title: ABCA1-induced cell surface binding sites for ApoA-I.
abstract: OBJECTIVE
  The purpose of this study was to understand the interactions of apoA-I with cells expressing ABCA1.
  
  
  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 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 (approximately 90%) band migrated as monomeric apoA-I. In contrast to WT apoA-I, the C-terminal deletion mutants delta190 to 243 and delta223 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.
authors.affiliation: Division of GI/Nutrition, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4318, USA.
authors.forename: Charulatha
authors.initials: C
authors.lastname: Vedhachalam
authors.forename: Amy B
authors.initials: AB
authors.lastname: Ghering
authors.forename: W Sean
authors.initials: WS
authors.lastname: Davidson
authors.forename: Sissel
authors.initials: S
authors.lastname: Lund-Katz
authors.forename: George H
authors.initials: GH
authors.lastname: Rothblat
authors.forename: Michael C
authors.initials: MC
authors.lastname: Phillips
mesh_terms: D000818:Animals; D016632:Apolipoprotein A-I / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D050356:Lipid Metabolism; D008264:Macrophages / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D012680:Sensitivity and Specificity
chemical_list: D016632:Apolipoprotein A-I