Peptide name : ATP-binding cassette sub-family C member 5

Source/Organism : Human

Linear/Cyclic : Not found

Chirality : Not found

Peptide length: 1437

C-terminal modification: Not found

N-terminal modification : Free

Non-natural peptide information: None

Assay type : Formate dehydrogenase–coupled PDF assay, Thymidine incorporation assay

Assay time : 48h

Activity : IC50 ± SD : 5.2 ± 2.2 μM

Cell line : Daudi lymphoma cells

Cancer type : Lymphoma

Other activity : Not found

Amino acid composition bar chart :

Molecular mass : 160658.490 Dalton

Aliphatic index : 0.994

Instability index : 39.3696

Hydrophobicity (GRAVY) : -0.016

Isoelectric point : 8.8667

Charge (pH 7) : 19.7445

Aromaticity : 0.078

Molar extinction coefficient (cysteine, cystine): (142670, 143795)

Hydrophobic/hydrophilic ratio : 1.09781021

hydrophobic moment : -0.033

Missing amino acid : None

Most occurring amino acid : L

Most occurring amino acid frequency : 167

Least occurring amino acid : W

Least occurring amino acid frequency : 17

Secondary structure fraction (Helix, Turn, Sheet): (0.3, 0.2, 0.3)

SMILES Notation: 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Paper title : Toward a comprehensive characterization of a human cancer cell phosphoproteome.

Doi : https://doi.org/10.1021/pr300630k

Abstract : Mass spectrometry (MS)-based phosphoproteomics has achieved extraordinary success in qualitative and quantitative analysis of cellular protein phosphorylation. Considering that an estimated level of phosphorylation in a cell is placed at well above 100,000 sites, there is still much room for improvement. Here, we attempt to extend the depth of phosphoproteome coverage while maintaining realistic aspirations in terms of available material, robustness, and instrument running time. We developed three strategies, where each provided a different balance between these three key parameters. The first strategy simply used enrichment by Ti(4+)-IMAC followed by reversed chromatography LC-MS (termed 1D). The second strategy incorporated an additional fractionation step through the use of HILIC (2D). Finally, a third strategy was designed employing first an SCX fractionation, followed by Ti(4+)-IMAC enrichment and additional fractionation by HILIC (3D). A preliminary evaluation was performed on the HeLa cell line. Detecting 3700 phosphopeptides in about 2 h, the 1D strategy was found to be the most sensitive but limited in comprehensivity, mainly due to issues with complexity and dynamic range. Overall, the best balance was achieved using the 2D based strategy, identifying close to 17,000 phosphopeptides with less than 1 mg of material in about 48 h. Subsequently, we confirmed the findings with the K562 cell sample. When sufficient material was available, the 3D strategy increased phosphoproteome allowing over 22,000 unique phosphopeptides to be identified. Unfortunately, the 3D strategy required more time and over 1 mg of material before it started to outperform 2D. Ultimately, combining all strategies, we were able to identify over 16,000 and nearly 24,000 unique phosphorylation sites from the cancer cell lines HeLa and K562, respectively. In summary, we demonstrate the need to carry out extensive fractionation for deep mining of the phosphoproteome and provide a guide for appropriate strategies depending on sample amount and/or analysis time.

Paper title : Characterization of the transport of nucleoside analog drugs by the human multidrug resistance proteins MRP4 and MRP5.

