Real-time Monitoring of the Interactions of Transforming Growth Factor-β (TGF-β) Isoforms with Latency-associated Protein and the Ectodomains of the TGF-β Type II and III Receptors Reveals Different Kinetic Models and Stoichiometries of Binding

Gregory De Crescenzo,Suzanne Grothe,John Zwaagstra,Monica Tsang,Maureen D O'Connor-Mccourt

doi:10.1074/jbc.m009765200

Abstract

Mature transforming growth factor-beta (TGF-beta) is proteolytically derived from the C terminus of a precursor protein. Latency-associated protein (LAP), the N-terminal remnant of the TGF-beta precursor, is able to bind and neutralize TGF-beta. Mature TGF-beta 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-beta 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-beta 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-beta isoforms with a 1:1 stoichiometry. In contrast, the type II ED interactions with TGF-beta 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-beta molecules. Further experiments revealed that LAP was able to block the interactions of TGF-beta 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-beta 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-beta-binding proteins (LAP, the type II and III receptor EDs, and the type II receptor/Fc chimera) than the apparent equilibrium constants.

Highlights

Peptides involved in the regulation of growth, development, tissue repair, tumorigenesis, inflammation, and host defense [1,2,3,4]
Using global fitting based on a numerical integration data analysis method, we demonstrated that Latency-associated protein (LAP) and the type II receptor/Fc chimera interacted with the TGF-␤ isoforms with a 1:1 stoichiometry
These results strongly support the existence of a cellsurface complex consisting of one type III receptor, two TGF-␤ molecules, and four type II receptors, prior to the recruitment of the type I receptor for signal transduction

Summary

Introduction

Peptides involved in the regulation of growth, development, tissue repair, tumorigenesis, inflammation, and host defense [1,2,3,4]. The isolated ectodomains (EDs) of the type II and III receptors are soluble and retain their ability to bind to TGF-␤ isoforms [15,16,17] They exert, to LAP, an antagonistic activity by binding to TGF-␤ and sequestering it away from cell-surface receptors [15, 18]. The ability of LAP, the type II ED, the type II ED/Fc chimera, and the type III ED to antagonize binding of TGF-␤ to its cell-surface receptors was tested using mink lung epithelial cells stably transfected with a TGF-␤sensitive luciferase reporter gene These results were correlated with the BIAcore data. Results of our kinetic analysis indicate that the apparent dissociation rate constant may be the best predictor of the antagonistic potency of TGF-␤-binding proteins

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