The multidrug resistance protein MRP1 mediates the release of glutathione disulfide from rat astrocytes during oxidative stress

The release of glutathione disulfide has been considered an important process for the maintenance of a reduced thiol redox potential in cells during oxidative stress. In cultured rat astrocytes, permanent hydrogen peroxide-induced oxidative stress caused a rapid increase in intracellular glutathione disulfide, which was followed by the appearance of glutathione disulfide in the medium. Under these conditions, the viability of the cells was not compromised. In the presence of cyclosporin A and the quinoline-derivative MK571, inhibitors of multidrug resistance proteins (MRP1 and MRP2), glutathione disulfide accumulated in cells and the release of glutathione disulfide from astrocytes during H2O2 stress was potently inhibited, suggesting a contribution of MRP1 or MRP2 in the release of glutathione disulfide from astrocytes. Using RT-PCR we amplified a cDNA from astroglial RNA with a high degree of homology to MRP1 from humans and mouse. In contrast, no fragment was amplified by using primers specific for rat MRP2. In addition, the presence of MRP1 protein in astrocytes was demonstrated by its immunolocalization in cells expressing the astroglial marker protein glial fibrillary acidic protein. Our data identify rat astrocytes as a MRP1-expressin, brain cell type and demonstrate that this transporter participates in the release of glutathione disulfide from astrocytes during oxidative stress.     

Effects of clotrimazole on transport mediated by multidrug resistance associated protein 1 (MRP1) in human erythrocytes and tumour cells.

Clotrimazole has been shown to have potent anti-malarial activity in vitro, one possible mechanism being inhibition of oxidized glutathione (GSSG) export from the infected human red blood cells or from the parasite itself. Efflux of GSSG from normal erythrocytes is mediated by a high affinity glutathione S-conjugate transporter. This paper shows that transport of the model substrate, 3 microm dinitrophenyl S-glutathione, across erythrocyte membranes is inhibited by multidrug resistance-associated protein 1 (MRP1)-specific antibody, QCRL-3, strongly suggesting that the high affinity transport is mediated by MRP1. The rates of transport observed with membrane vesicles prepared from erythrocytes or from multidrug resistant tumour cells show a similar pattern of responses to applied reduced glutathione, GSSG and MRP1 inhibitors (indomethacin, MK571) further supporting the conclusion that the high affinity transporter is MRP1. In both erythrocytes and MRP1-expressing tumour cells, MRP1-associated transport is inhibited by clotrimazole over the range 2-20 microm, and the inhibitory effect leads to increases in accumulation of MRP1 substrates, vincristine and calcein, and decreases in calcein efflux from intact MRP1-expressing human tumour cells. It also results in increased sensitivity to daunorubicin of the multidrug resistant cells, L23/R but not the sensitive parent L23/P cells. These results demonstrate that clotrimazole can inhibit the MRP1 which is present in human erythrocytes, an effect that may contribute to, though not fully account for, its anti-malarial action.     

hPop5, a protein subunit of the human RNase MRP and RNase P endoribonucleases.

The RNase MRP and RNase P particles both function as endoribonucleases. RNase MRP has been implicated in the processing of precursor-rRNA, whereas RNase P has been shown to function in the processing of pre-tRNA. Both ribonucleoprotein particles have an RNA component that can be folded into a similar secondary structure and share several protein components. We have identified human, rat, mouse, cow, and Drosophila homologues of the Pop5p protein subunit of the yeast RNase MRP and RNase P complexes. The human Pop5 cDNA encodes a protein of 163 amino acids with a predicted molecular mass of 18.8 kDa. Polyclonal antibodies raised against recombinant hPop5 identified a 19-kDa polypeptide in HeLa cells and showed that hPop5 is associated with both RNase MRP and RNase P. Using affinity-purified anti-hPop5 antibodies, we demonstrated that the endogenous hPop5 protein is localized in the nucleus and accumulates in the nucleolus, which is consistent with its association with RNase MRP and RNase P. Catalytically active RNase P was partially purified from HeLa cells, and hPop5 was shown to be associated with it. Finally, the evolutionarily conserved acidic C-terminal tail of hPop5 appeared to be required neither for complex formation nor for RNase P activity.     