Doi : https://doi.org/10.1124/mol.63.5.1094

Abstract : The human multidrug resistance proteins MRP4 and MRP5 are organic anion transporters that have the unusual ability to transport cyclic nucleotides and some nucleoside monophosphate analogs. Base and nucleoside analogs used in the chemotherapy of cancer and viral infections are potential substrates. To assess the possible contribution of MRP4 and MRP5 to resistance against these drugs, we have investigated the transport mediated by MRP4 and MRP5. In cytotoxicity assays, MRP4 conferred resistance to the antiviral agent 9-(2-phosphonomethoxyethyl)adenine (PMEA) and high-performance liquid chromatography analysis showed that, like MRP5, MRP4 transported PMEA in an unmodified form. MRP4 also mediated substantial resistance against other acyclic nucleoside phosphonates, whereas MRP5 did not. Apart from low-level MRP4-mediated cladribine resistance, the cytotoxicity of clinically used anticancer nucleosides was not influenced by overexpression of MRP4 or MRP5. In contrast, MRP5 mediated efflux of the pyrimidine-based antiviral 2',3'-dideoxynucleoside 2',3'-didehydro-2',3'-dideoxythymidine 5'-monophosphate (d4TMP) and its phosphoramidate derivative alaninyl-d4TMP from cells loaded with the 2',3'-didehydro-2',3'-dideoxythymidine prodrugs cyclosaligenyl-d4TMP and aryloxyphosphoramidate d4TMP (So324), respectively. Moreover, only inside-out membrane vesicles derived from MRP5-overexpressing cells accumulated alaninyl-d4TMP. Cellular efflux and vesicular uptake studies were carried out to further compare transport mediated by MRP4 and MRP5 and showed that dipyridamole, dilazep, nitrobenzyl mercaptopurine riboside, sildenafil, trequinsin and MK571 inhibited MRP4 more than MRP5, whereas cyclic nucleotides and monophosphorylated nucleoside analogs were equally poor inhibitors of both pumps. These results strongly suggest that the affinity of MRP4 and MRP5 for nucleotide-based substrates is low.

Paper title : Control of metazoan heme homeostasis by a conserved multidrug resistance protein.

Doi : https://doi.org/10.1016/j.cmet.2014.03.030

Abstract : Several lines of evidence predict that specific pathways must exist in metazoans for the escorted movement of heme, an essential but cytotoxic iron-containing organic ring, within and between cells and tissues, but these pathways remain obscure. In Caenorhabditis elegans, embryonic development is inextricably dependent on both maternally derived heme and environmentally acquired heme. Here, we show that the multidrug resistance protein MRP-5/ABCC5 likely acts as a heme exporter, and targeted depletion of mrp-5 in the intestine causes embryonic lethality. Transient knockdown of mrp5 in zebrafish leads to morphological defects and failure to hemoglobinize red blood cells. MRP5 resides on the plasma membrane and endosomal compartments and regulates export of cytosolic heme. Together, our genetic studies in worms, yeast, zebrafish, and mammalian cells identify a conserved, physiological role for a multidrug resistance protein in regulating systemic heme homeostasis. We envision other MRP family members may play similar unanticipated physiological roles in animal development.

Paper title : Characterization of MOAT-C and MOAT-D, new members of the MRP/cMOAT subfamily of transporter proteins.

Doi : https://doi.org/10.1093/jnci/90.22.1735

Abstract : BACKGROUND: Multidrug resistance-associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT) are transporter proteins that pump organic anions across cellular membranes and have been linked to resistance to cytotoxic drugs. We previously identified MOAT-B, an MRP/cMOAT-related transporter, by use of a polymerase chain reaction approach. However, analysis of expressed sequence tag (EST) databases indicated that there might be additional MRP/cMOAT-related transporters. To further define the MRP/cMOAT subfamily of transporters, we used EST probes to isolate complementary DNAs for two related transporter proteins, MOAT-C and MOAT-D. METHODS: MOAT-C and MOAT-D expression patterns in human tissues were determined by RNA blot analysis, and chromosomal localization of the genes was determined by fluorescence in situ hybridization. RESULTS: MOAT-C is predicted to encode a 1437-amino-acid protein that, among eukaryotic transporters, is most closely related to MRP, cMOAT, and MOAT-B (about 36% identity). However, MOAT-C is less related to MRP and cMOAT than MRP and cMOAT are to each other (about 48% identity). Like MOAT-B, MOAT-C lacks an N-terminal membrane-spanning domain, indicating that the topology of this protein is similarly distinct from that of MRP and cMOAT. MOAT-D is predicted to encode a 1527-amino-acid protein that is the closest known relative of MRP (about 58% identity). MOAT-D is also highly related to cMOAT (about 47% identity). The presence of an N-terminal membrane-spanning domain indicates that the topology of MOAT-D is quite similar to that of MRP and cMOAT. MOAT-C transcripts are widely expressed in human tissues; however, MOAT-D transcript expression is more restricted. The MOAT-C and MOAT-D genes are located at chromosomes 3q27 and 17q21.3, respectively. CONCLUSIONS: On the basis of amino acid identity and protein topology, the MRP/cMOAT transporter subfamily falls into two groups; the first group consists of MRP, cMOAT, and MOAT-D, and the second group consists of MOAT-B and MOAT-C.

