Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells.
Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to beta2 subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively.
Fu
Guo
G
State Key Laboratory of High Field Laser Physics, Shanghai Institute of Optics and Fine Mechanics, Chinese Academy of Sciences, Shanghai 201800, China.
Yang
Hua-yan
HY
Wang
Chen
C
Zhang
Feng
F
You
Zhen-dong
ZD
Wang
Gui-ying
GY
He
Cheng
C
Chen
Yi-zhang
YZ
Xu
Zhi-zhan
ZZ
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D016177:Macrophage-1 Antigen / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D016177:Macrophage-1 Antigen; D021122:Protein Subunits; D011994:Recombinant Proteins