Structure of AMH bound to AMHR2 provides insight into a unique signaling pair in the TGF-ß family.

Anti-Müllerian hormone (AMH), or Müllerian-inhibiting substance, is a protein hormone that promotes Müllerian duct regression during male fetal sexual differentiation and regulation of folliculogenesis in women. AMH is a member of the transforming growth factor beta (TGF-ß) family, which has evolved to signal through its own dedicated type II receptor, AMH receptor type II (AMHR2). Structures of other TGF-ß family members have revealed how ligands infer specificity for their cognate receptors; however, it is unknown how AMH binds AMHR2 at the molecular level. Therefore, in this study, we solved the X-ray crystal structure of AMH bound to the extracellular domain of AMHR2 to a resolution of 2.6Å. The structure reveals that while AMH binds AMHR2 in a similar location to Activin and BMP ligand binding to their type II receptors, differences in both AMH and AMHR2 account for a highly specific interaction. Furthermore, using an AMH responsive cell-based luciferase assay, we show that a conformation in finger 1 of AMHR2 and a salt bridge formed by K534 on AMH and D81/E84 of AMHR2 are key to the AMH/AMHR2 interaction. Overall, our study highlights how AMH engages AMHR2 using a modified paradigm of receptor binding facilitated by modifications to the three-finger toxin fold of AMHR2. Furthermore, understanding these elements contributing to the specificity of binding will help in the design of agonists or antagonists or the selection of antibody therapies.

Conditional steroidogenic cell-targeted deletion of TSPO unveils a crucial role in viability and hormone-dependent steroid formation.

Translocator protein (TSPO) is a key member of the mitochondrial cholesterol transport complex in steroidogenic tissues. To assess the function of TSPO, we generated two lines of Cre-mediated Tspo conditional knockout (cKO) mice. First, gonadal somatic cell-targeting Amhr2-Cre mice were crossed with Tspo-floxed mice to obtain F1 Tspo Amhr2 cKO mice (Tspo(fl/fl);Amhr2-Cre(/+)). The unexpected Mendelian ratio of 4.4% cKO mice was confirmed by genotyping of 12.5-day-postcoitum (dpc) embryos. As Amhr2-Cre is expressed in gonads at 12.5 dpc, these findings suggest preimplantation selection of embryos. Analysis of expression databases revealed elevated levels of Amhr2 in two- and eight-cell zygotes, suggesting ectopic Tspo silencing before the morula stage and demonstrating elevated embryonic lethality and involvement of TSPO in embryonic development. To circumvent this issue, steroidogenic cell-targeting Nr5a1-Cre mice were crossed with Tspo-floxed mice. The resulting Tspo(fl/fl);Nr5a1-Cre(/+) mice were born at a normal Mendelian ratio. Nr5a1-driven Tspo cKO mice exhibited highly reduced Tspo levels in adrenal cortex and gonads. Treatment of mice with human chorionic gonadotropin (hCG) resulted in increased circulating testosterone levels despite extensive lipid droplet depletion. In contrast, Nr5a1-driven Tspo cKO mice lost their ability to form corticosterone in response to adrenocorticotropic hormone (ACTH). Important for ACTH-dependent steroidogenesis, Mc2r, Stard1, and Cypa11a1 levels were unaffected, whereas Scarb1 levels were increased and accumulation of lipid droplets was observed, indicative of a blockade of cholesterol utilization for steroidogenesis. TSPO expression in the adrenal medulla and increased epinephrine production were also observed. In conclusion, TSPO was found necessary for preimplantation embryo development and ACTH-stimulated steroid biosynthesis.

Anti-Müllerian hormone is associated with extrauterine involvement and stage of disease in patients with endometrial cancer.

Our aim was to evaluate serum levels of anti-Müllerian hormone (AMH) and also immunohistochemical (IHC) staining properties of AMH receptor type II (AMHRII) in patients with endometrial cancer (EC) and a control group. Preoperatively, serum levels of AMH were assessed and AMHRII expression was evaluated by immunohistochemistry in a benign and malignant group. AMH serum levels of the control group and EC patients were comparable. For EC patients, there was no difference with respect to the AMH levels and tumour stage; grade; histological type; deep myometrial invasion; lymphovascular space invasion or lymph node involvement. However, AMH levels in patients with extrauterine involvement were higher than patients with disease confined to the uterus. EC samples were more likely to be stained positive for AMHRII than benign lesions. Also, as the stage of the lesion worsens, the rate of IHC staining of AMHRII decreases. In conclusion, AMHRII is expressed in normal endometrial cells as well as endometrial cancer cells. AMH levels increase in EC, with extrauterine involvement at least in locally advanced disease. Also AMH expression decreases as the disease is staged-up.

Molecular Mechanisms of AMH Signaling.

Anti-Müllerian Hormone (AMH) is a secreted glycoprotein hormone with critical roles in reproductive development and regulation. Its chemical and mechanistic similarities to members of the Transforming Growth Factor ß (TGF-ß) family have led to its placement within this signaling family. As a member of the TGF-ß family, AMH exists as a noncovalent complex of a large N-terminal prodomain and smaller C-terminal mature signaling domain. To produce a signal, the mature domain will bind to the extracellular domains of two type I and two type II receptors which results in an intracellular SMAD signal. Interestingly, as will be discussed in this review, AMH possesses several unique characteristics which set it apart from other ligands within the TGF-ß family. In particular, AMH has a dedicated type II receptor, Anti-Müllerian Hormone Receptor Type II (AMHR2), making this interaction intriguing mechanistically as well as therapeutically. Further, the prodomain of AMH has remained largely uncharacterized, despite being the largest prodomain within the family. Recent advancements in the field have provided valuable insight into the molecular mechanisms of AMH signaling, however there are still many areas of AMH signaling not understood. Herein, we will discuss what is known about the biochemistry of AMH and AMHR2, focusing on recent advances in understanding the unique characteristics of AMH signaling and the molecular mechanisms of receptor engagement.

Immunotherapy of ovarian cancer with a monoclonal antibody specific for the extracellular domain of anti-Müllerian hormone receptor II.

Epithelial ovarian carcinoma (EOC) is the most prevalent and lethal form of ovarian cancer. The low five-year overall survival after EOC diagnosis indicates an urgent need for more effective ways to control this disease. Anti-Müllerian hormone receptor 2 (AMHR2) is an ovarian protein overexpressed in the majority of human EOCs. We have previously found that vaccination against the ovarian-specific extracellular domain of AMHR2 (AMHR2-ED) significantly inhibits growth of murine EOCs through an IgG-mediated mechanism that agonizes receptor signaling of a Bax/caspase-3 dependent proapoptotic cascade. To determine if a single monoclonal antibody (mAb) could inhibit growth of human EOC, we generated a panel of mAbs specific for recombinant human AMHR2-ED and characterized a candidate mAb for humanization and use in clinical trials. We found that our candidate 4D12G1 mAb is an IgG1 that shows high affinity antigen-specific binding to the 7-mer 20KTLGELL26 sequence of AMHR2-ED that facilitates induction of programmed cell death in EOC cells. Most importantly, the 4D12G1 mAb significantly inhibits growth of primary human EOCs in patient-derived xenografts (PDXs) by inducing direct apoptosis of EOC tumors. Our results support the view that a humanized 4D12G1 mAb may be a much needed and effective reagent for passive immunotherapy of human EOC.