Paper title : cDNA cloning of a short type of multidrug resistance protein homologue, SMRP, from a human lung cancer cell line.

Doi : https://doi.org/10.1006/bbrc.1997.7346

Abstract : Members of the ATP binding cassette (ABC) superfamily are involved in the energy-dependent transport of a wide variety of substrates including anticancer agents across the membranes. We have cloned a cDNA fragment including a novel ABC sequence from a cisplatin-resistant human lung adenocarcinoma cell line, PC-14/CDDP, by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers and screened a cDNA library using the cDNA fragment as a probe. A full-length cDNA clone, BM4.8, was obtained. Sequence analysis showed that the cDNA encoded a short type of multidrug resistance protein homologue, SMRP, by computed homology search. SMRP was composed of 946 amino acids and had two ABCs with walker A and B motifs. This gene was mapped on chromosome 3 at band q27 by fluorescence in situ hybridization (FISH) analysis and was found to be expressed in various tissues by Northern blot analysis.

Paper title : Three novel ABCC5 splice variants in human retina and their role as regulators of ABCC5 gene expression.

Doi : https://doi.org/10.1186/1471-2199-8-42

Abstract : BACKGROUND: The ABCC5 gene encodes an organic anion pump of the ATP-binding cassette (ABC) transporter family, subclass C. The exact physiological function of ABCC5 however is not known. Here, we have isolated three novel ABCC5 splice variants and characterized their role in the regulation of ABCC5 gene expression. RESULTS: Two additional exons within intron 5 of the ABCC5 gene were identified; one of the exons exhibits alternative donor splice sites. Differential usage of these exons generates three short ABCC5 transcripts named ABCC5_SV1, ABCC5_SV2 and ABCC5_SV3. The variants share the first five exons with the ABCC5 gene but differ in their 3' sequences. ABCC5 and its novel isoforms are abundantly expressed in the human retina. Splice variant ABCC5_SV1 and ABCC5_SV2 contain premature stop codons. While inhibition of nonsense-mediated mRNA decay selectively stabilized ABCC5_SV1 but not ABCC5_SV2, the amount of full length ABCC5 mRNA was simultaneously reduced. A negative regulatory effect on full length ABCC5 expression was also observed when the ABCC5 isoforms were silenced with siRNA duplexes. Finally, we show that the evolutionarily conserved ABCC5_SV2 transcript is translated into a protein abundantly present in endothelial cells of inner retinal blood vessels and along RPE membranes. CONCLUSION: Our data suggest that alternative splicing of the ABCC5 gene has functional consequences by modulating ABCC5 gene expression. In addition, at least one ABCC5 splice variant is protein-coding and produces a truncated ABCC5 protein isoform with thus far unknown functional properties in the retina.

Paper title : The DNA sequence, annotation and analysis of human chromosome 3.

