Trafficking and functional defects by mutations of the ATP-binding domains in MRP2 in patients with Dubin-Johnson syndrome.
Dubin-Johnson syndrome (DJS) is a hereditary disease characterized by hyperbilirubinemia. We investigated the consequences of 2 missense mutations, R768W and Q1382R, of nucleotide-binding domains (NBDs) of the multidrug resistance protein 2 (MRP2; ABCC2) that were previously identified in patients with DJS. Pulse chase analysis revealed that the precursor form of the wild-type and Q1382R MRP2 were converted to the mature form, which is resistant to endoglycosidase H (Endo H) in about 60 minutes. However, the precursor form of the R768W MRP2, which is sensitive to endoglycosidase H, was degraded within 120 minutes and did not mature to the fully glycosylated form. Proteasome inhibitors inhibited the degradation of the precursor form of the R768W MRP2. Unlike the R768W MRP2, the Q1382R MRP2 was mainly localized on the apical membrane in the wild-type form. However, efflux of glutathione monochlorobimane (GS-MCLB) and ATP-dependent leukotriene C(4) (LTC(4)) uptake into plasma membrane vesicles from cells expressing the Q1382R MRP2 were markedly reduced, suggesting that the Q1382R MRP2 on the apical membrane was nonfunctional. Vanadate-induced nucleotide trapping with 8-azido-[alpha-32P]ATP in the wild-type MRP2 was stimulated by estradiol glucuronide (E(2)17betaG) in a concentration-dependent manner but that in the Q1382R MRP2 was not. In conclusion, the R768W mutation causes deficient maturation and impaired sorting, and the Q1382R mutation does not affect maturation or sorting but impairs the substrate-induced ATP hydrolysis.
Hashimoto
Kenkichi
K
Department of Medical Biochemistry, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Uchiumi
Takeshi
T
Konno
Toshikazu
T
Ebihara
Takuya
T
Nakamura
Takanori
T
Wada
Morimasa
M
Sakisaka
Shotaro
S
Maniwa
Fumio
F
Amachi
Teruo
T
Ueda
Kazumitsu
K
Kuwano
Michihiko
M
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007566:Jaundice, Chronic Idiopathic / Q000235:genetics* / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008099:Liver / Q000378:metabolism; D024101:Mitochondrial Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D020125:Mutation, Missense; D019920:Photoaffinity Labels; D046988:Proteasome Endopeptidase Complex; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000235:genetics*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013552:Swine; D014638:Vanadates / Q000494:pharmacology
C000715087:ABCC2 protein, human; C093947:MRP2 protein, S cerevisiae; D024101:Mitochondrial Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D009097:Multienzyme Complexes; D019920:Photoaffinity Labels; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D014638:Vanadates; D000255:Adenosine Triphosphate; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex