Peptide ligands that use a novel binding site to target both TGF-β receptors

The transforming growth factor beta (TGF-beta) signaling pathway plays myriad roles in development and disease. TGF-beta isoforms initiate signaling by organizing their cell surface receptors T beta RI and T beta RII. Exploration and exploitation of the versatility of TGF-beta signaling requires an enhanced understanding of structure-function relationships in this pathway. To this end, small molecule, peptide, and antibody effectors that bind key signaling components would serve as valuable probes. We focused on the extracellular domain of T beta R1 (T beta RI-ED) as a target for effector screening. The observation that T beta RI-ED can bind to a TGF-beta coreceptor (endoglin) suggests that the Tb beta I-ED may have multiple interaction sites. Using phage display, we identified two peptides LTGKNFPMFHRN (Pep1) and MHRMPSFLPTTL (Pep2) that bind the T beta RI-ED (K-d approximate to 10(-5) M). Although our screen focused on T beta RI-ED, the hit peptides interact with the T beta RII-ED with similar affinities. The peptide ligands occupy the same binding sites on T beta RI and T beta RII, as demonstrated by their ability to compete with each other for receptor binding. Moreover, neither interferes with TGF-beta binding. These results indicate that both T beta RI and T beta RII possess hot spots for protein-protein interactions that are distinct from those used by their known ligand TGF-beta. To convert these compounds into high affinity probes, we exploited the observation that T beta RI and T beta RII exist as dimers on the cell surface; therefore, we assembled a multivalent ligand. Specifically, we displayed one of our receptor-binding peptides on a dendrimer scaffold. We anticipate that the potent multivalent ligand that resulted can be used to probe the role of receptor assembly in TGF-beta function.