scholarly journals Molecular cloning and functional characterization of inhibitor-sensitive (mENT1) and inhibitor-resistant (mENT2) equilibrative nucleoside transporters from mouse brain

2000 ◽  
Vol 352 (2) ◽  
pp. 363-372 ◽  
Author(s):  
Andor KISS ◽  
Kalthoum FARAH ◽  
Joon KIM ◽  
Robert J. GARRIOCK ◽  
Thomas A. DRYSDALE ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Mammalian Cells ◽  
Transmembrane Domain ◽  
Functional Characterization ◽  
Phosphorylation Site ◽  
Functional Expression ◽  
Amino Acid Identity ◽  
Nucleoside Transporters ◽  
Intracellular Loop ◽  
Equilibrative Nucleoside Transporters

Mammalian cells express at least two subtypes of equilibrative nucleoside transporters, i.e. ENT1 and ENT2, which can be distinguished functionally by their sensitivity and resistance respectively to inhibition by nitrobenzylthioinosine. The ENT1 transporters exhibit distinctive species differences in their sensitivities to inhibition by dipyridamole, dilazep and draflazine (human>mouse>rat). A comparison of the ENT1 structures in the three species would facilitate the identification of the regions involved in the actions of these cardioprotective agents. We now report the molecular cloning and functional expression of the murine (m)ENT1 and mENT2 transporters. mENT1 and mENT2 encode proteins containing 458 and 456 residues respectively, with a predicted 11-transmembrane-domain topology. mENT1has 88% and 78% amino acid identity with rat ENT1 and human ENT1 respectively; mENT2 is more highly conserved, with 94% and 88% identity with rat ENT2 and human ENT2 respectively. We have also isolated two additional distinct cDNAs that encode proteins similar to mENT1; these probably represent distinct mENT1 isoforms or alternative splicing products. One cDNA encodes a protein with two additional amino acids (designated mENT1b) that adds a potential protein kinase CK2 phosphorylation site in the central intracellular loop of the transporter, and is similar, in this regard, to the human and rat ENT1 orthologues. The other cDNA has a 5′-untranslated region sequence that is distinct from that of full-length mENT1. Microinjection of mENT1, mENT1b or mENT2 cRNA into Xenopus oocytes resulted in enhanced uptake of [3H]uridine by the oocytes relative to that seen in water-injected controls. mENT1-mediated, but not mENT2-mediated, [3H]uridine uptake was inhibited by nitrobenzylthioinosine and dilazep. Dipyridamole inhibited both mENT1 and mENT2, but was significantly more effective against mENT1. Adenosine inhibited both systems with a similar potency, as did a range of other purine and pyrimidine nucleosides. These results are compatible with the known characteristics of the native mENT1 and mENT2 transporters.

scholarly journals Biophysical and functional characterization of Norrin signaling through Frizzled4

2018 ◽  
Vol 115 (35) ◽  
pp. 8787-8792 ◽  
Author(s):  
Injin Bang ◽  
Hee Ryung Kim ◽  
Andrew H. Beaven ◽  
Jinuk Kim ◽  
Seung-Bum Ko ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Wnt Signaling ◽  
Conformational Changes ◽  
Cellular Response ◽  
Transmembrane Domain ◽  
Functional Characterization ◽  
Intracellular Loop ◽  
Ligand Interaction ◽  
Rich Domain ◽  
Linker Domain

Wnt signaling is initiated by Wnt ligand binding to the extracellular ligand binding domain, called the cysteine-rich domain (CRD), of a Frizzled (Fzd) receptor. Norrin, an atypical Fzd ligand, specifically interacts with Fzd4 to activate β-catenin–dependent canonical Wnt signaling. Much of the molecular basis that confers Norrin selectivity in binding to Fzd4 was revealed through the structural study of the Fzd4CRD–Norrin complex. However, how the ligand interaction, seemingly localized at the CRD, is transmitted across full-length Fzd4 to the cytoplasm remains largely unknown. Here, we show that a flexible linker domain, which connects the CRD to the transmembrane domain, plays an important role in Norrin signaling. The linker domain directly contributes to the high-affinity interaction between Fzd4 and Norrin as shown by ∼10-fold higher binding affinity of Fzd4CRD to Norrin in the presence of the linker. Swapping the Fzd4 linker with the Fzd5 linker resulted in the loss of Norrin signaling, suggesting the importance of the linker in ligand-specific cellular response. In addition, structural dynamics of Fzd4 associated with Norrin binding investigated by hydrogen/deuterium exchange MS revealed Norrin-induced conformational changes on the linker domain and the intracellular loop 3 (ICL3) region of Fzd4. Cell-based functional assays showed that linker deletion, L430A and L433A mutations at ICL3, and C-terminal tail truncation displayed reduced β-catenin–dependent signaling activity, indicating the functional significance of these sites. Together, our results provide functional and biochemical dissection of Fzd4 in Norrin signaling.

scholarly journals Molecular cloning and characterization of a nitrobenzylthioinosine-insensitive (ei) equilibrative nucleoside transporter from human placenta

