Identification of a Functional Binding Site for Activin on the Type I Receptor ALK4

Abstract

Activins, like other members of the transforming growth factor-␤ (TGF-␤) superfamily, initiate signaling by assembling a complex of two types of transmembrane serine/threonine receptor kinases classified as type II (ActRII or ActRIIB) and type I (ALK4). A kinase-deleted version of ALK4 can form an inactive complex with activin and ActRII/IIB and thereby acts in a dominant negative manner to block activin signaling. Using the complex structure of bone morphogenetic protein-2 bound to its type I receptor (ALK3) as a guide, we introduced extracellular domain mutations in the context of the truncated ALK4 (ALK4-trunc) construct and assessed the ability of the mutants to inhibit activin function. We have identified five hydrophobic amino acid residues on the ALK4 extracellular domain (Leu 40 , Ile 70 , Val 73 , Leu 75 , and Pro 77 ) that, when mutated to alanine, have substantial effects on ALK4-trunc dominant negative activity. In addition, eleven mutants partially affected activin binding to ALK4. Together, these residues likely constitute the binding surface for activin on ALK4. Cross-linking studies measuring binding of 125 Iactivin-A to the ALK4-trunc mutants in the presence of ActRII implicated the same residues. Our results indicate that there is only a partial overlap of the binding sites on ALK4 and ALK3 for activin-A and bone morphogenetic protein-2, respectively. In addition three of the residues required for activin binding to ALK4 are conserved on the type I TGF-␤ receptor ALK5, suggesting the corresponding region on ALK5 may be important for TGF-␤ binding.Activins are members of the transforming growth factor-␤ (TGF-␤) 1 superfamily, which also includes the TGF-␤ (1) and bone morphogenetic protein (BMP) (2) families. These structurally related but functionally diverse polypeptides control the growth and differentiation of many cell types (3-5). In addition, activins regulate the production of follicle-stimulating hormone and other hormones by the anterior pituitary (6 -8) and have diverse endocrine, paracrine, and autocrine actions throughout the reproductive (9), immune (10, 11), hematopoietic (12, 13), endocrine (7), and central nervous systems (14). Activins also play key roles in numerous pathophysiologic processes including carcinogenesis (15). Activins (ϳ26 kDa) are disulfide-linked dimers of related polypeptides consisting of two ␤ chains (activin-A (␤A-␤A), activin-AB (␤A-␤B), and activin-B (␤B-␤B)) (16). The structure of these factors is determined by several conserved cysteine residues that form disulfide bonds in the tightly folded cystine-knot motif that is shared by TGF-␤ superfamily members (17).The signaling events initiated by activin require binding of two types of transmembrane serine/threonine receptor kinases classified as type II (ActRII or ActRIIB) and type I (ALK4). Both receptors are transmembrane proteins with ligand binding activity in the extracellular domain and serine/threonine kinase activity in the intracellular domain (15). The activin type II receptors are the primary li...

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