Doi : https://doi.org/10.1038/nature04728

Abstract : After the completion of a draft human genome sequence, the International Human Genome Sequencing Consortium has proceeded to finish and annotate each of the 24 chromosomes comprising the human genome. Here we describe the sequencing and analysis of human chromosome 3, one of the largest human chromosomes. Chromosome 3 comprises just four contigs, one of which currently represents the longest unbroken stretch of finished DNA sequence known so far. The chromosome is remarkable in having the lowest rate of segmental duplication in the genome. It also includes a chemokine receptor gene cluster as well as numerous loci involved in multiple human cancers such as the gene encoding FHIT, which contains the most common constitutive fragile site in the genome, FRA3B. Using genomic sequence from chimpanzee and rhesus macaque, we were able to characterize the breakpoints defining a large pericentric inversion that occurred some time after the split of Homininae from Ponginae, and propose an evolutionary history of the inversion.

Paper title : Correction for Barrio et al., In vivo characterization of chronic traumatic encephalopathy using [F-18]FDDNP PET brain imaging.

Doi : https://doi.org/10.1073/pnas.1508756112

Abstract : Not available

Paper title : Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis.

Doi : https://doi.org/10.1126/scisignal.2000475

Abstract : Eukaryotic cells replicate by a complex series of evolutionarily conserved events that are tightly regulated at defined stages of the cell division cycle. Progression through this cycle involves a large number of dedicated protein complexes and signaling pathways, and deregulation of this process is implicated in tumorigenesis. We applied high-resolution mass spectrometry-based proteomics to investigate the proteome and phosphoproteome of the human cell cycle on a global scale and quantified 6027 proteins and 20,443 unique phosphorylation sites and their dynamics. Co-regulated proteins and phosphorylation sites were grouped according to their cell cycle kinetics and compared to publicly available messenger RNA microarray data. Most detected phosphorylation sites and more than 20% of all quantified proteins showed substantial regulation, mainly in mitotic cells. Kinase-motif analysis revealed global activation during S phase of the DNA damage response network, which was mediated by phosphorylation by ATM or ATR or DNA-dependent protein kinases. We determined site-specific stoichiometry of more than 5000 sites and found that most of the up-regulated sites phosphorylated by cyclin-dependent kinase 1 (CDK1) or CDK2 were almost fully phosphorylated in mitotic cells. In particular, nuclear proteins and proteins involved in regulating metabolic processes have high phosphorylation site occupancy in mitosis. This suggests that these proteins may be inactivated by phosphorylation in mitotic cells.

Paper title : The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides.

Doi : https://doi.org/10.1074/jbc.M005463200

Abstract : Cellular export of cyclic nucleotides has been observed in various tissues and may represent an elimination pathway for these signaling molecules, in addition to degradation by phosphodiesterases. In the present study we provide evidence that this export is mediated by the multidrug resistance protein isoform MRP5 (gene symbol ABCC5). The transport function of MRP5 was studied in V79 hamster lung fibroblasts transfected with a human MRP5 cDNA. An MRP5-specific antibody detected an overexpression of the glycoprotein of 185 +/- 15 kDa in membranes from MRP5-transfected cells and a low basal expression of hamster Mrp5 in control membranes. ATP-dependent transport of 3',5'-cyclic GMP at a substrate concentration of 1 micrometer was 4-fold higher in membrane vesicles from MRP5-transfected cells than in control membranes. This transport was saturable with a K(m) value of 2.1 micrometer. MRP5-mediated transport was also detected for 3',5'-cyclic AMP at a lower affinity, with a K(m) value of 379 micrometer. A potent inhibition of MRP5-mediated transport was observed by several compounds, known as phosphodiesterase modulators, including trequinsin, with a K(i) of 240 nm, and sildenafil, with a K(i) value of 267 nm. Thus, cyclic nucleotides are physiological substrates for MRP5; moreover, MRP5 may represent a novel pharmacological target for the enhancement of tissue levels of cGMP.

Paper title : pABC11 (also known as MOAT-C and MRP5), a member of the ABC family of proteins, has anion transporter activity but does not confer multidrug resistance when overexpressed in human embryonic kidney 293 cells.

