N-terminal flank of the L2-beta loop. (D) Detailed view for hLH. The resulting binding cavity for sTyr331 formed by the L1-alpha loop (dark blue surface) and L2-beta loop (red surface) is more surrounded in hLH, and is flanked by the βR63 (red) and thus provides an additional H-bond donor for the interaction with the sTyr331 (yellow) of LHCGR compared to (E) detailed view for hCG (light green), where βR63 does not interact with sTyr331 and the binding pocket is much wider.
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Related In: Results  -  Collection

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Figure 3: There are differences in the L2-beta loop for hLH and hCG. This gives rise to tighter binding for hLH than for hCG at sTyr331, the second binding site at LHCGR. (A) Homology model details of superimposed hormones hLH and hCG extracellularly bound to LRRD and hinge region of LHCGR based on the crystal structure of FSH/FSHR [4AY9, Ref. (9)]. (B) Sequence differences in L2-beta loop of these glycoprotein hormones (cjCG: New world monkey Callithrix jacchus). Gray: positions representing interacting elements of the sTyr moiety of the LHCGR hinge region. White on black background: differences in prolines between FSH and hLH, hCG, and even between hLH (red boxed) and hCG. (C) hLH was modeled based on the hCG structure (PDB: 1HRP, light green). Close up view: the L1-alpha loop is similar for hLH (dark blue) and hCG (cyan). The differing conformations of the L2-beta loop for hLH is based on a fragment in the crystal structure of Insulysin (3TUV) with identical sequence PPLPQ. The resulting backbone conformation of hLH (red) clearly differs from that in hCG structure (light green). The additional proline in hLH (see red box in B) restricts the conformational degree of freedom of βR63 (red) located in the N-terminal flank of the L2-beta loop. (D) Detailed view for hLH. The resulting binding cavity for sTyr331 formed by the L1-alpha loop (dark blue surface) and L2-beta loop (red surface) is more surrounded in hLH, and is flanked by the βR63 (red) and thus provides an additional H-bond donor for the interaction with the sTyr331 (yellow) of LHCGR compared to (E) detailed view for hCG (light green), where βR63 does not interact with sTyr331 and the binding pocket is much wider.
Mentions: Structural homology models for the extracellular domains of LHCGR were built on the basis of the available FSHR crystal structures (27). The procedure for the homologous hTSHR modeling has been already described in detail (28). Disulfide bridges between cysteines of cysteine boxes Cb-2 and Cb-3 were built as suggested by the FSHR crystal structure. The sulfated sTyr335 of hFSHR was already known to be mandatory for hormone binding and signaling and interacts tightly with amino acids of the hormone subunits between the L2-beta loop and the L1-alpha loop (27). Both interaction models, hLHCGR/hLH and hLHCGR/hCG, were built (Figure 3). For the complex with bound hCG, hFSH in the template structure of the hFSH–hFSHR complex was substituted with the crystal structure of hCG [PDB-code: 1HRP, Ref. (29)]. In the hLH model, the alpha and beta-subunit of this hormone structure were used as template. Except for sequence 70PPLPQ74 that is different in the L2 loop of the beta-subunit, the corresponding fragment of another template 3TUV from PDB was used. On the basis of the N- and C-terminal fragments of the L2-beta loop of the crystal structures of CG (1QFW, 1HRP) and by overlapping superimposition with the elongated conformation of the 70PPLPQ74 fragment, the homology model of L2-beta loop was changed for hLH. Initially, side chains of the homology models were subjected to conjugate gradient minimizations [until they converged at a termination gradient of 0.05 kcal/(mol*Å)]. The AMBER F99 force field was used. Finally, the models were minimized without constraints. Structural modifications and homology modeling procedures were performed with Sybyl X2.0 (Certara, Inc., St. Louis, MI, USA).

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.