TGF-β isoforms, TGF-β1, 2, and 3, are disulfide-linked homodimers with molecular weights of approximately 25 kDa ().1, 2, 3
TGF-β signaling occurs upon formation of a quinary complex that consists of TGF-β and two copies each of the transmembrane Ser/Thr kinase receptors, TβRI and TβRII.2, 4
Signaling complex formation occurs when TGF-β1 or TGF-β3 binds with high affinity (Kd
~5-30 pM) to two copies of TβRII. The resulting TGF-β:TβRII complex then recruits two copies of TβRI to form a hetero-oligomeric complex. The TGF-β2 homolog is lacking two key arginine residues present in TGF-β1 and TGF-β3 that facilitate high affinity interactions with TβRII;5
therefore, TGF-β2 requires a coreceptor (β-glycan or TβRIII) to assemble a signaling complex. Once TβRI and TβRII are proximal, the cytoplasmic domain of TβRII catalyzes the phosphorylation of multiple TβRI threonine and serine residues within a conserved juxtamembrane GS domain (a 30-amino acid region that contains a characteristic SGSGSG sequence). GS domain phosphorylation promotes activation of the adjacent TβRI kinase domain. The activated enzyme then catalyzes the phosphorylation of the receptor-regulated Smad proteins (R-Smad),6
Smad2 and Smad3, with the help of an adaptor protein SARA (Smad anchor for receptor activation).7
R-Smads are critical regulators of TGF-β signaling that shuttle between the cytoplasm and nucleus. Upon growth factor stimulation, the phosphorylated Smad2 or Smad3 dissociates from SARA and binds to the common Smad (co-Smad), Smad4; this complex undergoes nuclear translocation. Once in the nucleus, the Smad complex interacts with various DNA binding partners to activate or repress the expression of hundreds of genes.
FIG. 1 (A) Schematic depiction of the TGF-β signaling pathway. The covalently linked TGF-β homodimer (orange) binds to two copies of TβRII (green) which forms non-covalent homodimers as well as higher order oligomers.18 The TGF-β/TβRII (more ...)
TGF-β-induced changes in gene expression elicit a wide range of cellular responses, including cell adhesion, migration, extracellular matrix deposition, proliferation, apoptosis, and differentiation.3, 8
Depending on the cellular context, TGF-β can play essential or deleterious roles in development, immunity, wound healing, or cancer. For example, the growth factor controls embryonic stem cell self-renewal as well as important developmental processes such as the epithelial to mesenchymal transition.9
Loss of TGF-β signaling is associated with autoimmunity, which highlights its role in immune suppression.10
TGF-β is crucial for wound healing, but its prolonged presence causes inflammation and scar formation.11
Another role for the growth factor is as a strong tumor suppressor, yet it is also implicated in the late stage metastasis of many cancer types.12
Because of the important and myriad roles of TGF-β, its ligands would be valuable tools. They could be used to probe its diverse cellular functions and facilitate the identification of potential therapeutics. Hence, TGF-β isoforms and their receptors are popular targets for small molecule screens and antibody-based therapeutics.13
Compounds that inhibit the TβRI kinase domain and the highly related kinase domains of another two type I receptors, Activin A and Nodal, have been sought.14
One such compound, the kinase inhibitor SB-431542, has become a powerful tool for assessing the involvement of TGF-β signaling in specific biological processes. TGF-β2 antisense oligonucleotides,15
and peptide ligands for the growth factor17
have been developed to dissect the roles of each individual TGF-β isoform. These investigations highlight the utility of compounds that act on targets within the TGF-β pathway for dissecting the function of TGF-β signaling components in development and disease.
These valuable tools, combined with structures of the TGF-β:TβRI-ED:TβRII-ED complex determined by X-ray crystallography,19, 24
have led to insight into the function of this canonical signaling complex. The growth factor and its receptors, however, have additional binding partners, and the roles of these interactions in TGF-β signaling are less explored. For example, TGF-β isoforms bind to β-glycan and endoglin. Endoglin is another type III receptor (sharing 71% amino acid identity in the transmembrane and cytoplasmic domain with β-glycan) that is highly expressed in proliferating endothelial cells. The receptors, TβRI and TβRII, also interact with endoglin through both their extracellular domains and cytoplasmic domains.26
Thus, although TβRI and TβRII have small extracellular domains (~150 residues) and large surface areas are buried in the TGF-β:TβRI-ED:TβRII-ED ternary complex,24
they possess other hot spots27
for protein-protein interactions. We found this feature of the receptors intriguing. Because phage-display has been used to identify preferred binding sites for protein–protein interactions, 28, 29
we envisioned applying this method to the TβR extracellular domains to indentify novel TβR ligands.
Phage-displayed peptide library screening is a technique used to identify ligands for protein targets.29, 30
Compounds that disrupt31
or promote protein–protein interactions,32
function as hormone or growth factor mimetics,33
or serve as ligands for whole cells34
have been discovered using this technology. We screened a phage-displayed peptide library using TβRI-ED as bait to identify TβR ligands. Intriguingly, this screen yielded peptide ligands that recognize both TβRI-ED and TβRII-ED, yet do not compete with TGF-β. Thus, our data indicate that TβRI and TβRII share a novel binding site that may serve as a target for probing and modulating TGF-β function.