Specific Docking of Apolipoprotein A-I at the Cell Surface Requires a Functional ABCA1 Transporter

Abstract

The identification of defects in ABCA1 as the molecular basis of Tangier disease has highlighted its crucial role in the loading with phospholipids and cholesterol of nascent apolipoprotein particles. Indeed the expression of ABCA1 affects apolipoprotein A-I (apoA-I)-mediated removal of lipids from cell membranes, and the possible role of ABCA1 as an apoA-I surface receptor has been recently suggested. In the present study, we have investigated the role of the ABCA1 transporter as an apoA-I receptor with the analysis of a panel of transfectants expressing functional or mutant forms of ABCA1. We provide experimental evidence that the forced expression of a functional ABCA1 transporter confers surface competence for apoA-I binding. This, however, appears to be dependent on ABCA1 function. Structurally intact but ATPase-deficient forms of the transporter fail to elicit a specific cell association of the ligand. In addition the diffusion parameters of membrane-associated apoA-I indicate an interaction with membrane lipids rather than proteins. These results do not support a direct molecular interaction between ABCA1 and apoA-I, but rather suggest that the ABCA1-induced modification of the lipid distribution in the membrane, evidenced by the phosphatidylserine exofacial flopping, generates a biophysical microenvironment required for the docking of apoA-I at the cell surface.The removal of cellular lipids is promoted by high density lipoproteins (HDL), 1 the plasma shuttle mediating reverse cholesterol transport from peripheral tissues to the liver for further uptake and metabolism (1). However, whether the interaction of the lipid-poor apoA-I particle, protein core of the nascent HDL, with cell membranes is mediated by a specific receptor and how its loading with phospholipids and cholesterol occurs is still a matter of debate (2). The recent discovery that a defective ABCA1 transporter leads to Tangier disease (3-9) has directly implicated this transmembrane protein in the active release of cellular lipids and prompted an investigation into its role as a candidate apoA-I receptor (10, 11). Indeed a correlation between the cAMP-induced cell surface apoA-I binding and the expression of ABCA1 in macrophage-like cell lines has been reported (12). Very recently, in addition, a direct molecular interaction between ABCA1 and apoA-I at the cell surface has been proposed on the basis of chemical cross-linking experiments (10, 11). To gain further insight into this issue, we developed an apoA-I binding assay based on the use of a fluorochrome-conjugated ligand. The analysis of apoA-I binding to a panel of transfectants expressing either functionally intact or defective ABCA1 proteins (13) led us to exclude that the transporter behaves as a bona fide receptor for apoA-I. Indeed, whereas surface binding increases with the expression of a functional ABCA1, the expression of structurally intact but functionally impaired ABCA1 proteins fails to elicit specific binding. Considering that, as previously demonstrated, ABCA1 promotes t...

Phosphorylation by Protein Kinase CK2 Modulates the Activity of the ATP Binding Cassette A1 Transporter

“…Apparent binding constants to the three cell populations were 7, 30, and 24 ϫ 10 Ϫ8 M for apoAI binding compared with 4, 16, and 17 ϫ 10 Ϫ8 M for apoAII binding. These values are comparable with those measured for apoAI/Cy5 binding to WT ABCA1 (27).…”

“…Cells were subdivided into three populations as a function of their level of ABCAI expression, reflected by the GFP fluorescence intensity. Apoprotein binding data were calculated for each cell population based upon the mean carboxyrhodamine fluorescence intensity (27). for [methyl-3 H]choline efflux or 6 h for [ 3 H]cholesterol efflux.…”

“…We analyzed apolipoprotein binding to three cell populations, with low, middle, and high levels of transfection, estimated from the GFP fluorescence, with cutoffs at Ͼ7 and Ͻ20 for population 1 (ABCA1ϩ), Ͼ21 and Ͻ90 for population 2 (ABCA1ϩϩ), and Ͼ91 for population 3 (ABCA1ϩϩϩ) (27). Subtraction of the carboxyrhodamine fluorescence intensity of Mock-transfected cells yielded saturation curves, with a plateau above 10 g/ml for apoAI/Rho and apoAII/Rho binding to WT ABCA1 (Fig.…”

“…ABCA1-We compared the binding of carboxyrhodamine-labeled apoAI and apoAII to the Hek-293 Tet-Off cells transfected with WT and ABCA1 phosphorylation mutants by flow cytometric analysis (27). We analyzed apolipoprotein binding to three cell populations, with low, middle, and high levels of transfection, estimated from the GFP fluorescence, with cutoffs at Ͼ7 and Ͻ20 for population 1 (ABCA1ϩ), Ͼ21 and Ͻ90 for population 2 (ABCA1ϩϩ), and Ͼ91 for population 3 (ABCA1ϩϩϩ) (27).…”

UV irradiation inhibits ABC transporters via generation of ADP-ribose by concerted action of poly(ADP-ribose) polymerase-1 and glycohydrolase

“…3 It has been suggested that the ABC transporter ABCA1 induces modifications in lipid distribution of plasma membrane, evidenced by exofacial flopping of phosphatidylserine. 6 Furthermore, ABC1 was shown to be involved in engulfment of apoptotic cells. 7,8 One of the physiological roles of ABC transporters is detoxification by extrusion of various toxic compounds from the cytoplasm.…”