Processing of Anti-Müllerian Hormone Regulates Receptor Activation by a Mechanism Distinct from TGF-β

Abstract

TGF-β family ligands are translated as prepropeptide precursors and are processed into mature C-terminal dimers that signal by assembling a serine/threonine kinase receptor complex containing type I and II components. Many TGF-β ligands are secreted in a latent form that cannot bind their receptor, due to the pro-region remaining associated with the mature ligand in a noncovalent complex after proteolytic cleavage. Here we show that anti-Müllerian hormone (AMH), a TGF-β family ligand involved in reproductive development, must be cleaved to bind its type II receptor (AMHRII), but dissociation of the pro-region from the mature C-terminal dimer is not required for this initial interaction. We provide direct evidence for this interaction by showing that the noncovalent complex binds to a soluble form of AMHRII in an ELISA format and to AMHRII immobilized on Sepharose. Binding of the noncovalent complex to Sepharose-coupled AMHRII induces dissociation of the pro-region from the mature C-terminal dimer, whereas no dissociation occurs after binding to immobilized AMH antibodies. The pro-region cannot be detected after binding of the AMH noncovalent complex to AMHRII expressed on COS cells, indicating that pro-region dissociation may occur as a natural consequence of receptor engagement on cells. Moreover, the mature C-terminal dimer is more active than the noncovalent complex in stimulating Sma- and Mad-related protein activation, suggesting that pro-region dissociation contributes to the assembly of the active receptor complex. AMH thus exemplifies a new mechanism for receptor engagement in which interaction with the type II receptor promotes pro-region dissociation to generate mature ligand.

Anti-Müllerian hormone inhibits growth of AMH type II receptor-positive human ovarian granulosa cell tumor cells by activating apoptosis

“…The used AMH concentrations (0-25 mg/ml or 0-179 nm) were similar to those used in epithelial ovarian carcinoma cell studies and in AMH bioactivity assays utilizing this particular AMH stock 20,22,[46][47][48][49] that is only partially cleaved (ie, activated) compared with fully cleaved AMH stocks that were recently established. 50,51 The K d of AMH binding to AMHRII in ovarian carcinoma cell lines (OVCAR5 and OVCAR8) was calculated to be 10.2 nM and 12 nM, respectively, 20,22 and we estimated the K d to be similar in this study. AMH levels in the culture medium of the control samples with 10% FFCS were analyzed with sensitive enzyme-linked immunosorbent assay (DSL-10-14400), according to instructions (Beckman Coulter, Webster, TX, USA).…”

“…Recent results show that endogenous and exogenous AMH needs to be cleaved to attain full activity upon binding to the type II receptor. 48,50,51,60 The AMH reagent used in this and earlier studies [24][25][26][27]61 is only partially cleaved. The production of purified, fully cleaved AMH has been achieved only recently, 51 and with this AMH one would need only 1:1000 of the dose compared with partially cleaved AMH.…”

Anti-Müllerian hormone is a gonadal cytokine with two circulating forms and cryptic actions

“…2; MacLaughlin et al 1992, Wilson et al 1993, Pankhurst & McLennan 2013. Circulating AMH has recently been discovered to contain a mixture of proAMH, which does not appear to activate AMHR2, and AMH N,C , which does (Pankhurst & McLennan 2013;see MacLaughlin et al (1992) and di Clemente et al (2010) for information relating to receptor binding). The implications of this are outlined in the paragraphs below.…”

“…Furthermore, the majority of loss-of-function mutations in human AMH are in the N-terminal domain, emphasising that the non-receptor binding component of AMH has function (Josso et al 2005). Free AMH C has yet to be detected in serum (Pankhurst & McLennan 2013), suggesting that AMH N,C only dissociates at or near the sites of AMH action (see also di Clemente et al (2010)). Experimental studies of AMH have typically used rAMH C , which may not precisely or invariably mimic AMH N,C .…”

Constitutive negative regulation in the processing of the anti-Müllerian hormone receptor II

“…Thus, all superfamily receptors signal through Smad effectors (Massagué and Gomis, 2006;Weiss and Attisano, 2013). In the case of AMH, the ligand-recruiting receptor (antiMüllerian hormone type-II receptor, AMHRII) binds to AMH (di Clemente et al, 2010) and activates a type-I receptor -ALK2, ALK3 or ALK6 (also known as ACVR1, BMPR1A and BMPR1B, respectively) (Clarke et al, 2001;Gouédard et al, 2000;Jamin et al, 2002;Orvis et al, 2008;Sèdes et al, 2013;Visser et al, 2001). The activated type-I receptor phosphorylates Smad1, Smad5 or Smad8 (R-Smads) and promotes their interaction with Smad4 and the translocation of the R-SmadSmad4 complex to the nucleus, where it regulates gene transcription.…”