Enhancement of the antagonistic potency of transforming growth factor-beta receptor extracellular domains by coiled coil-induced homo- and heterodimerization.
Transforming growth factor-beta (TGF-beta) plays a causal role in several human pathologies including fibrotic diseases and metastasis. TGF-beta 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-beta, making them attractive candidates to sequester TGF-beta 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-beta 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-beta 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.
De Crescenzo
Gregory
G
Cell Signaling and Proteomic Group, Health Sector, and Bioprocess Platform, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada.
Pham
Phuong L
PL
Durocher
Yves
Y
Chao
Heman
H
O'Connor-McCourt
Maureen D
MD
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D008168:Lung / Q000166:cytology; D008907:Mink; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018125:Receptors, Transforming Growth Factor beta / Q000737:chemistry*; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D013997:Time Factors
D018125:Receptors, Transforming Growth Factor beta; D008156:Luciferases