Cellular signaling and gene expression profiles evoked by a bivalent macrocyclic peptide that serves as an artificial MET receptor agonist

Wenyu Miao,Katsuya Sakai,Takumi Nishiuchi,Masataka Umitsu,Junichi Takagi,Tomomi Morioka,Naoya Ozawa,Yoshinori Suzuki,Hiroaki Suga,Kenichiro Ito,Kunio Matsumoto

doi:10.1038/s41598-018-34835-4

Abstract

Non-native ligands for growth factor receptors that are generated by chemical synthesis are applicable to therapeutics. However, non-native ligands often regulate cellular signaling and biological responses in a different manner than native ligands. Generation of surrogate ligands comparable to native ligands is a challenging need. Here we investigated changes in signal transduction and gene expression evoked by a bivalent macrocyclic peptide (aMD5-PEG11) capable of high-affinity binding to the MET/hepatocyte growth factor (HGF) receptor. Binding of aMD5-PEG11 to the MET extracellular region was abolished by deletion of the IPT3−IPT4 domain, indicating the involvement of IPT3−IPT4 in the binding of aMD5-PEG11 to the MET receptor. aMD5-PEG11 induced dimerization and activation of the MET receptor and promoted cell migration that was comparable to induction of these activities by HGF. Signal activation profiles indicated that aMD5-PEG11 induced phosphorylation of intracellular signaling molecules, with a similar intensity and time dependency as HGF. In 3-D culture, aMD5-PEG11 as well as HGF induced epithelial tubulogenesis and up-regulated the same sets of functionally classified genes involved in multicellular organism development. Thus, a non-native surrogate ligand that consisted of a bivalent macrocyclic peptide can serve as an artificial MET receptor agonist that functionally substitutes for the native ligand, HGF.

Highlights

Because growth factors and their receptors play critical roles in stem cell maintenance, tissue regeneration, and wound healing, growth factors have been developed as potent therapeutic drugs
The extracellular region of the MET receptor is composed of a semaphorin (SEMA) domain, a plexin-semaphorin-integrin (PSI) domain, and four immunoglobulin-like plexin transcription (IPT) factor domains[14,15]
A clear association between aMD5 and the fulllength MET receptor extracellular region was seen at a KD value of 11 nM, whereas the association was lost when the IPT3–IPT4 or IPT1–IPT4 domains were deleted from the full-length extracellular region (Fig. 1C)

Summary

Introduction

Because growth factors and their receptors play critical roles in stem cell maintenance, tissue regeneration, and wound healing, growth factors have been developed as potent therapeutic drugs. We demonstrated that crosslinking of MET receptor-binding peptides identified by flexizyme-based cell-free translation and an mRNA display–based screening method (Random non-standard Peptide Integrated Discovery: RaPID) can generate agonistic ligands capable of inducing MET receptor activation and MET receptor-mediated biological responses[13]. The details of intracellular signaling and the gene expression profile of HGF compared to macrocyclic peptides that are MET receptor agonists have not been addressed. If HGF and a MET receptor agonist that is a macrocyclic peptide evoke a similar intracellular signaling cascade, gene expression profile, and biological responses, RaPID-based macrocyclic peptide discovery and appropriate modification of bivalent/multivalent structures will provide a widely applicable strategy to obtain agonist macrocyclic peptides that substitute for growth factors and that activate cytokine receptors. We investigated changes in MET receptor dimerization, MET receptor activation, activation of intracellular signal transducers, and the gene expression profile that were triggered by HGF and the bivalent MET receptor agonist peptide

Methods

Results

Conclusion