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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 &gt;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\u2010binding interface, we generated a series of human AMH variants and assessed bioactivity. AMH\n                    <jats:sub>SCUT<\/jats:sub>\n                    potency (EC\n                    <jats:sub>50<\/jats:sub>\n                    4\u2009ng\/mL) was increased 5\u2010 or 10\u2010fold by incorporating Gln\n                    <jats:sup>484<\/jats:sup>\n                    Met\/Leu\n                    <jats:sup>535<\/jats:sup>\n                    Thr (EC\n                    <jats:sub>50<\/jats:sub>\n                    0.8\u2009ng\/mL) or Gln\n                    <jats:sup>484<\/jats:sup>\n                    Met\/Gly\n                    <jats:sup>533<\/jats:sup>\n                    Ser (EC\n                    <jats:sub>50<\/jats:sub>\n                    0.4\u2009ng\/mL) mutations, respectively. Furthermore, the Gln\n                    <jats:sup>484<\/jats:sup>\n                    Met\/Leu\n                    <jats:sup>535<\/jats:sup>\n                    Thr double mutant displayed enhanced efficacy, relative to AMH\n                    <jats:sub>SCUT<\/jats:sub>\n                    . Finally, we identified residues within the wrist pre\u2010helix of AMH (Trp\n                    <jats:sup>494<\/jats:sup>\n                    , Gln\n                    <jats:sup>496<\/jats:sup>\n                    , Ser\n                    <jats:sup>497<\/jats:sup>\n                    , and Asp\n                    <jats:sup>498<\/jats:sup>\n                    ) that likely mediate type I receptor binding. Mutagenesis of these residues generated gain\u2010 (Trp\n                    <jats:sup>494<\/jats:sup>\n                    Phe or Gln\n                    <jats:sup>496<\/jats:sup>\n                    Leu) or loss\u2010 (Ser\n                    <jats:sup>497<\/jats:sup>\n                    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\u2010wise receptor\u2010complex assembly mechanism, in which enhancement in the affinity of the ligand for either receptor can increase AMH activity beyond the natural level.\n                  <\/jats:p>","DOI":"10.1096\/fj.202301335rr","type":"journal-article","created":{"date-parts":[[2023,12,22]],"date-time":"2023-12-22T11:38:31Z","timestamp":1703245111000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Characterization of the molecular mechanisms that govern anti\u2010M\u00fcllerian hormone synthesis and activity"],"prefix":"10.1096","volume":"38","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-8334-2027","authenticated-orcid":false,"given":"William A.","family":"Stocker","sequence":"first","affiliation":[{"name":"Department of Physiology, Monash Biomedicine Discovery Institute Monash University  Clayton Victoria Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5020-9687","authenticated-orcid":false,"given":"James A.","family":"Howard","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Systems Physiology University of Cincinnati  Cincinnati Ohio USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shreya","family":"Maskey","sequence":"additional","affiliation":[{"name":"Department of Physiology, Monash Biomedicine Discovery Institute Monash University  Clayton Victoria Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haitong","family":"Luan","sequence":"additional","affiliation":[{"name":"Department of Physiology, Monash Biomedicine Discovery Institute Monash University  Clayton Victoria Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5332-3776","authenticated-orcid":false,"given":"Sophie G.","family":"Harrison","sequence":"additional","affiliation":[{"name":"Department of Physiology, Monash Biomedicine Discovery Institute Monash University  Clayton Victoria Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0484-1969","authenticated-orcid":false,"given":"Kaitlin N.","family":"Hart","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Systems Physiology University of Cincinnati  Cincinnati Ohio USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0731-1293","authenticated-orcid":false,"given":"Lucija","family":"Hok","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Biosciences University of Cincinnati  Cincinnati Ohio USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7041-5047","authenticated-orcid":false,"given":"Thomas 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