Immunological Detection of Isoforms of the Somatostatin Receptor Subtype, SSTR2

Abstract: Somatostatin (SRIF) induces its diverse physiological actions through interactions with different receptor subtypes. Multiple SRIF receptor subtypes have recently been cloned. To analyze the physical properties of receptor subtype SSTR2, two different peptide-directed antibodies were generated against SSTR2. Antibody “2e3,” directed against the peptide SSCTINWPGESGAWYT (residues 191–206), corresponding to a region in the predicted third extracellular domain of mouse SSTR2, and antibody “2i4,” directed against the peptide SGTEDGERSDS (residues 333–343) from the predicted cytoplasmic tail of mouse SSTR2, were developed. In Chinese hamster ovary (CHO) cells stably expressing the mouse SSTR2 gene (CHOB), the antibody 2e3 recognized specifically a protein of 93-kDa protein by immunoblotting. No specific immunoreactivity was detected by 2e3 in nontransfected CHO cells or CHO cells stably expressing vector alone or human SSTR1 or mouse SSTR3 genes. The antibody 2i4 specifically immunoprecipitated SSTR2 solubilized from CHOB cells that could be labeled with the SSTR2-specific ligand ¹²⁵I-MK-678. Furthermore, both 2e3 and 2i4 specifically immunoprecipitated 93-kDa [³⁵S]methionine-labeled proteins from CHOB cells, indicating that they recognize the same proteins. In contrast to studies in CHOB cells, immunoblotting studies showed that 2e3 detected specifically a single 148-kDa protein from different regions of the rat brain that have previously been shown to express high levels of SSTR2 mRNA and SRIF receptors with high affinity for ¹²⁵I-MK-678. In contrast, no immunoreactivity was detected in rat kidney, liver, or lung, which do not express SSTR2. No 93-kDa protein was detected specifically in the rat brain. The 148-kDa protein detected by 2e3 is an SRIF receptor because 2e3 and 2i4 specifically immunoprecipitated solubilized rat brain SRIF receptors that could be reversibly labeled with ¹²⁵I-MK-678. As in rat brain, 2e3 interacted specifically with a single 148-kDa protein in rat pituitary, in the rat pancreatic cell line AR42J, and in the HEK 293 cell line derived from human kidney, all of which express SSTR2 mRNA and SRIF receptors with high affinity for ¹²⁵I-MK-678. These findings indicate that rat brain and pituitary, as well as a pancreatic and a kidney cell line, express primarily a form of SSTR2 different from CHOB cells. The multiple forms of SSTR2 may result from differential post-translational processing of SSTR2 because 2e3 immunoprecipitated 41-kDa in vitro translation products generated from mRNA extracted from CHOB and AR42J cells. This 41-kDa protein has the predicted size of unprocessed SSTR2. These results demonstrate that 2e3 and 2i4 antibodies interact specifically with SSTR2. Detection of two different size proteins by the SSTR2 peptide-directed antibodies suggests the existence of multiple forms of SSTR2.     

Antiviral state against influenza virus neutralized by microinjection of antibodies to interferon-induced Mx proteins.

In mouse Mx+ cells, interferon alpha/beta induces the synthesis of the nuclear Mx protein, whose accumulation is correlated with specific inhibition of influenza viral protein synthesis. When Mx+ mouse cells are microinjected with the monoclonal anti-Mx antibody 2C12, interferon alpha/beta still induces Mx protein, but no longer inhibits efficiently the expression of influenza viral proteins as visualized by immunofluorescent labeling. However, interferon inhibition of an unrelated control virus, vesicular stomatitis virus, remains unchanged. Proteins with homology to mouse Mx protein are found in interferon-treated cells of a variety of mammalian species. In rat cells, for instance, rat interferon alpha/beta induces three Mx proteins which all cross-react with antibody 2C12 but differ in mol. wt and intracellular location, and it protects these cells well against influenza viruses. However, when rat cells are microinjected with antibody 2C12, interferon alpha/beta cannot induce an efficient antiviral state against influenza virus infection, whereas protection against vesicular stomatitis virus is not altered. These results show that both mouse and rat cells require functional Mx proteins for efficient protection against influenza virus. They further demonstrate that microinjection of antibodies is a promising way of elucidating the role of particular interferon-induced proteins in the intact cell.     

