Role of the C-terminus and the long cytoplasmic loop in reduced folate carrier expression and function

Sharina, Iraida; Zhao, Rongbao; Wang, Yanhua; Babani, Solomon; Goldman, I. David

doi:10.1016/s0006-2952(02)00955-3

Cited by 19 publications

(17 citation statements)

References 33 publications

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“…In striking contrast to the TMD regions, the 61 amino acids comprising the central connecting loop between TMDs 6 and 7 of hRFC are less conserved (44.3% homology between hRFC and rodent RFCs). Indeed, large deletions in this region of the murine (31 of 66 amino acids) and hamster (45 of 67 amino acids) RFCs preserved membrane targeting and transport activity, as long as a highly conserved stretch of 11 amino acids (corresponding to positions 204 -214 in hRFC) was present (29,30). In hRFC, deletions of 49 or 60 amino acids from the TMD6/7 linker (amino acids 215-263 and 204 -263, respectively) completely abolished transport activity for both Mtx and 5-formyl tetrahydrofolate (5-CHO-H 4 PteGlu) (31), suggesting that some minimal length for the linker is necessary.…”

mentioning

confidence: 99%

Restoration of Transport Activity by Co-expression of Human Reduced Folate Carrier Half-molecules in Transport-impaired K562 Cells

Witt

Stapels²,

Matherly

2004

Journal of Biological Chemistry

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Reduced folates such as 5-methyl tetrahydrofolate and classical antifolates such as methotrexate are actively transported into mammalian cells by the reduced folate carrier (RFC). RFC is characterized by 12 stretches of mostly hydrophobic, ␣-helix-promoting amino acids, internally oriented N and C termini, and a large central linker connecting transmembrane domains (TMDs) 1-6 and 7-12. Previous studies showed that deletion of the majority of the central loop domain between TMDs 6 and 7 abolished transport, but this segment could be replaced with mostly non-homologous sequence from the SLC19A2 thiamine transporter to restore transport function. In this report, we expressed RFC from separate TMD1-6 and TMD7-12 RFC halfmolecule constructs, each with a unique epitope tag, in RFC-null K562 cells to restore transport activity. Restored transport exhibited characteristic transport kinetics for methotrexate, a capacity for trans-stimulation by pretreatment with leucovorin, and inhibition by Nhydroxysuccinimide methotrexate, a documented affinity inhibitor of RFC. The TMD1-6 half-molecule migrated on SDS gels as a 38 -58 kDa glycosylated species and was converted to 27 kDa by N-glycosidase F or tunicamycin treatments. The 40 kDa TMD7-12 half-molecule was unaffected by these treatments. Using transfected cells expressing both TMDs 1-6 and TMDs 7-12 as separate polypeptides, the TMD7-12 half-molecule was covalently radiolabeled with N-hydroxysuccinimide [ 3 H]methotrexate. No radioactivity was incorporated into the TMD1-6 half-molecule. Digestion with endoproteinase GluC decreased the size of the radiolabeled 40 kDa TMD7-12 polypeptide to ϳ20 kDa. Our results demonstrate that a functional RFC can be reconstituted with RFC half-molecules and localize a critical substrate binding domain to within TMDs 7-12.

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confidence: 99%

Restoration of Transport Activity by Co-expression of Human Reduced Folate Carrier Half-molecules in Transport-impaired K562 Cells

Witt

Stapels²,

Matherly

2004

Journal of Biological Chemistry

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“…We used the bacterial two-hybrid system to screen a BacterioMatch II human intestinal cDNA library to search for proteins that may interact with the hRFC. The large intracellular loop between transmembranes 6 and 7 (amino acids 204 to 264) of hRFC was used as the bait in screening since it has been reported to play a critical role in function and expression of the RFC at the plasma membrane (23,32,36). The BacterioMatch II human intestinal cDNA library (primary size 1.67 ϫ 10 6 ; Stratagene) was screened with the generated recombinant pBT-hRFC plasmid.…”

Section: Resultsmentioning

confidence: 99%

“…The RFC protein is predicted to have 12 transmembrane domains with both the amino and carboxy termini predicted to be oriented intracellularly (7,14). The RFC protein is also predicted to contain a long intracellular loop between transmembrane domains 6 and 7, which is believed to play a role in function and expression of the carrier protein at the cell membrane (23,32,36). Furthermore, the backbone of the RFC polypeptide has been shown to be important for membrane targeting and expression of the protein (25).…”

