Abstract
ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux to apolipoprotein A1 (apoA-I) initiates the biogenesis of high density lipoprotein. Here we show that the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG bind to the C terminus of ABCA1 by a PDZ-PDZ interaction and prevent ABCA1 protein degradation by activating RhoA. ABCA1 is a protein with a short half-life, and apoA-I stabilizes ABCA1 protein; however, depletion of PDZ-RhoGEF/LARG by RNA interference suppressed the apoA-I stabilization of ABCA1 protein in human primary fibroblasts. Exogenous PDZ-RhoGEF expression activated RhoA and increased ABCA1 protein levels and cholesterol efflux activity. Likewise, forced expression of a constitutively active RhoA mutant significantly increased ABCA1 protein levels, whereas a dominant negative RhoA mutant decreased them. The constitutively active RhoA retarded ABCA1 degradation, thus accounting for its ability to increase ABCA1 protein. Moreover, stimulation with apoA-I transiently activated RhoA, and the pharmacological inhibition of RhoA or the dominant negative RhoA blocked the ability of apoA-I to stabilize ABCA1. Finally, depletion of RhoA or RhoGEFs/RhoA reduces the cholesterol efflux when transcriptional regulation via PPARgamma is eliminated. Taken together, our results have identified a novel physical and functional interaction between ABCA1 and PDZ-RhoGEF/LARG, which activates RhoA, resulting in ABCA1 stabilization and cholesterol efflux activity.
Highlights
The activity of ATP-binding cassette transporter A1 (ABCA1) is regulated both at the transcriptional level and at the post-translational level
We show that PDZ-RhoGEF (Rho guanine nucleotide exchange factor 11) and leukemia-associated RhoGEF (LARG, Rho guanine nucleotide exchange factor 12) bind ABCA1 and regulate ABCA1 protein levels and cholesterol efflux activity
Members of the RhoGEF family have a Dbl homology (DH) domain and a pleckstrin homology (PH) domain that are responsible for the guanine nucleotide exchange factor activity
Summary
Materials—The following reagents were purchased from the indicated suppliers: mouse monoclonal anti-Myc antibody (4A6) (Upstate); rabbit anti-HA (Clontech); Alexa Fluor 555 goat anti-mouse IgG, Lipofectamine 2000 (Invitrogen); M2 anti-FLAG mouse monoclonal agarose-conjugated antibody, mouse anti--actin antibody, protease inhibitor mixture, and cycloheximide (Sigma); mouse anti-GTRAP48 (rat PDZ-RhoGEF) (BD Biosciences); mouse monoclonal antiABCA1 (Abcam); rabbit polyclonal anti-ABCA1 antibody (Affinity Bioreagents); mouse monoclonal anti-RhoA, cell-permeable C3-transferase, and Rho activation assay biochemistry kit (Cytoskeleton Inc., Denver, CO); anti-calnexin (StressGen Biotechnologies); SMARTpool siRNA duplexes (Dharmacon); apoA-I (Intracell); radionucleotides (PerkinElmer Life Sciences); human dermal fibroblast Nucleofector kit (Amaxa); and TrueBlot anti-rabbit Ig IP beads (eBioscience). The differentiated cells were cultured in RPMI medium containing 0.2% bovine serum albumin for 24 h and used for the experiments [33]. The binding of full-length PDZ-RhoGEF to the ABCA1 C terminus was determined using the overlay technique as previously described [8]. The lysates were obtained from 293-EBNA-T cells transfected with cDNA of HA-tagged versions of full-length PDZ-RhoGEF for 2 h at room temperature. For the analysis of protein interactions in primary human fibroblasts, clarified lysates were incubated with rabbit anti-ABCA1 antibody (Affinity Bioreagents). 24 h of transfection, the cells were incubated in culture medium containing 0.5 Ci/ml [3H]cholesterol for 24 h. The cells were equilibrated in containing 2 mg/ml fatty acidfree bovine serum albumin and incubated with or without 10 g/ml apoA-I for 20 h [293] or 6 h (primary human fibroblasts). NaOH, and the percentage of cholesterol efflux was calculated by scintillation counting
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