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

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.

Loss-of-Function Mutations in the Human Luteinizing Hormone Receptor Predominantly Cause Intracellular Retention

“…Previous studies have shown that deletion of LHR Exon10 impairs signaling of LH but not hCG (36,37). However, for those extracellular mutants with measurable signaling, there was a strong correlation between the potency measured for both ligands, suggesting that none of the mutants (including Del Exon 10) displayed bias for either hormone (Supplemental Figure 6).…”

“…Thus, retention is unlikely to be the sole cause of nonfunctionality. Indeed, previous studies have attributed nonfunctionality of Del Exon10 to an impaired responsiveness to LH, but not hCG (36,37), supported by the fact that LHRs of the New World monkey lineage, in which CG␤ has functionally replaced LH␤, do not contain Exon 10. Furthermore, a homozygous Del Exon10 patient seemed to have normal hCG-mediated fetal gonadal development, but lacked LH-mediated pubertal development.…”

Human LH and hCG stimulate differently the early signalling pathways but result in equal testosterone synthesis in mouse Leydig cells in vitro

“…In fact, there is a specific, distinct site of interaction located within this region of the human receptor [15, 56, 57]. We could speculate that the mouse-specific amino acid sequence at the hinge region is not able to qualitatively discriminate between LH and hCG, reflecting the evolutionary differences embedded into the primate-specific LH and hCG dual ligand system.…”

Estrogen Modulates Specific Life and Death Signals Induced by LH and hCG in Human Primary Granulosa Cells In Vitro

“…These gonadotropin-specific effects were amplified in the presence of FSH [16], suggesting that different activity at the receptor level and intracellular signaling activation characterize the two hormones [30]. Indeed, different binding affinity of LH and hCG for the rodent LH receptor was demonstrated three decades ago [31], providing the first evidence that these hormones interact with different binding sites of the common receptor.…”