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.
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.
De Crescenzo
G
G
Biotechnology Research Institute (National Research Council Canada), Montreal, Quebec H4P 2R2, Canada.
Grothe
S
S
Zwaagstra
J
J
Tsang
M
M
O'Connor-McCourt
M D
MD
D000818:Animals; D001665:Binding Sites; D004847:Epithelial Cells / Q000378:metabolism; D007700:Kinetics; D008168:Lung / Q000166:cytology; D008907:Mink; D008956:Models, Chemical; D008962:Models, Theoretical; D011485:Protein Binding; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D011509:Proteoglycans / Q000378:metabolism; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000737:chemistry* / Q000378:metabolism*
D020033:Protein Isoforms; D011509:Proteoglycans; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta; C071736:betaglycan; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II