Visualization of CD146 dimerization and its regulation in living cells.
Our previous study showed that the adhesion molecule CD146 as a biomarker is over-expressed on activated endothelium during angiogenesis, which was induced by tumor conditional medium and inhibited by anti-CD146 monoclonal antibody (mAb AA98). However, the CD146 molecular organization on the cells is unknown. Here, using immunoprecipitation, we found that the dimerization of CD146 occurs in both normal and tumor cells. However, the dimer/monomer ratio was higher in tumor cells than in normal cells. Moreover, we found that CD146 dimerization was up-regulated by tumor conditional medium through the NF-kappa B pathway and down-regulated by mAb AA98. To further confirm that CD146 dimerization occurs in living cells, we used fluorescence resonance energy transfer (FRET) with melanoma Mel888 cells co-expressing CFP/YFP-tagged CD146 fusion proteins. By acceptor photobleaching, we observed a strong FRET signal produced by these two fluorescence-tagged proteins. The FRET efficiency reached 20.1%. Our data provide the first evidence that CD146 dimerization occurs in living cells and is regulated within the tumor microenvironment, implying that dimerization of CD146 may be associated with malignancy.
Bu
Pengcheng
P
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China.
Zhuang
Jie
J
Feng
Jing
J
Yang
Dongling
D
Shen
Xun
X
Yan
Xiyun
X
D000911:Antibodies, Monoclonal / Q000276:immunology; D051929:CD146 Antigen / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival; D017077:Culture Media, Conditioned; D019281:Dimerization; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D038761:Photobleaching; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D051929:CD146 Antigen; D017077:Culture Media, Conditioned; D016328:NF-kappa B; D011993:Recombinant Fusion Proteins