Phosphorylation of ABCB4 impacts its function: Insights from disease-causing mutations

Gautherot, Julien; Delautier, Danièle; Maubert, Marie-Anne; Aït‐Slimane, Tounsia; Bolbach, G.; Delaunay, Jean; Durand‐Schneider, Anne‐Marie; Firrincieli, Delphine; Barbu, Véronique; Chignard, Nicolas; Housset, Chantal; Maurice, Michèle; Falguières, Thomas

doi:10.1002/hep.27170

Cited by 46 publications

(70 citation statements)

References 36 publications

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“…PC secretion activity of ABCB4 mutants was examined in the nonpolarized epithelial HEK293 cells, which provide the best‐suited model for such studies. PC secretion was measured in the culture medium after transient transfection, as described . As in HepG2 cells, all mutants were localized predominantly at the plasma membrane (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Functional defect of variants in the adenosine triphosphate–binding sites of ABCB4 and their rescue by the cystic fibrosis transmembrane conductance regulator potentiator, ivacaftor (VX‐770)

et al. 2016

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ABCB4 (MDR3) is an adenosine triphosphate (ATP)‐binding cassette (ABC) transporter expressed at the canalicular membrane of hepatocytes, where it mediates phosphatidylcholine (PC) secretion. Variations in the ABCB4 gene are responsible for several biliary diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3), a rare disease that can be lethal in the absence of liver transplantation. In this study, we investigated the effect and potential rescue of ABCB4 missense variations that reside in the highly conserved motifs of ABC transporters, involved in ATP binding. Five disease‐causing variations in these motifs have been identified in ABCB4 (G535D, G536R, S1076C, S1176L, and G1178S), three of which are homologous to the gating mutations of cystic fibrosis transmembrane conductance regulator (CFTR or ABCC7; i.e., G551D, S1251N, and G1349D), that were previously shown to be function defective and corrected by ivacaftor (VX‐770; Kalydeco), a clinically approved CFTR potentiator. Three‐dimensional structural modeling predicted that all five ABCB4 variants would disrupt critical interactions in the binding of ATP and thereby impair ATP‐induced nucleotide‐binding domain dimerization and ABCB4 function. This prediction was confirmed by expression in cell models, which showed that the ABCB4 mutants were normally processed and targeted to the plasma membrane, whereas their PC secretion activity was dramatically decreased. As also hypothesized on the basis of molecular modeling, PC secretion activity of the mutants was rescued by the CFTR potentiator, ivacaftor (VX‐770). Conclusion: Disease‐causing variations in the ATP‐binding sites of ABCB4 cause defects in PC secretion, which can be rescued by ivacaftor. These results provide the first experimental evidence that ivacaftor is a potential therapy for selected patients who harbor mutations in the ATP‐binding sites of ABCB4. (Hepatology 2017;65:560‐570)

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Section: Resultsmentioning

confidence: 99%

“…Media were collected after 24 hours. Measurement of PC content in collected media was performed as described …”

Section: Methodsmentioning

confidence: 99%

Functional defect of variants in the adenosine triphosphate–binding sites of ABCB4 and their rescue by the cystic fibrosis transmembrane conductance regulator potentiator, ivacaftor (VX‐770)

et al. 2016

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“…PC secretion was measured in the culture medium of HEK293 cells, as described . Briefly, cells grown in six‐well plates were transfected with the plasmids encoding WT or mutant ABCB4.…”

Section: Methodsmentioning

confidence: 99%

A functional classification of ABCB4 variations causing progressive familial intrahepatic cholestasis type 3

et al. 2015

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Progressive familial intrahepatic cholestasis type 3 is caused by biallelic variations of ABCB4, most often ( 70%) missense. In this study, we examined the effects of 12 missense variations identified in progressive familial intrahepatic cholestasis type 3 patients. We classified these variations on the basis of the defects thus identified and explored potential rescue of trafficking-defective mutants by pharmacological means. Variations were reproduced in the ABCB4 complementary DNA and the mutants, thus obtained, expressed in HepG2 and HEK293 cells. Three mutants were either fully (I541F and L556R) or largely (Q855L) retained in the endoplasmic reticulum, in an immature form. Rescue of the defect, i.e., increase in the mature form at the bile canaliculi, was obtained by cell treatments with cyclosporin A or C and, to a lesser extent, B, D, or H. Five mutations with little or no effect on ABCB4 expression at the bile canaliculi caused a decrease (F357L, T775M, and G954S) or almost absence (S346I and P726L) of phosphatidylcholine secretion. Two mutants (T424A and N510S) were normally processed and expressed at the bile canaliculi, but their stability was reduced. We found no defect of the T175A mutant or of R652G, previously described as a polymorphism. In patients, the most severe phenotypes appreciated by the duration of transplant-free survival were caused by ABCB4 variants that were markedly retained in the endoplasmic reticulum and expressed in a homozygous status. Conclusion: ABCB4 variations can be classified as follows: nonsense variations (I) and, on the basis of current findings, missense variations that primarily affect the maturation (II), activity (III), or stability (IV) of the protein or have no detectable effect (V); this classification provides a strong basis for the development of genotype-based therapies.

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“…The PC secretion activities of both mutants in HEK293 cells are low compared with that of wild-type ABCB4 [44]. In the N-terminal domain of ABCB4, Thr34, Thr44, and Ser49 are phosphorylatable [44].…”

Section: Abcb4-mediated Efflux Of Endogenous Phospholipidsmentioning

confidence: 99%

Molecular Mechanisms for Biliary Phospholipid and Drug Efflux Mediated by ABCB4 and Bile Salts

Morita

Takato

2014

BioMed Research International

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On the canalicular membranes of hepatocytes, several ABC transporters are responsible for the secretion of bile lipids. Among them, ABCB4, also called MDR3, is essential for the secretion of phospholipids from hepatocytes into bile. The biliary phospholipids are associated with bile salts and cholesterol in mixed micelles, thereby reducing the detergent activity and cytotoxicity of bile salts and preventing cholesterol crystallization. Mutations in the ABCB4 gene result in progressive familial intrahepatic cholestasis type 3, intrahepatic cholestasis of pregnancy, low-phospholipid-associated cholelithiasis, primary biliary cirrhosis, and cholangiocarcinoma. In vivo and cell culture studies have demonstrated that the secretion of biliary phospholipids depends on both ABCB4 expression and bile salts. In the presence of bile salts, ABCB4 located in nonraft membranes mediates the efflux of phospholipids, preferentially phosphatidylcholine. Despite high homology with ABCB1, ABCB4 expression cannot confer multidrug resistance. This review summarizes our current understanding of ABCB4 functions and physiological relevance, and discusses the molecular mechanism for the ABCB4-mediated efflux of phospholipids.

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Phosphorylation of ABCB4 impacts its function: Insights from disease-causing mutations

Cited by 46 publications

References 36 publications

Functional defect of variants in the adenosine triphosphate–binding sites of ABCB4 and their rescue by the cystic fibrosis transmembrane conductance regulator potentiator, ivacaftor (VX‐770)

Functional defect of variants in the adenosine triphosphate–binding sites of ABCB4 and their rescue by the cystic fibrosis transmembrane conductance regulator potentiator, ivacaftor (VX‐770)

A functional classification of ABCB4 variations causing progressive familial intrahepatic cholestasis type 3

Molecular Mechanisms for Biliary Phospholipid and Drug Efflux Mediated by ABCB4 and Bile Salts

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