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confidence: 99%

Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier

Ashokkumar

Nabokina

Thomas³

et al. 2009

American Journal of Physiology-Gastrointestinal and Liver Physi

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Ashokkumar B, Nabokina SM, Ma TY, Said HM. Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier. Am J Physiol Gastrointest Liver Physiol 297: G480 -G487, 2009. First published July 1, 2009 doi:10.1152/ajpgi.00154.2009.-The reduced folate carrier (RFC) is a major folate transport system in mammalian cells. RFC is highly expressed in the intestine and believed to play a role in folate absorption. Studies from our laboratory and others have characterized different aspects of the intestinal folate absorption process, but little is known about possible existence of accessory protein(s) that interacts with RFC and influences its physiology and/or cell biology. We investigated this issue by employing a bacterial two-hybrid system to screen a BacterioMatch II human intestinal cDNA library using the large intracellular loop between transmembrane domains 6 and 7 of the human RFC (hRFC) as bait. Our screening has resulted in the identification of dynein light chain road block-1 (DYNLRB1) as an interacting partner with hRFC. Existence of a direct protein-protein interaction between hRFC and DYNLRB1 was confirmed by in vitro pull-down assay and in vivo mammalian two-hybrid luciferase assay and coimmunoprecipitation analysis. Furthermore, confocal imaging of live human intestinal epithelial HuTu-80 cells demonstrated colocalization of DYNLRB1 with hRFC. Coexpression of DYNLRB1 with hRFC led to a significant (P Ͻ 0.05) increase in folate uptake. On the other hand, inhibiting the endogenous DYNLRB1 with genespecific small interfering RNA or pharmacologically with a specific inhibitor (vanadate) led to a significant (P Ͻ 0.05) decrease in folate uptake. This study demonstrates for the first time the identification of DYNLRB1 as an interacting protein partner with hRFC. Furthermore, DYNLRB1 appears to influence the function and cell biology of hRFC. folate transport; protein-protein interactions; two-hybrid analysis; accessory proteins

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“…Both deletion and insertion mutagenesis strategies have been used to explore the functional and structural role of this region in RFC. Thus, deletion of 31 of the 66 amino acids from the TMD6/TMD7 connecting loop in murine RFC (Sharina et al, 2002), or deletion of up to 45 of the 67 amino acids in the TMD6/TMD7 loop domain from hamster RFC (Sadlish et al, 2002a) preserved membrane targeting and transport activity.…”

Section: Deletional and Insertional Mutagenesis Of Rfcmentioning

confidence: 99%

“…Thus, deletion of 27 N-terminal amino acids (residues 1-27) from hRFC (Marchant et al, 2002), or removal of 16 residues (amino acids 7-22) from hamster RFC (Sadlish et al, 2002a) had at most minor effects on membrane targeting or transport function. While deletion of 58 C-terminal residues from hamster RFC (residues 461-518) (Sadlish et al, 2002a) or 39 C-terminal residues from hRFC (residues 453-591) (Marchant et al, 2002) had a slight effect on transport function, for murine RFC, loss of the C-terminus (residues 445 to 512) resulted in a complete loss of membrane targeting (Sharina et al, 2002). As expected, larger deletions (e.g., 302-591, 1-301), including entire TMDs, completely abolished plasma membrane targeting of hRFC (Marchant et al, 2002).…”

Section: Deletional and Insertional Mutagenesis Of Rfcmentioning

confidence: 99%

Chapter 5 Structure and Function of the Reduced Folate Carrier

Matherly

Hou

2008

Vitamins &Amp; Hormones

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Folates are essential for life and folate deficiency contributes to a host of health problems including cardiovascular disease, fetal abnormalities, neurologic disorders, and cancer. Antifolates, represented by methotrexate, continue to occupy a unique niche among the modern day pharmacopoeia for cancer along with other pathologic conditions. This review focuses on the biology of the membrane transport system termed the "reduced folate carrier" or RFC with a particular emphasis on RFC structure and function. The ubiquitously expressed RFC is the major transporter for folates in mammalian cells and tissues. Loss of RFC expression or function portends potentially profound physiologic or developmental consequences. For chemotherapeutic antifolates used for cancer, loss of RFC expression or synthesis of mutant RFC protein with impaired function results in antifolate resistance due to incomplete inhibition of cellular enzyme targets and low levels of substrate for polyglutamate synthesis. The functional properties for RFC were first documented nearly 40 years ago in murine leukemia cells. Since 1994, when RFC was first cloned, tremendous advances in the molecular biology of RFC and biochemical approaches for studying the structure of polytopic membrane proteins have led to an increasingly detailed picture of the molecular structure of the carrier, including its membrane topology, its Nglycosylation, identification of functionally and structurally important domains and amino acids, and helix packing associations. Although no crystal structure for RFC is yet available, biochemical and molecular studies, combined with homology modeling, based on homologous bacterial Major Facilitator Superfamily transporters such as LacY, now permit the development of experimentally testable hypotheses designed to establish RFC structure and mechanism.

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Role of the C-terminus and the long cytoplasmic loop in reduced folate carrier expression and function

Cited by 19 publications

References 33 publications

Restoration of Transport Activity by Co-expression of Human Reduced Folate Carrier Half-molecules in Transport-impaired K562 Cells

Restoration of Transport Activity by Co-expression of Human Reduced Folate Carrier Half-molecules in Transport-impaired K562 Cells

Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier

Chapter 5 Structure and Function of the Reduced Folate Carrier

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