ABCA1-Induced Cell Surface Binding Sites for ApoA-I
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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 Δ190 to 243 and Δ223 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.