Doi : https://doi.org/10.1074/jbc.274.33.23541

Abstract : Several members of the ABC family of proteins have been implicated in multidrug resistance associated with cancer therapies. A novel member of this gene family, designated pABC11, has been identified using degenerate polymerase chain reaction. The full-length cDNA spans 5881 base pairs and encodes an open reading frame of 1437 amino acids predicted to contain two sets of transmembrane domains and two nucleotide binding domains characteristic of ABC proteins. The nucleotide sequence described herein extends that of three recently reported sequences, MRP5 (Kool, M., de Haas, M., Scheffer, G., Scheper, R., van Eijk, M., Juijn, J., Baas, F., and Borst, P. (1997) Cancer Res. 57, 3537-3547), SMRP (Suzuki, T., Nishio, K., Sasaki, H., Kurokawa, H., Saito-Ohara, F., Ikeuchi, T., Tanabe, S., Terada, M., and Saijo, N. (1997) Biochem. Biophys. Res. Commun. 238, 790-794), and MOAT-C (Belinsky, M., Bain, L., Balsara, B., Testa, J., and Kruh, G. (1998) J. Natl. Cancer Inst. 90, 1735-1741), in the 5' direction. Northern blot analysis detected five transcripts that were differentially expressed in several tissue types, and the gene encoding pABC11 was mapped to chromosome 3. Confocal imaging of HEK293 cells expressing a green fluorescent protein-pABC11 construct confirmed plasma membrane localization of the fusion protein. Overexpression of pABC11 resulted in reduced labeling with the fluorochromes 5-chloromethylfluorescein diacetate, fluorescein diacetate, and 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein acetoxymethyl ester but not with calcein or rhodamine derivatives, consistent with pABC11 being an anion transporter. Fluorochrome export was ATP-dependent but glutathione-independent. We also show that this export pump does not confer resistance to various classes of cytotoxic drugs but does provide small but significant resistance to CdCl(2) and potassium antimonyl tartrate.

Paper title : The human multidrug resistance protein MRP5 transports folates and can mediate cellular resistance against antifolates.

Doi : https://doi.org/10.1158/0008-5472.CAN-04-2810

Abstract : Members of the multidrug resistance protein family, notably MRP1-4/ABCC1-4, and the breast cancer resistance protein BCRP/ABCG2 have been recognized as cellular exporters for the folate antagonist methotrexate (MTX). Here we show that MRP5/ABCC5 is also an antifolate and folate exporter based on the following evidence: (a) Using membrane vesicles from HEK293 cells, we show that MRP5 transports both MTX (KM = 1.3 mmol/L and VMAX = 780 pmol per mg protein per minute) and folic acid (KM = 1.0 mmol/L and VMAX = 875 pmol per mg protein per minute). MRP5 also transports MTX-glu2 (KM = 0.7 mmol/L and VMAX = 450 pmol per mg protein per minute) but not MTX-glu3. (b) Both accumulation of total [3H]MTX and of MTX polyglutamates were significantly reduced in MRP5 overexpressing cells. (c) Cell growth inhibition studies with MRP5 transfected HEK293 cells showed that MRP5 conferred high-level resistance (>160-fold) against the antifolates MTX, GW1843, and ZD1694 (raltitrexed) in short-term (4 hours) incubations with high drug concentrations; this resistance was proportional to the MRP5 level. (d) MRP5-mediated resistance (8.5- and 2.1-fold) was also found in standard long-term incubations (72 hours) at low concentrations of ZD1694 and GW1843. These results show the potential of MRP5 to mediate transport of (anti)folates and contribute to resistance against antifolate drugs.

Paper title : Expression and immunolocalization of the multidrug resistance proteins, MRP1-MRP6 (ABCC1-ABCC6), in human brain.