1997 ◽  
Vol 328 (3) ◽  
pp. 739-743 ◽  
Author(s):  
Mark GRIFFITHS ◽  
Y. M. Sylvia YAO ◽  
Fatima ABIDI ◽  
E. V. Simon PHILLIPS ◽  
E. Carol CASS ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Human Placenta ◽  
Ovarian Tissue ◽  
Functional Expression ◽  
Nucleoside Transporter ◽  
Intact Cells ◽  
Equilibrative Nucleoside Transporter ◽  
Equilibrative Nucleoside Transporters ◽  
Sequence Deletion

Mammalian equilibrative nucleoside transporters are typically divided into two classes, es and ei, based on their sensitivity or resistance respectively to inhibition by nitrobenzylthioinosine (NBMPR). Previously, we have reported the isolation of a cDNA clone encoding a prototypic es-type transporter, hENT1 (human equilibrative nucleoside transporter 1), from human placenta. We now report the molecular cloning and functional expression in Xenopus oocytes of a cDNA from the same tissue encoding a homologous ei-type transporter, which we designate hENT2. This 456-residue protein is 46% identical in amino acid sequence with hENT1 and corresponds to a full-length form of the delayed-early proliferative response gene product HNP36, a protein of unknown function previously cloned in a form bearing a sequence deletion. In addition to placenta, hENT2 is found in brain, heart and ovarian tissue. Like hENT1, hENT2 mediates saturable transport of the pyrimidine nucleoside uridine (Km 0.2±0.03 mM) and also transports the purine nucleoside adenosine. However, in contrast with hENT1, which is potently inhibited by NBMPR (Ki 2 nM), hENT2 is NBMPR-insensitive (IC50 < 1 μM). It is also much less sensitive to inhibition by the coronary vasoactive drugs dipyridamole and dilazep and to the lidoflazine analogue draflazine, properties that closely resemble those reported for classical ei-type transport in studies with intact cells.

scholarly journals Cellular localization and functional characterization of the equilibrative nucleoside transporters of antitumor nucleosides

2007 ◽  
Vol 98 (10) ◽  
pp. 1633-1637 ◽  
Author(s):  
Yoshio Endo ◽  
Tohru Obata ◽  
Daigo Murata ◽  
Mariho Ito ◽  
Kazuki Sakamoto ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Functional Characterization ◽  
Nucleoside Transporters ◽  
Equilibrative Nucleoside Transporters

Identification of a nucleoside/nucleobase transporter from Plasmodium falciparum, a novel target for anti-malarial chemotherapy

2000 ◽  
Vol 349 (1) ◽  
pp. 67-75 ◽  
Author(s):  
Marie D. PARKER ◽  
Ralph J. HYDE ◽  
Sylvia Y. M. YAO ◽  
Louisa MCROBERT ◽  
Carol E. CASS ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
De Novo ◽  
Functional Expression ◽  
Nucleoside Transporters ◽  
Pyrimidine Nucleoside ◽  
Parasite Protein ◽  
Apparent Affinity ◽  
Equilibrative Nucleoside Transporters ◽  
Novel Target ◽  
Membrane Spanning

Plasmodium, the aetiologic agent of malaria, cannot synthesize purines de novo, and hence depends upon salvage from the host. Here we describe the molecular cloning and functional expression in Xenopus oocytes of the first purine transporter to be identified in this parasite. This 422-residue protein, which we designate PfENT1, is predicted to contain 11 membrane-spanning segments and is a distantly related member of the widely distributed eukaryotic protein family the equilibrative nucleoside transporters (ENTs). However, it differs profoundly at the sequence and functional levels from its homologous counterparts in the human host. The parasite protein exhibits a broad substrate specificity for natural nucleosides, but transports the purine nucleoside adenosine with a considerably higher apparent affinity (Km 0.32±0.05 mM) than the pyrimidine nucleoside uridine (Km 3.5±1.1 mM). It also efficiently transports nucleobases such as adenine (Km 0.32±0.10 mM) and hypoxanthine (Km 0.41±0.1 mM), and anti-viral 3ʹ-deoxynucleoside analogues. Moreover, it is not sensitive to classical inhibitors of mammalian ENTs, including NBMPR {6-[(4-nitrobenzyl)thio]-9-β-D-ribofuranosylpurine, or nitrobenzylthioinosine} and the coronary vasoactive drugs, dipyridamole, dilazep and draflazine. These unique properties suggest that PfENT1 might be a viable target for the development of novel anti-malarial drugs.

