Characterization of the molecular mechanisms that govern anti-Mullerian hormone synthesis and activity

The roles of anti-M & uuml;llerian hormone (AMH) continue to expand, from its discovery as a critical factor in sex determination, through its identification as a regulator of ovarian folliculogenesis, its use in fertility clinics as a measure of ovarian reserve, and its emerging role in hypothalamic-pituitary function. In light of these actions, AMH is considered an attractive therapeutic target to address diverse reproductive needs, including fertility preservation. Here, we set out to characterize the molecular mechanisms that govern AMH synthesis and activity. First, we enhanced the processing of the AMH precursor to >90% by introducing more efficient proprotein convertase cleavage sites (RKKR or ISSRKKRSVSS [SCUT]). Importantly, enhanced processing corresponded with a dramatic increase in secreted AMH activity. Next, based on species differences across the AMH type II receptor-binding interface, we generated a series of human AMH variants and assessed bioactivity. AMH(SCUT) potency (EC50 4 ng/mL) was increased 5- or 10-fold by incorporating Gln(484)Met/Leu(535)Thr (EC50 0.8 ng/mL) or Gln(484)Met/Gly(533)Ser (EC50 0.4 ng/mL) mutations, respectively. Furthermore, the Gln(484)Met/Leu(535)Thr double mutant displayed enhanced efficacy, relative to AMH(SCUT). Finally, we identified residues within the wrist pre-helix of AMH (Trp(494), Gln(496), Ser(497), and Asp(498)) that likely mediate type I receptor binding. Mutagenesis of these residues generated gain- (Trp(494)Phe or Gln(496)Leu) or loss- (Ser(497)Ala) of function AMH variants. Surprisingly, combining activating type I and type II receptor mutations only led to modest additive increases in AMH potency/efficacy. Our study is the first to characterize AMH residues involved in type I receptor binding and suggests a step-wise receptor-complex assembly mechanism, in which enhancement in the affinity of the ligand for either receptor can increase AMH activity beyond the natural level.