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Activin-A Binds Follistatin and Type II Receptors through Overlapping Binding Sites: Generation of Mutants with Isolated Binding Activities

Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168, Australia

Address all correspondence and requests for reprints to: Craig A. Harrison, Prince Henry’s Institute of Medical Research, 246 Clayton Road, Clayton, Victoria 3168, Australia. E-mail: craig.harrison{at}princehenrys.org.

Follistatin is a potent extracellular antagonist of members of the TGFß superfamily that use activin type II receptors (ActRII/IIB) as part of their signaling complex. A recent crystallographic study indicates that follistatin contacts activin-A residues at both the type I (ALK4) and type II receptor binding interfaces. However, the relative contribution of these two sites on human activin-A to follistatin binding has not been determined. Residues at these sites were mutated to alanine and mutants were screened for their ability to bind follistatin and ActRII and induce FSH secretion from a gonadotrope cell line. Despite extensive mutagenesis across the type I receptor interface, activin-A affinity for follistatin was not significantly diminished. In contrast, mutagenesis of residues at the type II binding interface had pronounced effects on activin’s interaction with follistatin. In particular, residues Leu⁹², Tyr⁹⁴, Ile¹⁰⁰, and Lys¹⁰² were critical for high-affinity follistatin binding. Interestingly, mutation of another primary determinant of ActRII/IIB binding, Ser⁹⁰, did not affect follistatin affinity, suggesting that the interaction surfaces for type II receptors and follistatin were overlapping but not identical. In support, mutation of Asp⁹⁵, on the opposite edge of the common ActRII/follistatin interface, was disruptive for follistatin binding without affecting ActRII/IIB interactions. Activin-S90A was able to compete with wild-type activin for follistatin binding, whereas activin-D95A, due to its 8-fold lower affinity for follistatin, is a potent activin agonist. These reagents could be used to modulate follistatin antagonism of activin and related ligands in processes such as cancer, wound healing, and reproduction.

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