scholarly journals Functional Characterization of Novel Human and Mouse Equilibrative Nucleoside Transporters (hENT3 and mENT3) Located in Intracellular Membranes

2005 ◽  
Vol 280 (16) ◽  
pp. 15880-15887 ◽  
Author(s):  
Stephen A. Baldwin ◽  
Sylvia Y. M. Yao ◽  
Ralph J. Hyde ◽  
Amy M. L. Ng ◽  
Sophie Foppolo ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Functional Characterization ◽  
Human Cells ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Transport Activity ◽  
Transporter Family ◽  
Human And Mouse

The first mammalian examples of the equilibrative nucleoside transporter family to be characterized, hENT1 and hENT2, were passive transporters located predominantly in the plasma membranes of human cells. We now report the functional characterization of members of a third subgroup of the family, from human and mouse, which differ profoundly in their properties from previously characterized mammalian nucleoside transporters. The 475-residue human and mouse proteins, designated hENT3 and mENT3, respectively, are 73% identical in amino acid sequence and possess long N-terminal hydrophilic domains that bear typical (DE)XXXL(LI) endosomal/lysosomal targeting motifs. ENT3 transcripts and proteins are widely distributed in human and rodent tissues, with a particular abundance in placenta. However, in contrast to ENT1 and ENT2, the endogenous and green fluorescent protein-tagged forms of the full-length hENT3 protein were found to be predominantly intracellular proteins that co-localized, in part, with lysosomal markers in cultured human cells. Truncation of the hydrophilic N-terminal region or mutation of its dileucine motif to alanine caused the protein to be relocated to the cell surface both in human cells and inXenopusoocytes, allowing characterization of its transport activity in the latter. The protein proved to be a broad selectivity, low affinity nucleoside transporter that could also transport adenine. Transport activity was relatively insensitive to the classical nucleoside transport inhibitors nitrobenzylthioinosine, dipyridamole, and dilazep and was sodium ion-independent. However, it was strongly dependent upon pH, and the optimum pH value of 5.5 probably reflected the location of the transporter in acidic, intracellular compartments.

Genomic analysis of nucleoside transporters in Diptera and functional characterization of DmENT2, a Drosophila equilibrative nucleoside transporter

2007 ◽  
Vol 28 (3) ◽  
pp. 337-347 ◽  
Author(s):  
Jerry Machado ◽  
Parween Abdulla ◽  
W. J. Brad Hanna ◽  
Arthur J. Hilliker ◽  
Imogen R. Coe
Keyword(s):  
Sequence Similarity ◽  
Functional Characterization ◽  
Genomic Analysis ◽  
Structure And Function ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Nucleoside Transport Inhibitors ◽  
And Function

The recent completion of genome sequencing projects in a number of eukaryotes allows comparative analysis of orthologs, which can aid in identifying evolutionary constraints on protein structure and function. Nucleoside transporters (NTs) are present in a diverse array of organisms and previous studies have suggested that there is low protein sequence similarity but conserved structure in invertebrate and vertebrate NT orthologs. In addition, most taxa possess multiple NT isoforms but their respective roles in the physiology of the organism are not clear. To investigate the evolution of the structure and function of NTs, we have extended our previous studies by identifying NT orthologs in the Dipteran Anopheles gambiae and comparing these proteins to human and Drosophila melanogaster (Dm) NTs. In addition, we have functionally characterized DmENT2, one of three putative D. melanogaster ENTs that we have previously described. DmENT2 has broad substrate specificity, is insensitive to standard nucleoside transport inhibitors and is expressed in the digestive tract of late stage embryos based on in situ hybridization. DmENT1 and DmENT2 are expressed in most stages during development with the exception of early embryogenesis suggesting specific physiological roles for each isoform. These data represent the first complete genomic analysis of Dipteran NTs and the first report of the functional characterization of any Dipteran NT.

scholarly journals Identification and functional characterization of the ZmCOPT copper transporter family in maize

PLoS ONE ◽  
2018 ◽  
Vol 13 (7) ◽  
pp. e0199081 ◽  
Author(s):  
Hongling Wang ◽  
Hanmei Du ◽  
Hongyou Li ◽  
Ying Huang ◽  
Jianzhou Ding ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Copper Transporter ◽  
Transporter Family

scholarly journals Decrease in equilibrative uridine transport during monocytic differentiation of HL-60 leukaemia: involvement of protein kinase C

1994 ◽  
Vol 300 (2) ◽  
pp. 407-412 ◽  
Author(s):  
C W Lee
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Nucleoside Transport ◽  
Protective Effects ◽  
Transport Activity ◽  
Monocytic Differentiation ◽  
Response Curves ◽  
Kinase C

The dose-response curves for the inhibition of equilibrative uridine transport by dilazep, dipyridamole and nitrobenzylthioinosine (NBMPR) in undifferentiated HL-60 cells were biphasic. Some 70% of the transport activity was inhibited with IC50 values of 0.7, 1 and 7 nM respectively. No inhibition of the remaining 30% of transport activity was observed until the dilazep, dipyridamole and NBMPR concentrations exceeded 1, 0.1 and 3 microM respectively. Exposure to phorbol 12-myristate 13-acetate (PMA) for 48 h, to induce monocytic differentiation, caused a 20-fold decrease in Vmax. of both NBMPR-sensitive and NBMPR-insensitive equilibrative uridine transport. The decrease in NBMPR-sensitive uridine transport induced by PMA corresponded to a decrease in NBMPR binding sites. A 30% decrease in specific NBMPR binding sites occurred within 6 h of PMA exposure, and could be prevented by uridine and thymidine at concentrations as low as 100 microM, and by staurosporine at 40 nM. However, the protective effects of these compounds diminished with prolonged PMA exposure. No protection was observed with uracil. Exogenous protein kinase C (PKC) in the presence of ATP and PMA decreased the number of specific NBMPR-binding sites in purified HL-60 cell plasma membranes. These results suggest that a PKC-induced conformational change in substrate-binding/transporting site may be responsible for the decrease in NBMPR-sensitive nucleoside transport during PMA-induced monocytic differentiation of HL-60 cells.

