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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:1603.)
© 2007 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

ABCA1-Induced Cell Surface Binding Sites for ApoA-I

Charulatha Vedhachalam; Amy B. Ghering; W. Sean Davidson; Sissel Lund-Katz; George H. Rothblat; Michael C. Phillips

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.

Correspondence to Dr Michael C. Phillips, The Children’s Hospital of Philadelphia, Abramson Research Center, Suite 1102, 3615 Civic Center Blvd, Philadelphia, PA 19104-4318. 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.

15-LO-I is expressed in rabbit aorta. 15-LO regulates vasodilatory eicosanoid synthesis and vascular tone. The current 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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