Monoclonal antibodies to monoamine oxidase B and another mitochondrial protein from human liver.

A monoclonal antibody has been generated to human liver monoamine oxidase (MAO) B by fusion of mouse myeloma cells with spleen cells from a mouse immunized with a mixture of semi-purified MAO A and MAO B. The antibody, 3F12/G10, an immunoglobulin G1, reacts with its antigen in cryostat sections of human liver, showing an intracellular particulate distribution as demonstrated by immunoperoxidase staining. The antibody indirectly precipitates [3H]pargyline-labelled human MAO B both from liver and platelet extracts but fails to precipitate MAO A from liver extracts. The antibody does not recognise rat liver MAO B, showing that the determinant is not universally expressed on MAO B. The antibody has no effect on the catalytic activity of MAO B. Other monoclonal antibodies were generated but they are directed to a protein with a subunit Mr of 54 000, a contaminant of the MAO preparation. One of these antibodies, A8/C2, an IgG2a, reacts with the same protein in both rat and human liver extracts.     

Identification of the apical membrane-targeting signal of the multidrug resistance-associated protein 2 (MRP2/MOAT)

The human canalicular multispecific organic anion transporter (cMOAT), known as the multidrug resistance-associated protein 2 (MRP2), is normally expressed in the liver and to a lesser extent in the kidney proximal tubules. In these tissues MRP2 specifically localizes to the apical membrane. The construction of MRP2 fused to the green fluorescent protein, and subsequent site-directed mutagenesis enabled the identification of a targeting signal in MRP2 that is responsible for its apical localization in polarized cells. The specific apical localization of MRP2 is due to a C-terminal tail that is not present in the basolaterally targeted MRP1. Deletion of three amino acids from the C-terminal of MRP2 (DeltaMRP2) causes the protein to be localized predominantly in the basolateral membrane in polarized Madin-Darby canine kidney cells. Interestingly, MRP2 expressed in a mouse leukemia cell line (L1210 cells) predominantly accumulates intracellularly with minimal cell membrane localization. In contrast, DeltaMRP2 was shown to predominantly localize in the cell membrane in L1210 cells. Increased transport of 2,4-dinitrophenyl glutathione from L1210 cells expressing DeltaMRP2 showed that the re-targeted protein retains its normal function.     

The use of monoclonal antibodies to study the proteins specified by the transforming region of human adenoviruses.

Monoclonal antibodies against two of the proteins specified by one of the transforming genes (early region 1B) of human adenovirus type 2 have been produced and characterized. Two clones (RA1 and PA6), generated by fusion of mouse myeloma NSO cells with splenocytes from rats immunized with whole-cell lysates of an adenovirus-transformed rat cell line (F19), secreted antibodies against a 58 kDa protein. Another clone (DC1) produced antibodies against the same protein, and resulted from fusion of immune rat splenocytes with the rat myeloma Y3.Ag.1.2.3. Immunoprecipitation studies showed that all three antibodies recognized [35S]-methionine-labelled 58 kDa protein, and phosphorylated derivatives of the 58 kDa protein labelled with [32P]orthophosphate present in infected human cells. One clone (EC3) produced antibody against a 19 kDa protein also encoded by early region 1B, but not sharing sequence homology with 58 kDa. The identity of the 19 kDa protein recognized by the EC3 antibody was established by immunoprecipitation from lysates of labelled-infected cells and from products of cell-free translation directed by mRNA isolated from adenovirus 2-infected cells. Indirect immunofluorescent-antibody staining of infected human cells using the RA1 and EC3 antibodies revealed a nuclear location of the 58 kDa protein and a mainly cytoplasmic location of the 19 kDa protein.     

hPop4: a new protein subunit of the human RNase MRP and RNase P ribonucleoprotein complexes.

