A common biosynthetic pathway governs the dimerization and secretion of inhibin and related transforming growth factor beta (TGFbeta) ligands

Abstract

The assembly and secretion of transforming growth factor beta 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 alpha-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 alpha-subunit. Homology modeling indicated that these mature residues are located at the interface between two beta-sheets of the alpha-subunit and that their side chains form a hydrophobic packing core. Mutation of these residues likely disturbs the conformation of this region, thereby disrupting non-covalent interactions with the prodomain. A similar hydrophobic interface was identified spanning the pro- and mature domains of the inhibin beta(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 beta(A)-propeptide reduced its ability to bind, or inhibit the activity of, activin A. Conservation of the identified hydrophobic motifs in the pro- and mature domains of other transforming growth factor beta superfamily ligands suggests that we have identified a common biosynthetic pathway governing dimer assembly.

FIGURE 1.

Effects of α-subunit prodomain (Pro) mutations on inhibin A biosynthesis. A, hydrophobic residues in the inhibin α-subunit prodomain were substituted with alanine using in vitro mutagenesis. To determine the effects of amino acid substitutions on inhibin A production, culture medium (B) and cell lysate (C) from CHO cells transfected with either wild type (lane 1) or mutant α-subunit (lanes 2–11), in combination with the β_A-subunit, were analyzed by Western blot. Samples were detected with the R1 mAb, specific for the inhibin αC (mature) domain. The 31-kDa inhibin A dimer, 52-kDa free α-subunit, and higher molecular mass inhibin precursors forms (65 and 95 kDa) are noted. D, the effect of α-subunit prodomain mutations on inhibin A expression in CHO culture medium was also determined by ELISA (^* = p < 0.05). WT, wild type.

FIGURE 2.

Sequence alignment of prodomains for human TGFβ ligands. The inhibin α-subunit (Inh α) prodomain was aligned with the prodomains of human TGFβ ligands using ClustalW. The residues are numbered according to the first residue of the signal peptide. The three residues determined in this study to be essential for inhibin dimer formation and secretion (Leu³⁰, Phe³⁷, and Leu⁴¹) are highlighted. The identified residues lie within a conserved hydrophobic motif (bottom of alignment). Act, activin.

FIGURE 3.

Effects of β_A prodomain (pro-βA) mutations on inhibin/activin biosynthesis. A, alanine substitutions were made in the hydrophobic residues of the inhibin β_A prodomain using in vitro mutagenesis. To determine the effects of these amino acid substitutions on inhibin A and activin A production, culture medium from CHO cells transfected with either wild type (lane 1) or mutant β_A-subunit (lanes 2–4), in combination with the α-subunit, were analyzed by Western blot. Blots were probed with the E4 mAb, specific for the inhibin/activin mature β_A domain (B) and the R1 mAb specific for the inhibin αC (mature) domain (C). D, Western blot analysis of the cell lysates of CHO cells transfected with either wild type (lane 1) or mutant β_A-subunit cDNAs (lanes 2–4) is shown. The 31-kDa inhibin A dimer, 24-kDa activin A dimer, 54-kDa free βA-subunit, and higher molecular mass precursor forms of inhibin and activin are noted. The effect of β_A-subunit prodomain mutations on activin A (E) and inhibin A (F) expression in CHO culture medium was also determined by ELISA (^* = p < 0.05). WT, wild type.

FIGURE 4.

Analysis of the interaction between the β_A-propeptide and mature inhibin and activin A dimers. A, ligand blot analysis of wild type (WT) and mutant β_A-propeptide binding to ¹²⁵I-inhibin A and ¹²⁵I-activin A dimers. Wild type and mutant β_A-propeptide (with C-terminal FLAG tag) were loaded at equivalent concentrations (as determined by Western blotting with the FLAG M2 mAb, top panel) onto SDS-PAGE and transferred to an ECL Hybond membrane. Membranes were probed with either ¹²⁵I-activin A (middle panel) or ¹²⁵I-inhibin A (bottom panel). B and C, the ability of the activin type II receptors (ActRIIA and ActRIIB) to compete with the β_A-propeptide (proβA) for binding to mature activin A was assessed by immunoprecipitation. Increasing concentrations of ActRIIA (B) and ActRIIB (C) extracellular domains (ECD) (25 ng–4 μg; R&D Systems) were added to samples containing wild type β_A-propeptide (400 ng) and activin A (12.5 ng). Samples were immunoprecipitated (IP) using FLAG M2 affinity resin and detected by immunoblot (IB) using the activin β_A subunit mAb (E4). To ensure that equal amounts of activin and β_A-propeptides were present in each of the samples, immunoblots using the FLAG M2 and E4 antibodies were also performed prior to immunoprecipitation. D, in vitro bioassay to assess the ability of wild type and mutant β_A-propeptides to block activin signaling. Adrenocortical cells were transfected with an activin responsive luciferase reporter and treated with 400 pm activin A (Act A) and increasing doses of either wild type or mutant β_A-propeptides (0.5–30 nm) (^* = p < 0.05).

FIGURE 5.

Effects of αC mutations on inhibin A biosynthesis. A, hydrophobic residues in the inhibin αC (mature) domain were substituted with alanine using in vitro mutagenesis. Pro, prodomain. To determine the effects of amino acid substitutions on inhibin A production, culture medium (B) and cell lysate (C) from CHO cells transfected with either wild type (lane 1) or mutant α-subunit (lanes 2–9), in combination with the β_A-subunit, were analyzed by Western blot. Samples were detected with the R1 mAb, specific for the inhibin αC domain. The 31-kDa inhibin A dimer, 52-kDa free α-subunit, and higher molecular mass inhibin precursors forms are noted. D, the effect of αC mutagenesis on inhibin A expression in CHO culture medium was also determined by ELISA (^* = p < 0.05). WT, wild type.

FIGURE 6.

Sequence alignment of the mature domains for the human TGFβ ligands. Residues comprising finger 1 (Ser²⁷⁰–Ile²⁸⁷) and finger 2 (Met³³⁵–Tyr³⁵²) of the mature inhibin α-subunit (Inh α) were aligned with the mature domains of human TGFβ ligands using ClustalW. The residues determined in this study to be essential for inhibin dimer formation and secretion (Phe²⁷¹, Ile²⁸⁰, Pro²⁸³, Leu³³⁸, and Val³⁴⁰) (highlighted) lie within the finger regions of the αC mature domain. Act, activin.

FIGURE 7.

Effects of mutations in the mature domain of the inhibin/activin A β_A-subunit on biosynthesis. A, key hydrophobic residues in the mature domain of the inhibin/activin β_A-subunit were substituted with alanine using in vitro mutagenesis. To determine the effects of these amino acid substitutions on inhibin A and activin A production, culture medium from CHO cells transfected with either wild type (lane 1) or mutant β_A-subunit (lanes 2–6), in combination with the α-subunit, was analyzed by Western blot. proβA, β_A-prodomain. Samples were detected with the E4 mAb specific for the inhibin/activin mature β_A domain (B) and the inhibin αC-specific R1 mAb (C). The 31-kDa inhibin A dimer, 24-kDa activin A dimer, 54-kDa free βA-subunit, and higher molecular mass precursor forms of inhibin and activin are noted. The effects ofβ_A-subunit mutations on activin A (D) and inhibin A (E) expression in CHO culture medium was also determined by ELISA (^* = p < 0.05). WT, wild type.

FIGURE 8.

Homology model of inhibin A. A, a homology model of the mature inhibin A dimer was generated in a previous study (38). The inhibin α-subunit is colored orange, whereas the inhibin β_A-subunit is green. The hydrophobic residues identified in the mature domains of the inhibin α-(magenta) and β_A-subunits (blue) that are required for inhibin biosynthesis were mapped onto the model. The identified residues lie on the outer convex surface of the finger regions, and the side chains of these residues form a hydrophobic packing core. Note that these residues are distant from the inhibin α/βA dimer interface. B, a model for the correct folding, dimerization, secretion, and activation of inhibin A.