title: Naturally occurring mutations in the largest extracellular loops of ABCA1 can disrupt its direct interaction with apolipoprotein A-I.
abstract: The ABCA1 transporter contains two large domains into which many of the genetic mutations in individuals with Tangier disease fall. To investigate the structural requirements for the cellular cholesterol efflux mediated by ABCA1, we have determined the topology of these two domains and generated transporters harboring five naturally occurring missense mutations in them. These mutants, unlike wild type ABCA1, produced little or no apoA-I-stimulated cholesterol efflux when transfected into 293 cells, establishing their causality in Tangier disease. Because all five mutant proteins were well expressed and detectable on the plasma membrane, their interaction with the ABCA1 ligand, apolipoprotein (apo) A-I, was measured using bifunctional cross-linking agents. Four of five mutants had a marked decline in cross-linking to apoA-I, whereas one (W590S) retained full cross-linking activity. Cross-linking of apoA-I was temperature-dependent, rapid in onset, and detectable with both lipid- and water-soluble cross-linking agents. These results suggest that apoA-I-stimulated cholesterol efflux cannot occur without a direct interaction between the apoprotein and critical residues in two extracellular loops of ABCA1. The behavior of the W590S mutant indicates that although binding of apoA-I by ABCA1 may be necessary, it is not sufficient for stimulation of cholesterol efflux.
authors.affiliation: Lipid Metabolism Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Fitzgerald
authors.forename: Andrea L
authors.initials: AL
authors.lastname: Morris
authors.forename: Jeongmi S
authors.initials: JS
authors.lastname: Rhee
authors.forename: Lorna P
authors.initials: LP
authors.lastname: Andersson
authors.forename: Armando J
authors.initials: AJ
authors.lastname: Mendez
authors.forename: Mason W
authors.initials: MW
authors.lastname: Freeman
mesh_terms: D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008051:Lipid Bilayers; D009154:Mutation; D020125:Mutation, Missense; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013696:Temperature; D013997:Time Factors; D014162:Transfection
chemical_list: C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D016632:Apolipoprotein A-I; D003432:Cross-Linking Reagents; D008024:Ligands; D008051:Lipid Bilayers; D004247:DNA; D002784:Cholesterol