Abstract
ATP-binding cassette transporter A1 (ABCA1) plays a critical role in HDL cholesterol metabolism, but the mechanism by which it transports lipid across membranes is poorly understood. Because growing evidence implicates accessory proteins in this process, we developed a method by which proteins interacting with the intact transporter could be identified. cDNAs encoding wild-type ABCA1 and a mutant lacking the C-terminal PDZ binding motif of ABCA1 were transfected into 293 cells, and the expressed proteins were solubilized using detergent conditions (0.75% CHAPS, 1 mg/ml phosphatidylcholine) predicted to retain high affinity protein-protein interactions. Proteins that co-purified with ABCA1 on an antibody affinity column were identified by liquid chromatographymass spectrometric analysis. A novel interaction with the PDZ protein beta1-syntrophin was identified using this approach, and this interaction was confirmed in human THP-1 macrophages and in mouse liver. Small interference RNA inhibition of beta1-syntrophin expression reduced cholesterol efflux from primary skin fibroblasts by 50% while decreasing efflux 30% in bone marrow-derived macrophages. Inhibition of beta1-syntrophin decreased ABCA1 protein levels, whereas overexpression of beta1-syntrophin increased ABCA1 cell-surface expression and stimulated efflux to apolipoprotein A-I. These findings indicate that beta1-syntrophin acts through a class-I PDZ interaction with the C terminus of ABCA1 to regulate the cellular distribution and activity of the transporter. The approach used to identify beta1-syntrophin as an ABCA1-binding protein should prove useful in elucidating other protein interactions upon which ABCA1 function depends.
Highlights
Apolipoproteins, primarily apolipoprotein A-I.4 The physiological importance of this process is demonstrated in patients with Tangier disease, a rare genetic condition caused by loss-of-function mutations in the ATP-binding cassette transporter A1 (ABCA1) transporter that eliminate apoA-1-stimulated cholesterol efflux [3,4,5,6]
Because HDL levels inversely correlate with cardiovascular disease, and cholesterol-engorged macrophages in the vessel wall are involved in the pathogenesis of atherosclerosis, a normally functioning ABCA1-mediated cholesterol efflux pathway is thought to play a critical role in preventing coronary artery disease [12, 13]
The ABCA1 C terminus (ESYV) conforms to the consensus motif (S/T-X-V-COOH) bound by class-I PDZ proteins, and we have previously shown that the PDZ domains of both PSD-95 and Dlg-1 bind to this sequence [28]
Summary
Apolipoproteins, primarily apolipoprotein A-I (apoA-I). The physiological importance of this process is demonstrated in patients with Tangier disease, a rare genetic condition caused by loss-of-function mutations in the ABCA1 transporter that eliminate apoA-1-stimulated cholesterol efflux [3,4,5,6]. Evidence for posttranslational regulation includes the finding that binding of apoA-I to the transporter prolongs its half-life by inhibiting a calpain-mediated degradation pathway [16, 17] This process is associated with alterations in the phosphorylation status of ABCA1 [18, 19]. Our search for cellular proteins involved in regulating ABCA1 activity was initially stimulated by our identification of a Tangier patient carrying a 46-amino acid deletion of the ABCA1 C terminus [26]. This mutation removes a putative PDZ protein-binding motif located in the terminal 3 residues of the cytoplasmic tail of the transporter. We further demonstrate that this interaction occurs under physiological conditions in murine liver and macrophages and that the interaction of -1 syntrophin with ABCA1 is involved in regulating the cellular distribution and activity of the transporter
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