RNase MRP is a ribonucleoprotein particle involved in the processing of pre-rRNA. The RNase MRP particle is structurally highly related to the RNase P particle, which is involved in pre-tRNA processing. Their RNA components fold into a similar secondary structure and they share several protein subunits. We have identified and characterised human and mouse cDNAs that encode proteins homologous to yPop4p, a protein subunit of both the yeast RNase MRP and RNase P complexes. The human Pop4 cDNA encodes a highly basic protein of 220 amino acids. Transfection experiments with epitope-tagged hPop4 protein indicated that hPop4 is localised in the nucleus and accumulates in the nucleolus. Immunoprecipitation assays using extracts from transfected cells expressing epitope-tagged hPop4 revealed that this protein is associated with both the human RNase MRP and RNase P particles. Polyclonal rabbit antibodies raised against recombinant hPop4 recognised a 30 kDa protein in total HeLa cell extracts and specifically co-immunoprecipitated the RNA components of the RNase MRP and RNase P complexes. Finally we showed that anti-hPop4 immunoprecipitates possess RNase P enzymatic activity. Taken together, these data show that we have identified a protein that represents the human counterpart of the yeast Pop4p protein.     

Evidence for the role of glycosylation in accessibility of the extracellular domains of human MRP1 (ABCC1)

To enable cell surface localization of the human multidrug resistance protein (MRP1, ABCC1) and to assess the role of the extracellular domains of this transporter, the FLAG epitope tag was introduced into different extracellular loops of the three membrane-spanning domains (MSDs) of the transporter. We constructed and expressed various partially and fully glycosylation-deficient, FLAG-tagged MRP1 proteins in a Vaccinia virus-based transient expression system, and the cell surface expression level of MRP1 on intact cells was followed by flow cytometry, using the FLAG tag specific monoclonal antibody M2. We also expressed the wild-type MRP1 protein and some of the FLAG-tagged mutants in stably transfected HEK293 cells, and followed the cell surface expression and the transport function of MRP1 both by monitoring the efflux of fluorescent substrate and by their ability to confer resistance to HEK293 transfectants to anticancer agents such as daunorubicin and etoposide. When we inserted the FLAG epitope in extracellular loops of the MSD1 or MSD3, the tag was accessible upon removal of N-glycosylation sites (N --> Q at positions 17, 23, and 1006, respectively), whereas the FLAG epitope placed in the MSD2 was not accessible even after removal of all three N-glycosylation sites, indicating that MSD2 region is deeply buried in the plasma membrane. However, all FLAG tagged MRP1 mutants were expressed at the cell surface to the same extent as the wild-type protein and also exhibited normal transport function. Our results demonstrate that the accessibility of the external FLAG epitope is strongly dependent on the position of the tag and the glycosylation state of the different FLAG-tagged MRP1s, and the conformation of extracellular loops in MSD1 and MDS3 does not appear to contribute to the functional status of MRP1.     

Cytoplasmic retraction of the amino terminus of human multidrug resistance protein 1

Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette (ABC) transport superfamily which also includes human multidrug resistance 1 (MDR1) gene product P-glycoprotein (Pgp). Overexpression of MRP1 or Pgp causes multidrug resistance in cancer cells. Different from Pgp, MRP1 contains an extra membrane-spanning domain (MSD1) with a putative extracellular amino terminus in addition to the core structure of two MSDs and two NBDs (nucleotide-binding domains). The structural and functional significance of the additional MSD1 in MRP1 remains elusive. In this study, we generated an IgG1 subclass monoclonal antibody, IU2H10, specific to the amino terminus of human MRP1 and mapped its epitope to 10 amino acids (S8ADGSDPLWD17). It can be used for Western blot, immunoprecipitation, and indirect immunofluorescence studies of human MRP1. However, surprisingly we found that IU2H10 cannot react with MRP1 unless cells are permeabilized. Furthermore, the IU2H10 epitope is exposed extracellularly when the carboxyl-terminal core domain of human MRP1 is deleted. Examination of the amino-terminal sequence of human MRP1 suggests that it consist of mainly coiled structures. These observations provide evidence for a model that is different from the prevailing extracellular location of the amino terminus of human MRP1. It is possible that part of the amino terminus of human MRP1, following exposure to the lumen of the endoplasmic reticulum, is retracted to the cytoplasm.     

