Liver X receptor beta (LXRbeta) interacts directly with ATP-binding cassette A1 (ABCA1) to promote high density lipoprotein formation during acute cholesterol accumulation.

Cells have evolved multiple mechanisms for maintaining cholesterol homeostasis, and, among these, ATP-binding cassette protein A1 (ABCA1)-mediated cholesterol efflux is highly regulated at the transcriptional level through the activity of the nuclear receptor liver X receptor (LXR). Here, we show that in addition to its well defined role in transcription, LXRß directly binds to the C-terminal region ((2247)LTSFL(2251)) of ABCA1 to mediate its post-translational regulation. In the absence of cholesterol accumulation in the macrophage-like cell line THP-1, the ABCA1-LXRß complex stably localizes to the plasma membrane, but apolipoprotein A-I (apoA-I) binding or cholesterol efflux does not occur. Exogenously added LXR ligands, which mimic cholesterol accumulation, cause LXRß to dissociate from ABCA1, thus freeing ABCA1 for apoA-I binding and subsequent cholesterol efflux. Photoaffinity labeling experiments with 8-azido-[α-(32)P]ATP showed that the interaction of LXRß with ABCA1 inhibits ATP binding by ABCA1. This is the first study to show that a protein-protein interaction with the endogenous protein suppresses the function of ABC proteins by inhibiting ATP binding. LXRß can cause a post-translational response by binding directly to ABCA1, as well as a transcriptional response, to maintain cholesterol homeostasis.

Direct interaction of nuclear liver X receptor-beta with ABCA1 modulates cholesterol efflux.

Cholesterol is an essential component of eukaryotic cells; at the same time, however, hyperaccumulation of cholesterol is harmful. Therefore, the ABCA1 gene, the product of which mediates secretion of cholesterol, is highly regulated at both the transcriptional and post-transcriptional levels. The transcription of ABCA1 is regulated by intracellular oxysterol concentration via the nuclear liver X receptor (LXR)/retinoid X receptor (RXR); once synthesized, ABCA1 protein turns over rapidly with a half-life of 1-2 h. Here, we show that the LXRbeta/RXR complex binds directly to ABCA1 on the plasma membrane of macrophages and modulates cholesterol secretion. When cholesterol does not accumulate, ABCA1-LXRbeta/RXR localizes on the plasma membrane, but is inert. When cholesterol accumulates, oxysterols bind to LXRbeta, and the LXRbeta/RXR complex dissociates from ABCA1, restoring ABCA1 activity and allowing apoA-I-dependent cholesterol secretion. LXRbeta can exert an immediate post-translational response, as well as a rather slow transcriptional response, to changes in cellular cholesterol accumulation. Thus, we provide the first demonstration that protein-protein interaction suppresses ABCA1 function. Furthermore, we show that LXRbeta is involved in both the transcriptional and post-transcriptional regulation of the ABCA1 transporter.

Alpha1-syntrophin modulates turnover of ABCA1.

ABCA1 (ATP-binding cassette transporter A1) mediates the release of cellular cholesterol and phospholipid to form high density lipoprotein. Functions of ABCA1 are highly regulated at the transcriptional and post-transcriptional levels, and the synthesized ABCA1 protein turns over rapidly with a half-life of 1-2 h. To examine whether the functions of ABCA1 are modulated by associated proteins, a yeast two-hybrid library was screened with the C-terminal 120 amino acids of ABCA1. Two PDZ (PSD95-Discs large-ZO1) proteins, alpha1-syntrophin and Lin7, were found to interact with ABCA1. Immunoprecipitation revealed that alpha1-syntrophin interacted with ABCA1 strongly and that the interaction was via the C-terminal three amino acids SYV of ABCA1. Co-expression of alpha1-syntrophin in human embryonic kidney 293 cells retarded degradation of ABCA1 and made the half-life of ABCA1 five times longer than in the cells not expressing alpha1-syntrophin. This effect is not common among PDZ-containing proteins interacting with ABCA1, because Lin7, which was also found to interact with the C terminus region of ABCA1, did not have a significant effect on the half-life of ABCA1. Co-expression of alpha1-syntrophin significantly increased the apoA-I-mediated release of cholesterol. ABCA1 was co-immunoprecipitated with alpha1-syntrophin from mouse brain. These results suggest that alpha1-syntrophin is involved in intracellular signaling, which determines the stability of ABCA1 and modulates cellular cholesterol release.

Posttranscriptional regulation of human ABCA7 and its function for the apoA-I-dependent lipid release.

ABCA7 is expressed predominantly in myelo-lymphatic tissues or reticuloendothelial cells. Physiological role and function of this protein are not fully understood. We isolated the full-length cDNA (type I) and a splicing variant cDNA (type II) of human ABCA7, and developed monoclonal antibodies against extracellular domain (ECD)1 of ABCA7. RT-PCR experiments suggested that human ABCA7 gene produced the type II mRNA in a tissue-specific manner. Immunostaining revealed that the type I ABCA7, expressed in HEK293 cells, was localized to the plasma membrane and ECD1 was exposed to the extracellular space as was the case for ABCA1. HEK293 cells expressing type I ABCA7 showed apoA-I-dependent cholesterol and phospholipid release. In contrast, type II ABCA7 appeared to be localized mainly in endoplasmic reticulum and did not show apoA-I-dependent cholesterol and phospholipid release. Alternative splicing could be involved in the post-transcriptional regulation of the expression and function of human ABCA7.