Enhancement of the Antagonistic Potency of Transforming Growth Factor-β Receptor Extracellular Domains by Coiled Coil-induced Homo- and Heterodimerization

Abstract

Transforming growth factor-␤ (TGF-␤) plays a causal role in several human pathologies including fibrotic diseases and metastasis. TGF-␤ signaling is mediated through its interaction with three types of cell surface receptors, RI, RII, and RIII. The soluble ectodomains of RII and RIII bind to TGF-␤, making them attractive candidates to sequester TGF-␤ and inhibit its activity. To optimize the activity of the ectodomains, we studied the effect of artificially dimerizing them upon their kinetics of binding to TGF-␤ using an optical biosensor and studied their antagonistic potencies using an in vitro signaling assay. We fused the RII ectodomain and the membrane-proximal ligand-binding domain of the RIII ectodomain to de novo designed heterodimerizing coil strands and demonstrated that the coil strands within the fusion proteins were capable of promoting the dimerization of the coil-tagged ectodomains. Our results indicate that coiled coil-induced dimerization of the ectodomains stabilized their interaction with TGF-␤ as compared with the monomeric ectodomains. Also, in contrast to the monomeric ectodomains, which did not block signaling, the coiled coil-induced dimers were characterized by antagonistic potencies in the low nanomolar range.Mammalian transforming growth factor-␤ isoforms (TGF-␤1, -␤2, and -␤3) 1 mediate signaling by binding to and complexing three types of cell surface receptors known as the TGF-␤ type I (RI), type II (RII), and type III (RIII) receptors (1). In the absence of ligand, both RI and RII can form homooligomers (2). TGF-␤1 and TGF-␤3 bind simultaneously to two RII ectodomains (RIIEDs) (3). This binding event is thought to reorient the RIIs at the cell surface, allowing for the formation of a signaling-competent RI-RII complex (4). RIII was first thought to be an "accessory" receptor whose role is to present TGF-␤2 to RI and RII (5). Recent studies suggest, however, that the role of RIII is more complex. Firstly, it was demonstrated that RIII is required for TGF-␤1-promoted mesenchymal transformation during chick embryonic heart development (6). Furthermore, it was shown that the RIII cytoplasmic domain is important for signaling (7) and that the role of RIII in signaling may be modulated by its glycosylation state (8). Two independent TGF-␤-binding domains have been identified within the RIII ectodomain (9 -11), further emphasizing the complexity of this receptor.TGF-␤ is involved in the regulation of many physiological processes including cell growth and differentiation. TGF-␤ overexpression plays a key role in several human pathologies including fibrotic diseases (12) and metastasis (13). Recently, two reports provided encouraging results showing that a TGF-␤ antagonist, corresponding to the RIIED artificially dimerized through the Fc portion of an antibody (RIIED-Fc), reduces metastasis in vivo without any adverse side effects (14, 15). Other soluble TGF-␤-binding proteins, including the ectodomain of RIII, have also been evaluated for their antagonistic potency (16 -19). We reported th...

In the Absence of Type III Receptor, the Transforming Growth Factor (TGF)-β Type II-B Receptor Requires the Type I Receptor to Bind TGF-β2

“…Corroborating data from a biological inhibition assay of TGF-␤ activity in cells were consistent with the selectivity and specificity of sT␤RII.Fc and sT␤RII-B.Fc seen in the binding data. Our measured binding affinities (30 to 500 pM) are consistent with previously published affinities for the ECD of T␤RII (31,36,37), and compare well with indirect estimates for membrane-bound complexes (1).…”

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications

“…the generation of mini-antibodies [41,42], the assembly of receptor ectodomain subunits [9,19,[43][44][45], and as an affinity tag system for purification and biosensor applications [8][9][10][46][47][48]. Immobilized EGF has been shown to be more mitogenic for CHO cells than soluble EGF [29], perhaps due to decreased ligand inter- nalization and degradation [30].…”

“…as a tag for purification purposes or for induced protein-protein interaction studies. We have shown previously using SPR-based biosensor studies that E5 or K5-coil-tagged receptor ectodomains (from the transforming growth factor (TGF)-b Types II and III receptors) bind TGF-b similarly to untagged receptor ectodomains, and that the coil tags can be used for receptor capture on the biosensor surface [9,10]. More recently, we used the E/K coiled-coil dimerization system to establish a virus retargeting scheme in which E5-coil-EGF was utilized to retarget K4-coil-expressing adenovirus to EGFR expressing cells [11].…”

Reconstitution and Analysis of Soluble Inhibin and Activin Receptor Complexes in a Cell-free System

“…However, modeling studies of TGF-␤ superfamily receptors suggest that the type I and type II receptors exist as multimers in cells even in the absence of ligand (15). It was recently shown that, when obligated to form dimers through addition of heterologous coiledcoil domains, the affinity of sT␤RII and sT␤RIII for TGF-␤ was increased such that biological antagonism could be observed (36). Interestingly, the expression system used for the present studies utilizes a portion of the human Fc chain that contains a free cysteine, resulting in the receptor-Fc fusion proteins being secreted as dimers.…”