on May 3, 2007
Published online before print May 3, 2007, doi: 10.1161/ATVBAHA.107.145789
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Submitted on February 22, 2007
Accepted on April 23, 2007
ABCA1-Induced Cell Surface Binding Sites for ApoA-I
Charulatha Vedhachalam ;
From the Division of GI/Nutrition (C.V., S.L.-K., G.H.R., M.C.P.), The Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia; and the Department of Pathology and Laboratory Medicine (A.B.G., W.S.D.), University of Cincinnati, Ohio.
* To whom correspondence should be addressed. E-mail: Phillipsmi{at}email.chop.edu.
Objective--The purpose of this study was to understand the interactions of apoA-I with cells expressing ABCA1.
Methods and Results--The binding of wild-type (WT) and mutant forms of human apoA-I to mouse J774 macrophages was examined. Analysis of total binding at 37°C of 125I-WT apoA-I to the cells and specifically to ABCA1, as determined by covalent cross-linking, revealed saturable high affinity binding in both cases. Determination of the level of cell-surface expression of ABCA1 showed that only about 10% of the apoA-I associated with the cell surface was bound directly to ABCA1. Furthermore, when 125I -apoA-I was cross-linked to ABCA1-upregulated cells and examined by SDS-PAGE, the major (
90%) band migrated as monomeric apoA-I. In contrast to WT apoA-I, the C-terminal deletion mutants 
190 to 243 and 223 to 243 that have reduced lipid affinity, exhibited marked reductions (50 and 70%, respectively) in their abilities to bind to the surface of ABCA1-upregulated cells. However, these C-terminal deletion mutants cross-linked to ABCA1 as effectively as WT apoA-I.
Conclusions--This study demonstrates that ABCA1 activity creates 2 types of high affinity apoA-I binding sites at the cell surface. The low capacity site formed by direct apoA-I/ABCA1 interaction functions in a regulatory role, whereas the much higher capacity site generated by apoA-I/lipid interactions functions in the assembly of nascent HDL particles.
Key words: ABCA1 • apoA-I • phospholipids • binding
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