The 23-kilodalton protein, a substrate of protein kinase C, in bovine neutrophil cytosol is a member of the S100 family.

A bovine neutrophil protein termed p23 because of an apparent molecular mass of 23 kDa in SDS-PAGE is present in large amounts both in a soluble form in the cytosolic fraction of bovine neutrophil homogenates and associated to the cytoskeleton. P23 is accompanied during the first steps of the purification procedure by a smaller size protein termed p7 on the basis of a rate of migration in SDS-PAGE corresponding to a 7-kDa protein [Stasia, M. J., Dianoux, A. C., & Vignais, P. V. (1989) Biochemistry 28, 9659-9667]. The two proteins, p23 and p7, have been purified to homogeneity by an improved procedure consisting of two chromatographic steps. The electrospray mass spectrometry technique applied to p23 and p7 indicated molecular masses close to 17 and 10 kDa, respectively, significantly different from the masses derived by SDS-PAGE. Bovine neutrophil p23 and p7 presented large primary structure homologies with two human proteins, MRP14 and MRP8, which are expressed in large amounts in macrophages under conditions of chronic inflammation. In addition, p23 and p7 cross-reacted with monoclonal antibodies specific of MRP14 and MRP8. Bovine p23 and p7 bound Ca2+, and their amino acid sequences contained two Ca(2+)-binding domains per protein, largely identical to those of human MRP14 and MRP8. Bovine p23 and p7 associated together to form a heterodimeric complex, which largely escaped attack by trypsin, whereas the isolated p23 and p7 components were readily digested. These features are typical of Ca(2+)-binding proteins belonging to the S100 family.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of interferon action. Production and characterization of monoclonal and polyclonal antibodies to the interferon-induced phosphoprotein P1

Monoclonal and polyclonal antibodies to the interferon-induced phosphoprotein P1 were prepared using protein P1 purified from human amnion U cells as the immunogen. Rabbit antiserum to protein P1 recognized with comparable efficiency P1 both from human U cells and from mouse L929 cells. Immunoprecipitates that contained protein P1 also possessed a protein kinase activity that catalyzed the phosphorylation of protein P1 and the alpha subunit of initiation factor eIF-2. Three BALB/C mouse monoclonal antibodies efficiently recognized human protein P1, but either did not recognize or recognized very poorly P1 from mouse cells. A fourth monoclonal antibody against human P1 recognized mouse P1 with nearly equal efficiency. Immunoprecipitation of human P1 with different sequential combinations of the monoclonal antibodies suggest that two antigenic classes of protein P1 may exist.     

ADAMTS1 cleaves aggrecan at multiple sites and is differentially inhibited by metalloproteinase inhibitors

ADAMTS1 is a secreted protein that belongs to the recently described ADAMTS (a disintegrin and metalloprotease with thrombospondin repeats) family of proteases. Evaluation of ADAMTS1 catalytic activity on a panel of extracellular matrix proteins showed a restrictive substrate specificity which includes some proteoglycans. Our results demonstrated that human ADAMTS1 cleaves aggrecan at a previously shown site by its mouse homolog, but we have also identified additional cleavage sites that ultimately confirm the classification of this protease as an 'aggrecanase'. Specificity of ADAMTS1 activity was further verified when a point mutation in the zinc-binding domain abolished its catalytic effects, and latency conferred by the prodomain was also demonstrated using a furin cleavage site mutant. Suppression of ADAMTS1 activity was accomplished with a specific monoclonal antibody and some metalloprotease inhibitors, including tissue inhibitor of metalloproteinases 2 and 3. Finally, we developed an activity assay using an artificial peptide substrate based on the interglobular domain cleavage site (E(373)-A) of rat aggrecan.     