Doi : https://doi.org/10.1016/j.neuroscience.2004.07.051

Abstract : Multidrug resistance proteins (MRPs, symbol ABCC) are membrane glycoproteins that mediate the ATP-dependent export of organic anions, including cytotoxic and antiviral drugs, from cells. To identify MRP family members possibly involved in the intrinsic resistance of human brain to cytotoxic and antiviral drugs, we analyzed the expression and localization of MRP1-MRP6 in rapidly frozen perilesional samples of several regions of adult human brain obtained during neurosurgery. Quantitative polymerase chain reaction analysis showed expression of MRP1, MRP2, MRP3, MRP4, and MRP5 mRNA, whereas MRP6 mRNA was below detectability. However, immunofluorescence microscopy of cryosections from human brain showed no reactivity for the MRP2 or MRP3 proteins. The proteins MRP1, MRP4, and MRP5 were clearly localized by confocal laser scanning microscopy to the luminal side of brain capillary endothelial cells. The MRP4 and MRP5 proteins were also detected in astrocytes of the subcortical white matter. Notably, MRP5 protein was present in pyramidal neurons. MRP proteins may, thus, contribute to the cellular efflux of endogenous anionic glutathione or glucuronate conjugates (substrates for MRP1), cyclic nucleotides (substrates for MRP4 and MRP5), or glutathione (co-substrate for MRP1 and MRP4); in addition, they may play an important role in the resistance of the brain to several cytotoxic and antiviral drugs.

Paper title : ATP-binding Cassette Subfamily C Member 5 (ABCC5) Functions as an Efflux Transporter of Glutamate Conjugates and Analogs.

Doi : https://doi.org/10.1074/jbc.M115.692103

Abstract : The ubiquitous efflux transporter ABCC5 (ATP-binding cassette subfamily C member 5) is present at high levels in the blood-brain barrier, neurons, and glia, but its in vivo substrates and function are not known. Using untargeted metabolomic screens, we show that Abcc5(-/-) mice accumulate endogenous glutamate conjugates in several tissues, but brain in particular. The abundant neurotransmitter N-acetylaspartylglutamate was 2.4-fold higher in Abcc5(-/-) brain. The metabolites that accumulated in Abcc5(-/-) tissues were depleted in cultured cells that overexpressed human ABCC5. In a vesicular membrane transport assay, ABCC5 also transported exogenous glutamate analogs, like the classic excitotoxic neurotoxins kainic acid, domoic acid, and NMDA; the therapeutic glutamate analog ZJ43; and, as previously shown, the anti-cancer drug methotrexate. Glutamate conjugates and analogs are of physiological relevance because they can affect the function of glutamate, the principal excitatory neurotransmitter in the brain. After CO2 asphyxiation, several immediate early genes were expressed at lower levels in Abcc5(-/-) brains than in wild type brains, suggesting altered glutamate signaling. Our results show that ABCC5 is a general glutamate conjugate and analog transporter that affects the disposition of endogenous metabolites, toxins, and drugs.

Paper title : A quantitative atlas of mitotic phosphorylation.

Doi : https://doi.org/10.1073/pnas.0805139105

Abstract : The eukaryotic cell division cycle is characterized by a sequence of orderly and highly regulated events resulting in the duplication and separation of all cellular material into two newly formed daughter cells. Protein phosphorylation by cyclin-dependent kinases (CDKs) drives this cycle. To gain further insight into how phosphorylation regulates the cell cycle, we sought to identify proteins whose phosphorylation is cell cycle regulated. Using stable isotope labeling along with a two-step strategy for phosphopeptide enrichment and high mass accuracy mass spectrometry, we examined protein phosphorylation in a human cell line arrested in the G(1) and mitotic phases of the cell cycle. We report the identification of >14,000 different phosphorylation events, more than half of which, to our knowledge, have not been described in the literature, along with relative quantitative data for the majority of these sites. We observed >1,000 proteins with increased phosphorylation in mitosis including many known cell cycle regulators. The majority of sites on regulated phosphopeptides lie in [S/T]P motifs, the minimum required sequence for CDKs, suggesting that many of the proteins may be CDK substrates. Analysis of non-proline site-containing phosphopeptides identified two unique motifs that suggest there are at least two undiscovered mitotic kinases.

