Single-Molecule Force Spectroscopy Study of Interaction between Transforming Growth Factor beta 1 and Its Receptor in Living Cells

摘要

Transforming growth factor beta1 (TGF-beta1) regulates many important cellular processes such as cell proliferation, differentiation, and apoptosis, etc. Its signaling is initiated by binding to and bringing together TGF-beta type II receptor (T beta RII) and type I receptor (T beta RI). However, it is not fully understood how the TGF-beta1 ligand-receptor interaction occurs in living cells and what is the molecular mechanism of the signaling complex TGF-beta1/T beta RII/T beta RI formation. In this study, we have investigated the interaction between TGF-beta 1 and its receptors in living cells with single-molecule force spectroscopy for the first time. By positioning TGF-beta1-modified atomic force microscope (AFM) tips on the cells expressing fluorescent protein tagged TGF-beta receptors, the living-cell force measurement was realized with a combined fluorescence microscope and AFM. We found that coexpression of T beta RI with T beta RII enhanced the binding force of TGF-beta 1 with its receptors, whereas the expressed T beta RI itself exhibited no binding affinity to TGF-beta 1. Moreover, the unbinding dynamics of TGF-beta 1/T beta RII and TGF-beta1/T beta RI/T beta RII were investigated with dynamic force spectroscopy under different AFM loading rates. The dissociation rate constants of TGF-beta 1 with its receptors as well as other parameters characterizing their dissociation pathways were obtained. The results suggested a more stable binding of TGF-beta 1 with the receptor after T beta RI is recruited and the important contribution of T beta RI to the signaling complex formation during TGF-beta 1 signaling.