Poster Presentation ESA-SRB 2023 in conjunction with ENSA

Characterisation of inhibin-binding to its novel co-receptor, TGFBR3L (#231)

Haitong Luan 1 , William A Stocker 2 , Evan RS Buddle 3 , Daniel J Bernard 3 , Craig A Harrison 1 , Kelly L Walton 4
  1. Department of Physiology, Monash Biomedicine Discovery Institute,Monash University, Clayton, Victoria, Australia
  2. Monash University, Clayton, VIC, Australia
  3. Department of Pharmacology and Therapeutics, McGill University, Montreal, QC , Canada
  4. School of Biomedical Science, University of Queensland, Brisbane, QLD, Australia

The gonadotrope-restricted transmembrane protein, transforming growth factor-β receptor 3-like (TGFBR3L), has emerged as an inhibin B-specific co-receptor involved in the regulation of follicle-stimulating hormone (FSH) synthesis. This novel co-receptor shares a high degree of similarity with known inhibin co-receptor, betaglycan (TGFBR3). As TGFBR3L specifically binds inhibin B (α/βB dimers) and not inhibin A (α/βA dimers), it is hypothesised that inhibin B binding to TGFBR3L is mediated via discrete residues in the bB-subunit. Here, we aimed to identify the key residues in the βB-subunit that permit binding of inhibin B, and not inhibin A, to TGFBR3L. Using site-directed mutagenesis and luciferase reporter assays, we identified a cluster of residues within the inhibin βB-subunit (Leu340, Thr352 and Ala353) critical for TGFBR3L interactions. Substitution of these βB-subunit residues with the corresponding residues from the βA-subunit disrupted the ability of inhibin B to dock onto TGFBR3L and suppress activin-mediated signalling in COV434 granulosa cells. Interestingly, the Leu340Ile inhibin B variant retained in vitro bioactivity in the presence of betaglycan, despite being non-functional in the presence of TGFBR3L. Conversely, Thr352Ser and Ala353Thr βB-subunit mutations disrupted inhibin B in vitro bioactivity in both the presence of TGFBR3L and betaglycan, likely indicating cross-over with ligand/co-receptor contact sites. Importantly, mutation of these βB-subunit residues did not impair the ability of resultant activin B forms (βB/B dimers) to signal via the activin type II receptors in these cells. Introducing the identified key residues in the βB-subunit into the inhibin βA-subunit permitted inhibin A activity via TGFBR3L (IC50=1.9nM), albeit at lower potency than wild-type inhibin B (IC50=0.15nM). Together, our results have identified key residues in the inhibin βB-subunit that mediate the specific binding of inhibin B to itsnewly identified co-receptor, TGFBR3L. Understanding the mechanisms by which inhibin B modulates FSH will advance the development of reproductive technologies.