ABCMdb

ABCA1 p.Leu2247Ala

Predicted by SNAP2:	A: N (61%), C: N (57%), D: D (63%), E: D (53%), F: N (72%), G: D (59%), H: N (57%), I: N (78%), K: N (61%), M: N (82%), N: N (61%), P: N (57%), Q: N (61%), R: N (66%), S: N (66%), T: N (66%), V: N (78%), W: N (53%), Y: N (72%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N,

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[hide] Liver X receptor beta (LXRbeta) interacts directly... J Biol Chem. 2011 Jun 3;286(22):20117-24. Epub 2011 Apr 20. Hozoji-Inada M, Munehira Y, Nagao K, Kioka N, Ueda K
Liver X receptor beta (LXRbeta) interacts directly with ATP-binding cassette A1 (ABCA1) to promote high density lipoprotein formation during acute cholesterol accumulation.
J Biol Chem. 2011 Jun 3;286(22):20117-24. Epub 2011 Apr 20., [PMID:21507939]

Abstract [show]
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, LXRbeta 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-LXRbeta 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 LXRbeta to dissociate from ABCA1, thus freeing ABCA1 for apoA-I binding and subsequent cholesterol efflux. Photoaffinity labeling experiments with 8-azido-[alpha-(32)P]ATP showed that the interaction of LXRbeta 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. LXRbeta can cause a post-translational response by binding directly to ABCA1, as well as a transcriptional response, to maintain cholesterol homeostasis.

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No. Sentence Comment
90 However, mutation of Leu2247 and Leu2251 to alanine abrogated the co-precipitation of LXRbeta despite comparable levels of expression among these mutants and WT ABCA1. Login to comment
108 HEK293 cells stably expressing FLAG-tagged LXRbeta were transfected with HA-tagged WT ABCA1 L2247A mutant or L2251A mutant. Login to comment
113 When the mutant constructs were expressed in HEK293 cells, L2247A or L2251A substitution did not affect the surface expression of ABCA1, but LXRbeta no longer co-localized with ABCA1 (Fig. 2). Login to comment
120 Interestingly, apoA-I bound to cells expressing L2247A or L2251A mutant ABCA1 even when LXRbeta was co-expressed. Login to comment
122 When ABCA1-L2247A, ABCA1-L2251A, or ABCA1-L2247A/L2251A was expressed alone, cholesterol efflux to apoA-I was comparable with cells expressing WT ABCA1 in the absence or presence of TO901317 (black bars and empty bars, respectively, in Fig. 3B). Login to comment
126 Co-expression of LXRbeta doubled the plasma membrane levels of WT ABCA1 as shown previously (16), but surface levels of the mutants (L2247A, L2251A, and L2247/L2251A) were not affected by LXRbeta expression (Fig. 3C). Login to comment
149 ABCA1-L2247A/L2251A double mutant was labeled as efficiently as WT ABCA1 (lane 9), but this was not inhibited by LXRbeta co-expression (lanes 11 and 12), suggesting that the interaction of LXRbeta with ABCA1 via Leu2247 and Leu2251 prevents tight ATP binding. Login to comment
89 However, mutation of Leu2247 and Leu2251 to alanine abrogated the co-precipitation of LXRbeta despite comparable levels of expression among these mutants and WT ABCA1. Login to comment
107 HEK293 cells stably expressing FLAG-tagged LXRbeta were transfected with HA-tagged WT ABCA1 L2247A mutant or L2251A mutant. Login to comment
112 When the mutant constructs were expressed in HEK293 cells, L2247A or L2251A substitution did not affect the surface expression of ABCA1, but LXRbeta no longer co-localized with ABCA1 (Fig. 2). Login to comment
119 Interestingly, apoA-I bound to cells expressing L2247A or L2251A mutant ABCA1 even when LXRbeta was co-expressed. Login to comment
121 When ABCA1-L2247A, ABCA1-L2251A, or ABCA1-L2247A/L2251A was expressed alone, cholesterol efflux to apoA-I was comparable with cells expressing WT ABCA1 in the absence or presence of TO901317 (black bars and empty bars, respectively, in Fig. 3B). Login to comment
125 Co-expression of LXRbeta doubled the plasma membrane levels of WT ABCA1 as shown previously (16), but surface levels of the mutants (L2247A, L2251A, and L2247/L2251A) were not affected by LXRbeta expression (Fig. 3C). Login to comment
148 ABCA1-L2247A/L2251A double mutant was labeled as efficiently as WT ABCA1 (lane 9), but this was not inhibited by LXRbeta co-expression (lanes 11 and 12), suggesting that the interaction of LXRbeta with ABCA1 via Leu2247 and Leu2251 prevents tight ATP binding. Login to comment

[hide] Direct interaction of nuclear liver X receptor-bet... J Biol Chem. 2008 Oct 31;283(44):30057-63. Epub 2008 Sep 9. Hozoji M, Munehira Y, Ikeda Y, Makishima M, Matsuo M, Kioka N, Ueda K
Direct interaction of nuclear liver X receptor-beta with ABCA1 modulates cholesterol efflux.
J Biol Chem. 2008 Oct 31;283(44):30057-63. Epub 2008 Sep 9., [PMID:18782758]

Abstract [show]
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.

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No. Sentence Comment
192 The coexpression of LXRbeta did not retard the degradation of ABCA1(L2247A), whereas the coexpression of ␣1-syntrophin retarded the degradation of ABCA1(L2247A) (supplemental Fig. 3, b and c). Login to comment
193 These results suggest that the amino acid substitution L2247A does not alter total protein conformation but rather specifically affects the interaction with LXRbeta. Login to comment
194 ABCA1(L2247A) showed significant apoA-I-dependent cholesterol efflux, and the addition of TO901317 did not affect it even when LXRbeta was coexpressed (supplemental Fig. 3d). Login to comment
191 The coexpression of LXRbeta did not retard the degradation of ABCA1(L2247A), whereas the coexpression of ॷ1-syntrophin retarded the degradation of ABCA1(L2247A) (supplemental Fig. 3, b and c). Login to comment