Characterisation of the bioactivity of the naturally occurring Inhibin Proα-peptide. (#211)
Inhibin A and activin A reciprocally regulate follicle stimulating hormone synthesis by the pituitary. Similar to other TGF-β proteins, inhibin A and activin A are synthesised as large precursor molecules, comprising N-terminal pro- and C-terminal mature domains. The pro-inhibin heterodimer comprises α- and βA-subunit chains, whereas pro-activin is a homodimer of two βA chains. We have previously shown that key residues in an α-helix (α1) within the prodomain promote the folding and dimerisation of pro-inhibin A and activin A. Following dimerisation, the inhibin α- and β-subunit prodomains are enzymatically cleaved from the mature domains at consensus RXXR sites (site1). The inhibin α-subunit is a unique TGF-β ligand, comprising a second cleavage site (site2) within its prodomain. Using site-directed mutagenesis we showed that silencing of site2 in the inhibin α-subunit prodomain, abrogated the synthesis of inhibin A. Thus, processing at site2 limits the bioavailability of inhibin. Cleavage at site2 in the inhibin α-subunit prodomain releases a 43-amino acid Proα-peptide, comprising the α1-helix. We aimed to determine the influence of this peptide on inhibin and activin bioactivity. Ligand blot analysis and solid-phase binding assays indicated that the proα-peptide binds specifically to mature inhibin, and was unable to bind activin. Furthermore, the proα-peptide was capable of suppressing inhibin A bioactivity in primary rat pituitary cells. To ascertain the contribution of Proα in naturally occurring inhibin forms, we silenced the cleavage sites in the inhibin α-subunit and β-subunits using in vitro mutagenesis to promote the production of Pro-inhibin forms. Interestingly, we found that the Pro-inhibin forms had suppressed immunoreactivity in inhibin ELISAs. Activation of the Proα-containing inhibin forms using dissociating agents rescued the immunoreactivity. The present results indicate that the naturally occurring inhibin Proα-peptide is an inhibin-specific binding protein that limits the immuno- and bioactivity of inhibin forms.