Paper title : Detailed structural analysis on both human MRP5 and mouse mrp5 transcripts.

Doi : https://doi.org/10.1016/s0378-1119(99)00529-6

Abstract : The multidrug-resistant phenotype in tumor cells is attributed in part to anti-cancer drug efflux transporters such as the MRP family. The amino-terminal structure of MRP5 has not been refined. To determine the amino-terminal structure of a major transcript of the MRP5 gene, we performed primer extension analysis to determine a major transcriptional start site of this gene and compared the structure of human MRP5 and that of mouse mrp5. We successfully determined the structures of human MRP5 and mouse mrp5. Estimated amino acid sequences are 1437 and 1436 amino acids for human MRP5 and mouse mrp5 respectively, and were highly conserved (94.1%). We further showed that our previously identified SMRP mRNA was a splicing variant of the MRP5 gene, which was expressed in various human tissues, suggesting that a short form of MRP5 protein encoded by the SMRP mRNA may have a physiological role.

Paper title : Thiopurine metabolism and identification of the thiopurine metabolites transported by MRP4 and MRP5 overexpressed in human embryonic kidney cells.

Doi : https://doi.org/10.1124/mol.62.6.1321

Abstract : Mercaptopurines have been used as anticancer agents for more than 40 years, and most acute lymphoblastic leukemias are treated with 6-mercaptopurine (6MP) or 6-thioguanine (TG). Overexpression of the two related multidrug resistance proteins MRP4 and MRP5 has been shown to confer some resistance against mercaptopurines, which has been attributed to extrusion of mercaptopurine metabolites by these transporters. We have analyzed the mercaptopurine metabolites formed in human embryonic kidney cells and determined which metabolites are extruded by MRP4 and MRP5. Incubation with 6MP led to the formation of thioinosine and thioxanthosine metabolites and we found that thio-IMP was transported by both MRP4 and MRP5; MRP5 showed the highest transport rate. In contrast, only MRP5 transported thioxanthosine monophosphate (tXMP). During incubation with TG, the monophosphorylated form of thioguanosine was transported by both MRP4 and MRP5; the highest transport rate was for MRP4. Similarly, only 6-methyl-thio-IMP was formed during incubation with 6-methyl mercaptopurine riboside. This compound was a substrate for both MRP4 and MRP5; MRP4 showed the highest transport rate. Our results show that all major thiopurine monophosphates important in the efficacy of mercaptopurine treatment are transported by MRP4 and MRP5, although the substrate specificity of the two transporters differs in detail.

Paper title : The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

Doi : https://doi.org/10.1101/gr.2596504

Abstract : The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

Paper title : cGMP transport by vesicles from human and mouse erythrocytes.

Doi : https://doi.org/10.1111/j.1742-4658.2006.05591.x

Abstract : cGMP secretion from cells can be mediated by ATP-binding cassette (ABC) transporters ABCC4, ABCC5, and ABCC11. Indirect evidence suggests that ABCC4 and ABCC5 contribute to cGMP transport by erythrocytes. We have re-investigated the issue using erythrocytes from wild-type and transporter knockout mice. Murine wild-type erythrocyte vesicles transported cGMP with an apparent Km that was 100-fold higher than their human counterparts, the apparent Vmax being similar. Whereas cGMP transport into human vesicles was efficiently inhibited by the ABCC4-specific substrate prostaglandin E1, cGMP transport into mouse vesicles was inhibited equally by Abcg2 and Abcc4 inhibitors/substrates. Similarly, cGMP transport into vesicles from Abcc4-/- and Abcg2-/- mice was 42% and 51% of that into wild-type mouse vesicles, respectively, whereas cGMP transport into vesicles from Abcc4(-/-)/Abcg2(-/-) mice was near background. The knockout mice were used to show that Abcg2-mediated cGMP transport occurred with lower affinity but higher Vmax than Abcc4-mediated transport. Involvement of Abcg2 in cGMP transport by Abcc4-/- erythrocyte vesicles was supported by higher transport at pH 5.5 than at pH 7.4, a characteristic of Abcg2-mediated transport. The relative contribution of ABCC4/Abcc4 and ABCG2/Abcg2 in cGMP transport was confirmed with a new inhibitor of ABCC4 transport, the protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride.

