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*

Mature 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.