A covalently dimerized recombinant human bone morphogenetic protein-15 variant identifies bone morphogenetic protein receptor type 1B as a key cell surface receptor on ovarian granulosa cells.

Genetic studies have identified bone morphogenetic protein-15 (BMP15) as an essential regulator of female fertility in humans and in sheep. Oocyte-derived BMP15 is a noncovalently linked dimeric growth factor mediating its effects to ovarian somatic cells in a paracrine manner. Although receptor ectodomains capable of binding BMP15 have previously been reported, no cell surface receptor complex involved in BMP15 signaling has previously been characterized. Here we have expressed and purified recombinant human BMP15 noncovalent and covalent dimer variants. The biological effects of these BMP15 variants were assessed in cultured human granulosa-luteal cells or COV434 granulosa cell tumor cells using BMP-responsive transcriptional reporter assays and an inhibin B ELISA. Biochemical characterization of ligand-receptor interactions was performed with affinity-labeling experiments using [(125)I]iodinated BMP15 variants. Both ligand variants were shown to form homodimers and to stimulate Smad1/5/8 signaling and inhibin B production in human granulosa cells in a similar manner. [(125)I]Iodination of both ligands was achieved, but only the covalent dimer variant retained receptor binding capacity. The [(125)I]BMP15(S356C) variant bound preferentially to endogenous BMP receptor 1B (BMPR1B) and BMPR2 receptors on COV434 cells. Binding experiments in COS cells with overexpression of these receptors confirmed that the [(125)I]BMP15(S356C) variant binds to BMPR1B and BMPR2 forming the BMP15 signaling complex. The results provide the first direct evidence in any species on the identification of specific cell surface receptors for a member of the GDF9/BMP15 subfamily of oocyte growth factors. The fact that BMP15 uses preferentially BMPR1B as its type I receptor suggests an important role for the BMPR1B receptor in human female fertility. The result is well in line with the demonstration of ovarian failure in a recently reported human subject with a homozygous BMPR1B loss-of-function mutant.