Differential effects of progressive familial intrahepatic cholestasis type 1 and benign recurrent intrahepatic cholestasis type 1 mutations on canalicular localization of ATP8B1

Abstract

Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis type 1 (BRIC1), forming a spectrum of cholestatic disease. Whereas PFIC1 is a progressive, endstage liver disease, BRIC1 patients suffer from episodic periods of cholestasis that resolve spontaneously. At present it is not clear how the type and location of the mutations relate to the clinical manifestations of PFIC1 and BRIC1. ATP8B1 localizes to the canalicular membrane of hepatocytes where it mediates the inward translocation of phosphatidylserine. ATP8B1 interacts with CDC50A, which is required for endoplasmic reticulum exit and plasma membrane localization. In this study we analyzed a panel of missense mutations causing PFIC1 (G308V, D554N, G1040R) or BRIC1 (D70N, I661T). In addition, we included two mutations that have been associated with intrahepatic cholestasis of pregnancy (ICP) (D70N, R867C). We examined the effect of these mutations on protein stability and interaction with CDC50A in Chinese hamster ovary cells, and studied the subcellular localization in WIF-B9 cells. Protein stability was reduced for three out of six mutations studied. Two out of three PFIC1 mutant proteins did not interact with CDC50A, whereas BRIC1/ICP mutants displayed reduced interaction. Importantly, none of the PFIC1 mutants were detectable in the canalicular membrane of WIF-B9 cells, whereas all BRIC1/ICP mutants displayed the same cellular staining pattern as wild-type ATP8B1. Our data indicate that PFIC1 mutations lead to the complete absence of canalicular expression, whereas in BRIC1/ICP residual protein is expressed in the canalicular membrane. Conclusion: These data provide an explanation for the difference in severity between the phenotypes of PFIC1 and BRIC1. (HEPATOLOGY 2009;50: 1597-1605

Phospholipid Flippase Activities and Substrate Specificities of Human Type IV P-type ATPases Localized to the Plasma Membrane

“…Incubation of the stable cell lines with MG-132, a proteasome inhibitor, resulted in modest recovery of the protein levels of these mutants (Fig. 8C) as reported previously (38,39), suggesting that the mutated proteins are degraded by the proteasome. However, MG-132 treatment did not significantly increase the cell surface levels of these mutants (Fig.…”

“…However, the relationships between types of missense mutations and the flippase activity have not been explored, and many missense mutants of ATP8B1 found in PFIC1 are degraded before delivery to the plasma membrane (38,39). Stone et al (44) introduced PFIC1-type mutations into the yeast plasma membrane P4-ATPase Dnf2p and examined whether the mutations affected its PCflipping activity.…”

“…However, the relationships between types of missense mutations and flippase activity have not been explored in part because the substrate specificity of ATP8B1 was uncertain. Many missense mutant ATP8B1 proteins found in PFIC1 are degraded before delivery to the plasma membrane (38,39) (Fig. 8A).…”

“…8, A and B). The D70N and L127P mutants are properly localized to the plasma membrane and are able to interact with CDC50 protein (38,39). Therefore, we next explored the flippase activities of these mutants toward PC and PS.…”

Hepatobiliary Transport in Health and Disease

“…PFIC1 patients in Inuit families in Greenland and Canada have a G-A mutation, resulting in the substitution of aspartate with asparagine at amino acid 554 [57]. The G308V missense mutation in Byler disease and the D554N missense mutation in Greenland familial cholestasis lead to an absence of canalicular expression of ATP8B1 [59]. Other ATP8B1 mutations that affect splicing or the protein structure have been identified in different populations [49].…”