Real-time monitoring of the interactions of transforming growth factor-beta (TGF-beta) isoforms with latency-associated protein and the ectodomains of the TGF-beta type II and III receptors reveals different kinetic models and stoichiometries of binding

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DOIResolve DOI: http://doi.org/10.1074/jbc.M009765200
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TypeArticle
Journal titleThe Journal of Biological Chemistry
Volume276
Issue32
Pages2963229643; # of pages: 12
Subjectpha
AbstractMature transforming growth factor-β (TGF-β) is proteolytically derived from the C terminus of a precursor protein. Latency-associated protein (LAP), the N-terminal remnant of the TGF-β precursor, is able to bind and neutralize TGF-β. Mature TGF-β exerts its activity by binding and complexing members of two subfamilies of receptors, the type I and II receptors. In addition to these signaling receptors, TGF-β can also interact with an accessory receptor termed the type III receptor. Using a surface plasmon resonance-based biosensor (BIAcore), we determined the mechanisms of interaction of four binding proteins (LAP, the type II and III receptor ectodomains (EDs), and a type II receptor ED/Fc chimera) with three TGF-β isoforms, and we quantified their related kinetic parameters. Using global fitting based on a numerical integration data analysis method, we demonstrated that LAP and the type II receptor/Fc chimera interacted with the TGF-β isoforms with a 1:1 stoichiometry. In contrast, the type II ED interactions with TGF-β were best fit by a kinetic model assuming the presence of two independent binding sites on the ligand molecule. We also showed that the type III ED bound two TGF-β molecules. Further experiments revealed that LAP was able to block the interactions of TGF-β with the two EDs, but that the two EDs did not compete or cooperate with each other. Together, these results strongly support the existence of a cell-surface complex consisting of one type III receptor, two TGF-β molecules, and four type II receptors, prior to the recruitment of the type I receptor for signal transduction. Additionally, our results indicate that the apparent dissociation rate constants are more predictive of the neutralizing potency of these TGF-β-binding proteins (LAP, the type II and III receptor EDs, and the type II receptor/Fc chimera) than the apparent equilibrium constants.
Publication date
LanguageEnglish
AffiliationNRC Biotechnology Research Institute; National Research Council Canada
Peer reviewedNo
NRC number44825
NPARC number3539965
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Record identifier5e6ea850-8d96-44f3-a06c-53fd274cc1bc
Record created2009-03-01
Record modified2016-05-09
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