scholarly journals Functional characterization of human equilibrative nucleoside transporter 1

2016 ◽  
Vol 8 (4) ◽  
pp. 284-295 ◽  
Author(s):  
Weiyun Huang ◽  
Xin Zeng ◽  
Yigong Shi ◽  
Minhao Liu
Keyword(s):  
Functional Characterization ◽  
Nucleoside Transporter ◽  
Equilibrative Nucleoside Transporter

scholarly journals Isolation and Functional Characterization of the PfNT1 Nucleoside Transporter Gene fromPlasmodium falciparum

2000 ◽  
Vol 275 (14) ◽  
pp. 10683-10691 ◽  
Author(s):  
Nicola S. Carter ◽  
Choukri Ben Mamoun ◽  
Wei Liu ◽  
Edilene O. Silva ◽  
Scott M. Landfear ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Nucleoside Transporter ◽  
Transporter Gene

scholarly journals Na+-dependent nucleoside transport in liver: two different isoforms from the same gene family are expressed in liver cells

1998 ◽  
Vol 330 (2) ◽  
pp. 997-1001 ◽  
Author(s):  
Antonio FELIPE ◽  
Raquel VALDES ◽  
Belén del SANTO ◽  
Jorge LLOBERAS ◽  
Javier CASADO ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Polyclonal Antibodies ◽  
High Specificity ◽  
Liver Cells ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Nucleoside Transporter ◽  
Transport Activity ◽  
Plasma Membrane Vesicles

Hepatocytes show a Na+-dependent nucleoside transport activity that is kinetically heterogeneous and consistent with the expression of at least two independent concentrative Na+-coupled nucleoside transport systems (Mercader et al. Biochem. J. 317, 835-842, 1996). So far, only a single nucleoside carrier-related cDNA (SPNT) has been isolated from liver cells (Che et al. J. Biol. Chem. 270, 13596-13599, 1995). This cDNA presumably encodes a plasma membrane protein responsible for Na+-dependent purine nucleoside transport activity. Thus, the liver must express, at least, a second nucleoside transporter which should be pyrimidine-preferring. Homology cloning using RT-PCR revealed that a second isoform is indeed present in liver. This second isoform turned out to be identical to the ‘epithelial-specific isoform’ called cNT1, which shows in fact high specificity for pyrimidine nucleosides. Although cNT1 mRNA is present at lower amounts than SPNT mRNA, the amounts of cNT1 protein, when measured using isoform-specific polyclonal antibodies, were even higher than the SPNT protein levels. Moreover, partially purified basolateral plasma membrane vesicles from liver were enriched in the SPNT but not in the cNT1 protein, which suggests that the subcellular localization of these carrier proteins is different. SPNT and cNT1 protein amounts in crude membrane extracts from 6 h-regenerating rat livers are higher than in the preparations from sham-operated controls (3.5- and 2-fold, respectively). These results suggest that liver parenchymal cells express at least two different isoforms of concentrative nucleoside carriers, the cNT1 and SPNT proteins, which show differential regulation and subcellular localization.

scholarly journals Functional characterization of a recombinant sodium-dependent nucleoside transporter with selectivity for pyrimidine nucleosides (cNT1rat) by transient expression in cultured mammalian cells

1996 ◽  
Vol 317 (2) ◽  
pp. 457-465 ◽  
Author(s):  
Xiao FANG ◽  
Fiona E. PARKINSON ◽  
Delores A. MOWLES ◽  
James D. YOUNG ◽  
Carol E. CASS
Keyword(s):  
Mammalian Cells ◽  
Expression System ◽  
Functional Characterization ◽  
Transport Processes ◽  
Nucleoside Transport ◽  
Single Type ◽  
Nucleoside Transporter ◽  
Pyrimidine Nucleosides ◽  
Transporter Protein ◽  
Sodium Dependent

We have demonstrated that monkey kidney (COS-1) cells have a single type of nucleoside transport process, which, because it was equilibrative, sodium-independent and could be inhibited by nitrobenzylthioinosine (NBMPR), was identified as the ‘equilibrative sensitive’ or ‘es’ transporter. Using NBMPR or dilazep to inhibit the endogenous nucleoside transport activity, we have transiently expressed a cDNA that encodes an inhibitor-insensitive, concentrative nucleoside transporter protein (cNT1rat) of rat intestine in COS-1 cells. The production of recombinant cNT1rat was examined by immunoblotting using an epitope-tagged construct and by analysis of inward fluxes of 3H-labelled nucleosides. Recombinant cNT1rat was sodium-dependent and selective for pyrimidine nucleosides, with approximate Km values of 21 μM, 12.5 μM and 15 μM for uridine, thymidine and adenosine, respectively. Although adenosine exhibited high affinity for the recombinant transporter, its Vmax value was low. A variety of anti-viral and anti-cancer nucleoside drugs inhibited cNT1rat-mediated uptake of uridine by transfected COS-1 cells although to different extents (Floxidine > Idoxuridine > Zidovudine > Zalcitabine > Cytarabine > Gemcitabine), suggesting that the concentrative pyrimidine-selective nucleoside transporters, of which cNT1rat is a representative, may play a role in cellular uptake of these drugs. The cNT1rat/COS-1 expression system is a useful tool for analysis of cNT1rat-mediated transport processes.

Sign in / Sign up

Export Citation Format

Share Document