Paper title : Characterization of the MRP4- and MRP5-mediated transport of cyclic nucleotides from intact cells.

Doi : https://doi.org/10.1074/jbc.M212723200

Abstract : Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues. Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3',5'-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles. We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5. The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively. MRP4- and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner. In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport. Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20-600 microm, indicating low affinity transport. In addition to several classic inhibitors of organic anion transport, prostaglandins A(1) and E(1), the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux. The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour. This was also the case when phosphodiesterase-mediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal. These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting.

Paper title : Multidrug-resistance protein 5 is a multispecific organic anion transporter able to transport nucleotide analogs.

Doi : https://doi.org/10.1073/pnas.120159197

Abstract : Two prominent members of the ATP-binding cassette superfamily of transmembrane proteins, multidrug resistance 1 (MDR1) P-glycoprotein and multidrug resistance protein 1 (MRP1), can mediate the cellular extrusion of xenobiotics and (anticancer) drugs from normal and tumor cells. The MRP subfamily consists of at least six members, and here we report the functional characterization of human MRP5. We found resistance against the thiopurine anticancer drugs, 6-mercaptopurine (6-MP) and thioguanine, and the anti-HIV drug 9-(2-phosphonylmethoxyethyl)adenine (PMEA) in MRP5-transfected cells. This resistance is due to an increased extrusion of PMEA and 6-thioinosine monophosphate from the cells that overproduce MRP5. In polarized Madin-Darby canine kidney II (MDCKII) cells transfected with an MRP5 cDNA construct, MRP5 is routed to the basolateral membrane and these cells transport S-(2,4-dinitrophenyl)glutathione and glutathione preferentially toward the basal compartment. Inhibitors of organic anion transport inhibit transport mediated by MRP5. We speculate that MRP5 might play a role in some cases of unexplained resistance to thiopurines in acute lymphoblastic leukemia and/or to antiretroviral nucleoside analogs in HIV-infected patients.

Paper title : Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines.

Doi : https://doi.org/Not available

Abstract : By screening databases of human expressed sequence tags, we have identified three new homologues of MRP1, the gene encoding the multidrug resistance-associated protein, and cMOAT (or MRP2), the canalicular multispecific organic anion transporter gene. We call these new genes MRP3, MRP4, and MRP5. MRP3, like cMOAT, is mainly expressed in the liver. MRP4 is expressed only at very low levels in a few tissues, and MRP5, like MRP1, is expressed in almost every tissue tested. To assess a possible role of these new MRP homologues in multidrug or cisplatin resistance, a large set of resistant cell lines was examined for the (over)expression of MRP1, cMOAT, MRP3, MRP4, and MRP5. We find that even in cells selected for a low level of resistance, several MRP-related genes can be up-regulated simultaneously. However, MRP4 is not overexpressed in any of the cell lines we analyzed; MRP3 and MRP5 are only overexpressed in a few cell lines, and the RNA levels do not seem to correlate with resistance to either doxorubicin or cisplatin. cMOAT is substantially overexpressed in several cell lines, and cMOAT RNA levels correlate with cisplatin but not doxorubicin resistance in a subset of resistant cell lines. Our results emphasize the need for gene-specific blocks in gene expression to define which transporter contributes to resistance in each resistant cell line.