A Common Biosynthetic Pathway Governs the Dimerization and Secretion of Inhibin and Related Transforming Growth Factor β (TGFβ) Ligands

Abstract

The assembly and secretion of transforming growth factor ␤ superfamily ligands is dependent upon non-covalent interactions between their pro-and mature domains. Despite the importance of this interaction, little is known regarding the underlying regulatory mechanisms. In this study, the binding interface between the pro-and mature domains of the inhibin ␣-subunit was characterized using in vitro mutagenesis. Three hydrophobic residues near the N terminus of the prodomain (Leu 30 , Phe 37 , Leu 41 ) were identified that, when mutated to alanine, disrupted heterodimer assembly and secretion. It is postulated that these residues mediate dimerization by interacting non-covalently with hydrophobic residues (Phe 271 , Ile 280 , Pro 283 , Leu 338 , and Val 340 ) on the outer convex surface of the mature ␣-subunit. Homology modeling indicated that these mature residues are located at the interface between two ␤-sheets of the ␣-subunit and that their side chains form a hydrophobic packing core. Mutation of these residues likely disturbs the conformation of this region, thereby disrupting noncovalent interactions with the prodomain. A similar hydrophobic interface was identified spanning the pro-and mature domains of the inhibin ␤ A -subunit. Mutation of key residues, including Ile 62 , Leu 66 , Phe 329 , and Pro 341 , across this interface was disruptive for the production of both inhibin A and activin A. In addition, mutation of Ile 62 and Leu 66 in the ␤ A -propeptide reduced its ability to bind, or inhibit the activity of, activin A. Conservation of the identified hydrophobic motifs in the proand mature domains of other transforming growth factor ␤ superfamily ligands suggests that we have identified a common biosynthetic pathway governing dimer assembly.Inhibin A and B, members of the transforming growth factor ␤ (TGF␤) 3 superfamily, negatively regulate the production and secretion of follicle-stimulating hormone from the anterior pituitary (1, 2), control ovarian follicle development and steroidogenesis (3), and act as tumor suppressors in the gonads (4). Outside the hypothalamic pituitary gonadal axis, inhibins contribute to the endocrine regulation of bone metabolism (5) and play critical roles in adrenal gland growth and function (6, 7). It is recognized that inhibins regulate these processes by inhibiting the stimulatory actions of the structurally related proteins, activins (8). Inhibins are heterodimers of an 18-kDa ␣-subunit disulfide linked to one of two 13-kDa ␤-subunits (␤ A and ␤ B ), resulting in inhibin A or inhibin B, respectively. Activins are composed of two ␤-subunits: ␤ A -␤ A (activin A), ␤ A -␤ B (activin AB), and ␤ B -␤ B (activin B). Inhibin antagonism of activin-related ligands is dependent upon interactions with betaglycan, a cell surface proteoglycan that also acts as a TGF␤2 co-receptor (9). Betaglycan binds inhibin A directly and promotes the formation of a stable high affinity complex involving activin type II receptors (10). Sequestration of type II receptors in this way prevents their ...

Active immunization against the proregions of GDF9 or BMP15 alters ovulation rate and litter size in mice

“…However, the physiological significance of secreted BMP15 and GDF9 precursor proteins is yet unknown. The N-terminus proregion peptides used for immunization includes a hydrophobic motif identified as binding to the mature protein at the same site that also interacts with BMPR2 (Walton et al 2009). Antibodies generated against this region in BMP15 altered CL number, but there was no effect on CL number when mice were immunized with the corresponding GDF9 N-terminus peptide, suggestive of the in vivo interactions between the proregion and the mature proteins of mouse GDF9 and BMP15 being functionally different.…”

“…Intracellularly, the proregions of TGFB proteins are known to facilitate protein folding, dimerization, secretion, and stability while interacting noncovalently with the respective mature protein (Gray & Mason 1990, Constam & Robertson 1999, Hashimoto et al 2005, Walton et al 2009). Additionally, a wide range of extracellular functions for proregions of several TGFB family members interacting with mature domains has been observed (Bottinger et al 1996, Hill et al 2002, Haramoto et al 2004, Jiang et al 2004, Brown et al 2005, Le Good et al 2005, Anderson et al 2008, Sengle et al 2011.…”

Two Distinct Regions of Latency-associated Peptide Coordinate Stability of the Latent Transforming Growth Factor-β1 Complex

“…For other family members (e.g. activins and bone morphogenetic proteins), affinity for receptors is greater than affinity for propeptides, and they are secreted in an "active" form (6,22).…”

“…For other family members (e.g. activins and bone morphogenetic proteins), affinity for receptors is greater than affinity for propeptides, and they are secreted in an "active" form (6,22).LTBPs associate with LAP via signature 8-Cys domains, which are unique to these proteins and the structurally related molecules, fibrillin-1 and fibrillin-2 (23-25). Once secreted, LTBPs target latent TGF-␤1 to fibrillin microfibrils within the extracellular matrix.…”

“…Initially, hydrophobic residues near the N terminus of LAP bind to mature TGF-␤1 (6), and this interaction is necessary to maintain the molecule in a conformation competent for dimerization. Two precursors are then covalently linked at sites within both the mature growth factor and LAP to form the small latent complex (SLC) (7)(8)(9)(10).…”