Molecular Cloning of the Human Placental Folate Transporter

Prasad, Puttur D.; Ramamoorthy, Sammanda; Leibach, F. H.; Ganapathy, Vadivel

doi:10.1006/bbrc.1995.1096

Cited by 155 publications

(95 citation statements)

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“…Using these cells we then tested the possible involvement of specific protein kinase-and Ca 2ϩ /calmodulin-mediated pathways in the regulation of folic acid uptake by colonocytes. We focused on pathways that involve protein kinases (PKC and PKA) for which consensus sequences have been shown to exist in the recently cloned folate carriers (13)(14)(15)32) as well as those pathways that have been shown to play an important role in the regulation of uptake of other nutrients by epithelial cells (PTK-and Ca 2ϩ /calmodulin-mediated pathways) (33)(34)(35)(36)(37)(38)(39)(40)(41). When specific modulators of these pathways were used, we found that PKC-mediated pathways had no role in regulating folic acid uptake by NCM460 cells.…”

Section: Discussionmentioning

confidence: 99%

A Protein-tyrosine Kinase-regulated, pH-dependent, Carrier-mediated Uptake System for Folate in Human Normal Colonic Epithelial Cell Line NCM460

Kumar

Moyer

Dudeja

et al. 1997

Journal of Biological Chemistry

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A significant proportion of the bacterially synthesized folate in the large intestine exists in the form of folate monoglutamate. Recent studies in our laboratory using human colonic apical membrane vesicles have shown the existence of an efficient carrier-mediated system for folate uptake. Nothing, however, is known about the cellular regulation of the colonic uptake process. In this study, we used a recently established human normal colonic epithelial cell line NCM460 to address this issue. Uptake of folic acid by NCM460 cells was: 1) linear with time for 4 min of incubation and occurred with minimal metabolic alterations, 2) temperature-and pH-(but not Na ؉ ) dependent, 3) saturable as a function of concentration (apparent K m of 1.4 M), 4) inhibited by structural analogs and anion transport inhibitors, and 5) energydependent. These characteristics of folic acid uptake by NCM460 cells are similar to those seen with apical membrane vesicles derived from human native colonic tissue. Using these cells, we found that protein kinase Cand Ca 2؉ /calmodulin-mediated pathways have no role in regulating folic acid uptake. On the other hand, cAMP (through a mechanism independent of protein kinase A) and protein-tyrosine kinase-mediated pathways were found to play a role in the regulation of folic acid uptake by these cells. These results establish the suitability of NCM460 cells as an in vitro model system for investigating the details of the mechanism of colonic folate uptake and its regulation. Folic acid uptake by these cells appears to involve a carrier-mediated system, which is temperature-, pH-, and energy-dependent and appears to be under the regulation of cAMP and protein tyrosine kinase.Folate is an essential micronutrient, which acts as a coenzyme in the synthesis of DNA and RNA and the interconversion and degradation of several amino acids (1-4). An adequate supply of folate is therefore necessary for normal cellular function, growth, and development. Folate deficiency has been suggested as one of the most common vitamin deficiencies in the Western Hemisphere (5, 6). Humans and other mammals cannot synthesize folate and rely on exogenous sources to meet their metabolic requirements. Folates are presented to the host from the diet and are also synthesized in the large intestine by normal microflora. The mechanism of absorption of dietary folate has been intensively examined over the past two decades at the tissue, cellular, subcellular, and more recently, molecular levels (6 -15). Absorption of dietary folate has been shown to occur mainly in the proximal small intestine and involves a specialized, carrier-mediated system (6 -12).As to the bacterially synthesized folate in the large intestine, a significant amount of that folate exists in the monoglutamate, i.e. the absorbable form. Using [ 3 H]p-aminobenzoic acid to label the newly synthesized folate by the intestinal flora, Rong et al. (16) have shown that a portion of this folate is indeed absorbed by the rat and is incorporated into its various tissues. ...

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Section: Discussionmentioning

confidence: 99%

A Protein-tyrosine Kinase-regulated, pH-dependent, Carrier-mediated Uptake System for Folate in Human Normal Colonic Epithelial Cell Line NCM460

Kumar

Moyer

Dudeja

et al. 1997

Journal of Biological Chemistry

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“…Resistance to antifolates R Zhao and ID Goldman (Dixon et al, 1994;Williams et al, 1994;Moscow et al, 1995;Prasad et al, 1995;Williams and Flintoff, 1995;Wong et al, 1995). The validity of this secondary structure has been strengthened by hemaglutatinin epitope insertion analysis (Ferguson and Flintoff, 1999) although there remains a question regarding the topology of TMDs 9-12 (Liu and Matherly, 2002).…”

Section: The Reduced Folate Carriermentioning

confidence: 99%

Resistance to antifolates

2003

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“…The incidence of mutations in the first transmembrane domain of the RFC (amino acids 28-48), 11 in general, and the inciLeukemia dence of the E45K mutation in particular in ALL samples at relapse (three T-cell and 17 B-precursor) was examined by automated DNA sequencing and XmnI restriction enzyme digestion, respectively. The E45K mutation was not identified in any of 20 relapse ALL specimens ( Figure 5) and no other sequence mutations were identified among amino acids 28 to 48 (data not shown).…”

Section: Hrfc Mutations In Diagnosis and Relapsed Allmentioning

confidence: 99%

“…The RFC is an integral membrane protein with 12 putative transmembrane domains. 9,11,14 A large number of RFC gene mutations leading to an antifolate-resistant phenotype have been described in rodent and human cell lines. [15][16][17][18][19][20][21] Although mutations have been identified throughout the RFC coding region, they appear to be disproportionately clustered within the first putative transmembrane domain (eg G44E, E45K, S46N, I48F).…”

Section: Introductionmentioning

confidence: 99%

Role of the E45K-reduced folate carrier gene mutation in methotrexate resistance in human leukemia cells

Gifford

Haber

et al. 2002

Leukemia

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Collectively, the results demonstrate that expression of E45K hRFC leads to increased MTX resistance due to decreased membrane transport and, secondarily, from alterations in binding affinities and transport of folate substrates. However, despite these findings, we could find no evidence of this mutation in 121 childhood ALL samples, suggesting that it does not contribute to clinical MTX resistance in this disease.

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Molecular Cloning of the Human Placental Folate Transporter

Cited by 155 publications

References 0 publications

A Protein-tyrosine Kinase-regulated, pH-dependent, Carrier-mediated Uptake System for Folate in Human Normal Colonic Epithelial Cell Line NCM460

A Protein-tyrosine Kinase-regulated, pH-dependent, Carrier-mediated Uptake System for Folate in Human Normal Colonic Epithelial Cell Line NCM460

Resistance to antifolates

Role of the E45K-reduced folate carrier gene mutation in methotrexate resistance in human leukemia cells

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