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A more recent version of this article appeared on June 11, 2004
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Papers In Press, published online ahead of print March 30, 2004
J. Biol. Chem, 10.1074/jbc.M311256200
Submitted on October 13, 2003
Revised on March 30, 2004
Accepted on March 30, 2004

Apolipoprotein E4 domain interaction occurs in living neuronal cells as determined by fluorescence resonance energy transfer

Qin Xu, Walter J. Brecht, Karl H. Weisgraber, Robert W. Mahley, and Yadong Huang

Gladstone Institute of Neurological Disease, University of California, San Francisco, San Francisco, CA 94110

Corresponding Author: yhuang{at}gladstone.ucsf.edu

Apolipoprotein (apo) E4 is a major risk factor for Alzheimer’s disease (AD). Although the mechanisms remain to be determined, the detrimental effects of apoE4 in neurobiology must be based on its unique structural and biophysical properties. One such property is domain interaction, mediated by a salt bridge between Arg-61 in the N-terminal domain and Glu-255 in the C-terminal domain of apoE4. This interaction, which does not occur in apoE3 or apoE2, causes apoE4 to bind preferentially to certain lipoprotein particles in vitro and in vivo. Here we used fluorescence resonance energy transfer (FRET) to determine if apoE4 domain interaction occurs in living neuronal cells. Neuro-2a cells were transfected with constructs encoding apoE3 or apoE4 in which yellow fluorescent protein (YFP) was fused to the N-terminus and cyan fluorescent protein (CFP) to the C-terminus. To generate a FRET signal that can be detected by spectrum confocal microscopy, the labeled N- and C- termini must be in close proximity (<100 Å). FRET occurred in cells transfected with YFP-apoE4-CFP, but not in those transfected with YFP-apoE3-CFP, suggesting that the N- and C-termini of apoE4 are in close proximity in living cells, and those of apoE3 are not. FRET did not occur in cells cotransfected with YFP-apoE4 and apoE4-CFP, suggesting that the FRET in YFP-apoE4-CFP-transfected cells was intramolecular. Mutation of Arg-61 to Thr or Glu-255 to Ala in apoE4, which disrupts domain interaction, abolished FRET in Neuro-2a cells, strongly suggesting that the FRET in YFP-apoE4-CFP cells was caused by domain interaction. ApoE4-producing cells secreted less phospholipid than apoE3-producing cells, but after disruption of domain interaction in apoE4, phospholipid secretion increased to the levels seen with apoE3, suggesting that domain interaction decreases the phospholipid-binding capacity of apoE4. Thus, apoE4 domain interaction occurs in living neuronal cells and might be a molecular basis for apoE4-related neurodegeneration.


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