[Analysis of Toll-like receptor 4 and myeloid differentiation protein-2 interaction with fluorescence resonance energy transfer].
OBJECTIVE
To study the interaction between Toll-like receptor (TLR) 4 and myeloid differentiation protein-2 (MD-2) in living cells using fluorescence resonance energy transfer (FRET) technology.


METHODS
The coding sequences of TLR4 and MD-2 (without the signal peptide sequence) were amplified by PCR and cloned into enhanced cyan fluorescence protein (CFP) and enhanced yellow fluorescence protein (YFP) expression vectors carrying TLR4 signal peptides (pECFP-C1-SP and pEYFP-C1-SP). HEK293 cells were transfected respectively or together with the reconstructed plasmids verified by enzyme digestion and sequence analysis, and the expression and sublocalization of these fluorescence proteins in the cells were observed using fluorescence microscope. FRET in the cells coexpressing CFP-TLR4 and YFP-MD-2 was detected using routine and acceptor photobleaching method.


RESULTS
The reconstructed plasmids were expressed in HEK293 cells. The cyan or yellow fluorescence was located in the cytoplasm, mainly around the nucleus in the cells transfected with pECFP/TLR4 or pEYFP/MD-2, and both the cyan and yellow fluorescence located mainly in the membrane and occasional in the cytoplasm of cells cotransfected with pECFP/TLR4 and pEYFP/MD-2. Routine or acceptor photobleaching detected FRET phenomena in cells coexpressing CFP-TLR4 and YFP-MD-2, suggesting direct interaction between TLR4 and MD-2.


CONCLUSIONS
This study provides direct evidence of the interaction between TLR4 and MD-2 in living cells.
Liu
Ya-wei
YW
College of Basic Medicine, Southern Medical University, Guangzhou 510515, China.
Liu
Jing-hua
JH
Tang
Jing
J
Zhao
Qing
Q
Li
Jian-jun
JJ
Zhao
Ming-zhe
MZ
Li
Zhi-jie
ZJ
Wang
Guo-jun
GJ
Zhong
Tian-yu
TY
Deng
Peng
P
Jiang
Yong
Y
D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051218:Lymphocyte Antigen 96 / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
C495377:LY96 protein, human; D008164:Luminescent Proteins; D051218:Lymphocyte Antigen 96; D011993:Recombinant Fusion Proteins; D051197:Toll-Like Receptor 4