Abstract
Inhibins A and B negatively regulate the production and secretion of follicle-stimulating hormone from the anterior pituitary, control ovarian follicle development and steroidogenesis, and act as tumor suppressors in the gonads. Inhibins regulate these reproductive events by forming high affinity complexes with betaglycan and activin or bone morphogenetic protein type II receptors. In this study, the binding site of inhibin A for betaglycan was characterized using inhibin A mutant proteins. An epitope for high affinity betaglycan binding was detected spanning the outer convex surface of the inhibin alpha-subunit. Homology modeling indicates that key alpha-subunit residues (Tyr(50), Val(108), Thr(111), Ser(112), Phe(118), Lys(119), and Tyr(120)) form a contiguous epitope in this region of the molecule. Disruption of betaglycan binding by the simultaneous substitution of Thr(111), Ser(112), and Tyr(120) to alanine yielded an inhibin A variant that was unable to suppress activin-induced follicle-stimulating hormone release by rat pituitary cells in culture. Together these results indicate that a high affinity interaction between betaglycan and residues Val(108)-Tyr(120) of the inhibin alpha-subunit mediate inhibin A biological activity.
Highlights
The inhibin residues involved in binding to the type II receptors can be inferred from the crystal structure of activin A bound to ActRIIB [12]
Similar results were obtained using the more physiologically relevant rat pituitary cell culture system, where wild type inhibin A (IC50 ϭ 25 pM) was 4-fold more potent than the K119A/Y120A variant (IC50 ϭ 100 pM) and at least 40-fold more potent than the T111A/S112A/Y120A variant (Fig. 6B). Together these results indicate that inhibin A biological activity is dependent upon a high affinity interaction with its co-receptor, betaglycan
We show that the high affinity interaction of inhibin A with betaglycan, necessary for biological activity, is mediated by residues on the outer convex surface of the ␣-subunit
Summary
Production of Inhibin A Mutants—An overlapping PCR strategy was utilized to incorporate mutations in the mature region of the full-length human inhibin ␣-subunit cDNA. Wild type or selected mutant inhibin ␣-subunit cDNAs, amplified from pcDNA3.1 inhibin vectors, were inserted into the first multiple cloning site at the NheI/EcoRI sites of pIRES. CHO cells were transfected with the pIRES-Inhibin A vectors, and cells stably expressing wild type or mutant inhibin A were selected with 500 g/ml G418. To produce wild type and mutant inhibin A, stable CHO cell lines were grown to confluence, and the medium was changed to serum-free Opti-MEM. ActRII Binding Assay—To ensure that point mutations that disrupted inhibin A binding to betaglycan did not affect overall protein structure, we utilized an activin type II receptor binding assay. The dimer was made by superposing the coordinates of Betaglycan Binding of Inhibin A Mutants—Based on the the inhibin ␣- and A-subunits onto the dimer of Protein Data structural location of the mutated ␣-subunit residues, the. The differential affinity of the inhibin forms for betaglycan [9]
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