ABCA1-induced cell surface binding sites for ApoA-I

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 degrees C of I-125-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 I-125 - apoA- I was cross- linked to ABCA1- upregulated cells and examined by SDS- PAGE, the major ( approximate to 90%) band migrated as monomeric apoA- I. In contrast to WT apoA- I, the C- terminal deletion mutants Delta 190 to 243 and Delta 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.