Two distinct cytotoxic T lymphocyte subpopulations in patients with Vogt-Koyanagi-Harada disease that recognize human melanoma cells

The functional properties of cytotoxic lymphocytes from patients with Vogt-Koyanagi-Harada disease ( VKH ) specific for human melanoma cells (P-36 melanoma cell line established from a patient with malignant melanoma) were investigated by using monoclonal antibodies specific for human T cell subsets. Peripheral blood lymphocytes (PBL) from patients with VKH showed significant cytotoxic activity against the P-36 (SK-MEL-28) human melanoma cell line, but not against a human cervical carcinoma of the uterus cell line (HeLa-S3 cell line) or against a mouse melanoma cell line (B-16 cell line) originating from a C57BL/6 strain mouse or against the EL-4 mouse lymphoma cell line from a C57BL/6 mouse. The cytotoxic activity of the patients' PBL against the P-36 melanoma cell line was markedly reduced by pretreatment of the PBL with monoclonal anti-human Leu-1 antibody plus rabbit complement, but it was reduced to much less extent by pretreatment with either monoclonal anti-human Leu-2a or Leu-3a antibody plus rabbit complement. The specific cytotoxic activity of the patients' PBL against the P-36 human melanoma cell line is, therefore, mediated by T cells bearing Leu-1+ Leu-2a+ or Leu-1+ Leu-3a+ antigens. Furthermore, the cytotoxic activity was shown to be blocked not only by anti-Leu-2a antibody specific to human cytotoxic/suppressor T cells but also unexpectedly by anti-Leu-3a antibody which has previously been considered to be specific to human inducer/helper T cells. The results of this study suggest that at least two distinct subpopulations of cytotoxic T cells specific for P-36 human melanoma cells are present in the peripheral blood of VKH patients. These cytotoxic T cells have different surface antigens, Leu-2a and Leu-3a.     

Expression and localization of the multidrug resistance-associated protein 3 in rat small and large intestine

Multidrug resistance-associated protein 3 (MRP3; symbol ABCC3), has been shown to mediate ATP-dependent transport of organic anions including 17beta-glucuronosyl estradiol, glucuronosyl bilirubin, monovalent, and sulfated bile salts. MRP3 mRNA expression was reported in rat intestine suggesting a role of MRP3 in the intestinal transport. We examined the expression and localization of MRP3 in rat small and large intestine by RT-PCR, immunofluorescence, and immunoblot analysis. MRP3 was identified in all intestinal segments by RT-PCR. MRP3 expression was low in duodenum and jejunum but markedly increased in ileum and colon. With the use of a rat MRP3 specific antibody, MRP3 was localized to the basolateral domains of enterocytes. Immunofluorescence analysis and immunoblot analysis confirmed a strong expression of rat MRP3 in ileum and colon. In contrast, MRP2 was predominantly expressed in the proximal segments of rat small intestine. Our findings demonstrate a high expression of rat MRP3 in ileum and colon and provide evidence for an involvement of MRP3 in the ATP-dependent transport of organic anions, including bile salts from the enterocyte into blood.     

Dynamic localization of RNase MRP RNA in the nucleolus observed by fluorescent RNA cytochemistry in living cells

The dynamic intra-nuclear localization of MRP RNA, the RNA component of the ribonucleoprotein enzyme RNase MRP, was examined in living cells by the method of fluorescent RNA cytochemistry (Wang, J., L.-G. Cao, Y.-L. Wang, and T. Pederson. 1991. Proc. Natl. Acad. Sci. USA. 88:7391-7395). MRP RNA very rapidly accumulated in nucleoli after nuclear microinjection of normal rat kidney (NRK) epithelial cells. Localization was specifically in the dense fibrillar component of the nucleolus, as revealed by immunocytochemistry with a monoclonal antibody against fibrillarin, a known dense fibrillar component protein, as well as by digital optical sectioning microscopy and 3-D stereo reconstruction. When MRP RNA was injected into the cytoplasm it was not imported into the nucleus. Nuclear microinjection of mutant MRP RNAs revealed that nucleolar localization requires a sequence element (nucleotides 23-62) previously implicated as a binding site for a nucleolar protein, the To antigen. These results demonstrate the dynamic localization of MRP RNA in the nucleus and provide important insights into the nucleolar targeting of MRP RNA.