Comparison of the interaction of uridine, cytidine, and other pyrimidine nucleoside analogues with recombinant human equilibrative nucleoside transporter 2 (hENT2) produced in Saccharomyces cerevisiae

2002 ◽  
Vol 80 (5) ◽  
pp. 639-644 ◽  
Author(s):  
Mark F Vickers ◽  
Rakesh Kumar ◽  
Frank Visser ◽  
Jing Zhang ◽  
Jahangir Charania ◽  
...  
Keyword(s):  
Functional Expression ◽  
Amino Acid Identity ◽  
Hydroxyl Group ◽  
Nucleoside Transporter ◽  
Expression Systems ◽  
Equilibrative Nucleoside Transporter ◽  
Pyrimidine Nucleosides ◽  
Equilibrative Nucleoside Transporters ◽  
Lower Ability ◽  
Ribose Moiety

The human equilibrative nucleoside transporters 1 and 2 (hENT1, hENT2) share 50% amino acid identity and exhibit broad selectivities, accepting purine and pyrimidine nucleosides as permeants. The permeant selectivity of hENT2 is less well understood because of the low abundance of the native transporter in cells amenable to functional analysis. Recent studies of hENT2 produced in recombinant form in functional expression systems have shown that it differs from hENT1 in that it transports nucleobases. To further understand the structural requirements for permeant interaction with hENT2, we compared the relative abilities of uridine, cytidine, and their analogues to inhibit transport of [3H]uridine by recombinant hENT1 and hENT2 produced in yeast. hENT1 and hENT2 tolerated halogen modification at the 5 position of the base and the 2' and 5' positions of the ribose moieties of uridine whereas removal of the hydroxyl group at the 3' position of the ribose moiety of uridine eliminated interaction with both transporters. hENT2 displayed a lower ability, compared with hENT1, to interact with cytidine and cytidine analogues, suggesting a low tolerance for the presence of the amino group at the 4 position of the base.Key words: hENT2, hENT1, araC, uridine, NBMPR.

scholarly journals Sulphonation of dehydroepiandrosterone and neurosteroids: molecular cloning, expression, and functional characterization of a novel zebrafish SULT2 cytosolic sulphotransferase

2003 ◽  
Vol 375 (3) ◽  
pp. 785-791 ◽  
Author(s):  
Takuya SUGAHARA ◽  
Yuh-Shyong YANG ◽  
Chau-Ching LIU ◽  
T. Govind PAI ◽  
Ming-Cheh LIU
Keyword(s):  
Molecular Cloning ◽  
Expressed Sequence Tag ◽  
Expression System ◽  
Functional Characterization ◽  
Amino Acid Identity ◽  
Cdna Clones ◽  
Reverse Transcription Pcr ◽  
Prokaryotic Expression System ◽  
Partial Cdna

By searching the zebrafish EST (expressed-sequence tag) database, we have identified two partial cDNA clones encoding the 5′ and 3′ regions of a putative zebrafish sulphotransferase (ST). Using the reverse transcription-PCR technique, a full-length cDNA encoding this zebrafish ST was successfully cloned. Sequence analysis revealed that this novel zebrafish ST displays 44%, 43% and 40% amino acid identity with mouse SULT2B1, human SULT2B1b and human SULT2A1 ST respectively. This zebrafish ST therefore appears to belong to the SULT2 cytosolic ST gene family. Recombinant zebrafish ST, expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as a 34 kDa protein upon SDS/PAGE. Purified zebrafish ST displayed a strong sulphonating activity toward DHEA (dehydroepiandrosterone), with a optimum pH of 9.5. The enzyme also exhibited activities toward several neurosteroids with differential Km and Vmax values. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 °C and 43 °C. Among ten different divalent metal cations tested, Fe2+ and Cd2+ exhibited small, but significant, stimulatory effects, whereas Hg2+ and Cu2+ displayed considerably stronger inhibitory effects on the DHEA-sulphonating activity of the enzyme. These results constitute the first study on the molecular cloning, expression, and characterization of a zebrafish cytosolic SULT2 ST.

Iron transport by Nramp2/DMT1: pH regulation of transport by 2 histidines in transmembrane domain 6

Blood ◽  
2003 ◽  
Vol 101 (9) ◽  
pp. 3699-3707 ◽  
Author(s):  
Steven Lam-Yuk-Tseung ◽  
Gregory Govoni ◽  
John Forbes ◽  
Philippe Gros
Keyword(s):  
Mammalian Cells ◽  
Iron Homeostasis ◽  
Transmembrane Domain ◽  
Ph Regulation ◽  
Functional Characterization ◽  
Yeast Cells ◽  
Natural Resistance ◽  
Loss Of Function ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter

Mutations at natural resistance-associated macrophage protein 1(Nramp1) impair phagocyte function and cause susceptibility to infections while mutations at Nramp2 (divalent metal transporter 1 [DMT1]) affect iron homeostasis and cause severe microcytic anemia. Structure-function relationships in the Nramp superfamily were studied by mutagenesis, followed by functional characterization in yeast and in mammalian cells. These studies identify 3 negatively charged and highly conserved residues in transmembrane domains (TM) 1, 4, and 7 as essential for cation transport by Nramp2/DMT1. The introduction of a charged residue (Gly185Arg) in TM4 found in the naturally occurring microcytic anemiamk (mouse) and Belgrade (rat) mutants is shown to cause a partial or complete loss of function in mammalian and yeast cells, respectively. A pair of mutation-sensitive and highly conserved histidines (His267, His272) was identified in TM6. Surprisingly, inactive His267 and His272 mutants could be rescued by lowering the pH of the transport assay. This indicates that His267/His272 are not directly involved in metal binding but, rather, play an important role in pH regulation of metal transport by Nramp proteins.

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