Single-molecule force spectroscopy study of interaction between transforming growth factor beta1 and its receptor in living cells.
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 (TbetaRII) and type I receptor (TbetaRI). 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/TbetaRII/TbetaRI formation. In this study, we have investigated the interaction between TGF-beta1 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 TbetaRI with TbetaRII enhanced the binding force of TGF-beta1 with its receptors, whereas the expressed TbetaRI itself exhibited no binding affinity to TGF-beta1. Moreover, the unbinding dynamics of TGF-beta1/TbetaRII and TGF-beta1/TbetaRI/TbetaRII were investigated with dynamic force spectroscopy under different AFM loading rates. The dissociation rate constants of TGF-beta1 with its receptors as well as other parameters characterizing their dissociation pathways were obtained. The results suggested a more stable binding of TGF-beta1 with the receptor after TbetaRI is recruited and the important contribution of TbetaRI to the signaling complex formation during TGF-beta1 signaling.
Yu
Junping
J
Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, People's Republic of China.
Wang
Qiang
Q
Shi
Xiaoli
X
Ma
Xinyong
X
Yang
Huayan
H
Chen
Ye-Guang
YG
Fang
Xiaohong
X
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D006801:Humans; D018625:Microscopy, Atomic Force; D011485:Protein Binding; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D015398:Signal Transduction; D013057:Spectrum Analysis / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D017931:DNA Primers; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta