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Multidrug resistance protein 4 (MRP4; ABCC4) is a member of the MRP/ATP-binding cassette family serving as a transmembrane transporter involved in energy-dependent efflux of anticancer/antiviral nucleotide agents and of physiological substrates, including cyclic nucleotides and prostaglandins (PGs). Phenotypic consequences of mrp4 deficiency were investigated using mrp4-knockout mice and derived immortalized mouse embryonic fibroblast (MEF) cells. Mrp4 deficiency caused decreased extracellular and increased intracellular levels of cAMP in MEF cells under normal and forskolin-stimulated conditions. Mrp4 deficiency and RNA interference-mediated mrp4 knockdown led to a pronounced reduction in extracellular PGE 2 but with no accumulation of intracellular PGE 2 in MEF cells. This result was consistent with attenuated cAMP-dependent protein kinase activity and reduced cyclooxygenase-2 (Cox-2) expression in mrp4-deficient MEF cells, suggesting that PG synthesis is restrained along with a lack of PG transport caused by mrp4 deficiency. Mice lacking mrp4 exhibited no outward phenotypes but had a decrease in plasma PGE metabolites and an increase in inflammatory pain threshold compared with wild-type mice. Collectively, these findings imply that mrp4 mediates the efflux of PGE 2 and concomitantly modulates cAMP mediated signaling for balanced PG synthesis in MEF cells. Abrogation of mrp4 affects the regulation of peripheral PG levels and consequently alters inflammatory nociceptive responses in vivo.MRP4 (ABCC4) is a member of the multidrug resistance proteins (MRPs) belonging to the C group of the ATP-binding cassette (ABC) protein superfamily. To date, nine total MRP members, MRP1

An expression vector, pIN‐GT, encoding the soluble form of beta 1,4‐galactosyltransferase (GT) has been constructed from human GT cDNAs and the pIN‐III‐ompA2 expression vector. Escherichia coli strain SB221 harboring the pIN‐GT plasmid produces and secretes a fusion protein consisting of the ompA signal and GT. The expression of GT was detected by assaying enzymatic activity as well as by Western blotting using anti‐GT antibodies. The recombinant GT was purified to homogeneity by N‐acetylglucosamine‐Sepharose affinity chromatography. The NH2‐terminal peptide sequence of purified GT confirmed the cleavage site of the fusion protein by bacterial signal peptidase. This expression system was utilized to produce mutant forms of GT in order to identify specific amino acids involved in substrate binding sites. Photoaffinity labeling of GT with UDP‐galactose analog, 4‐azido‐2‐nitrophenyluridylylpyrophosphate (ANUP), followed by cyanogen bromide (CNBr) cleavage revealed that ANUP bound to a fragment of GT composed of amino acid residues from Asp276 to Met328. Within this peptide segment, Tyr284, Tyr287, Tyr309, Trp310 and Trp312 were separately substituted into Gly and Tyr287 into Phe by site‐directed mutagenesis. Enzymatic activity assay showed drastic reduction of the activity in all of the mutants except that Tyr287––Phe remained as active as wild‐type GT. Kinetic studies of the mutated GT showed that Tyr284, Tyr309 and Trp310 are critically involved in the N‐acetyglucosamine binding and Tyr309 is involved in UDP‐galactose binding as well.(ABSTRACT TRUNCATED AT 250 WORDS)

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