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Functional interactions between arginine-133 and aspartate-88 in the human reduced folate carrier: evidence for a charge-pair association.

ABSTRACT: The human reduced folate carrier (hRFC) is an integral membrane protein that mediates cellular uptake of reduced folates and antifolates. hRFC contains several highly conserved charged residues predicted to lie in the transmembrane domains (TMDs). To explore the possible roles of the conserved arginine-133, located in TMD 4, in hRFC structure and function, this residue was systematically mutagenized to histidine, leucine, lysine and glutamate. When transfected into transport-impaired K562 cells, the mutant hRFC constructs were expressed at high levels; however, only lysine-133 hRFC was able to transport methotrexate and (6S)-5-formyl tetrahydrofolate. Substitution of aspartate-453 (in hRFC TMD 12) by valine largely preserved transport activity for both substrates. Although mutagenesis of aspartate-88 (in TMD 2) to leucine completely abolished transport activity in transfected cells, substitution with a glutamate preserved low levels ( approximately 12%) of transport. To assess the possibility that arginine-133 and aspartate-88 may form a charge-pair to stabilize hRFC tertiary structure, both charges were neutralized (by substituting leucine and valine, respectively) in the same construct. In contrast to the singly mutated hRFCs, the double mutant exhibited high levels of transport with both methotrexate and 5-formyl tetrahydrofolate. These results strongly suggest that arginine-133 and aspartate-88 form a charge-pair and that TMD 4 lies next to TMD 2 in the hRFC tertiary structure.

SUBMITTER: Liu XY

PROVIDER: S-EPMC1222086 | biostudies-other | 2001 Sep

REPOSITORIES: biostudies-other

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Project description:The ubiquitously expressed reduced folate carrier (RFC) or SLC19A1 is recognized to be an essential transport system for folates in mammalian cells and tissues. In addition to its generalized role as a folate transporter, RFC provides specialized tissue functions including absorption across intestinal/colonic epithelia, transport across the basolateral membrane of renal proximal tubules, transplacental transport of folates, and folate transport across the blood-brain barrier. The human RFC (hRFC) gene is regulated by five major upstream non-coding regions (designated A1/A2, A, B, C, and D), each transcribed from a unique promoter. Altogether, at least 14 distinct hRFC transcripts can be envisaged in which different 5' untranslated regions (UTRs) are fused to a common splice acceptor region (positions -1 to -49) within the first coding exon with a common 1776bp coding sequence. The 5' non-coding regions are characterized by alternate transcription start sites, multiple splice forms, and selective tissue distributions. Alternate 5' UTRs impact mRNA stabilities and translation efficiencies, and result in synthesis of modified hRFC proteins translated from upstream AUGs. In this report, we describe production and characterization of transgenic mice (TghRFC1) containing a functional hRFC gene and of humanized mice in which the mRFC gene is inactivated and an active hRFC gene has been introduced. The mice appear to be healthy and to breed well. Analysis of tissue specificity of expression in both the TghRFC1 and humanized hRFC mice by real-time RT-PCR demonstrates that the hRFC gene is expressed with a specificity closely resembling that seen in human tissues. For the humanized hRFC mice, levels of B and A1/A2 5' UTRs predominated in all mice/tissues, thus resembling results in normal human tissues. Lower levels of A and C 5' UTRs were also detected. The availability of humanized mouse models for hRFC will permit investigators to address critical unanswered questions pertinent to human health and disease. These include the ability to analyze the hRFC gene in vivo, to control dietary and other environmental conditions that may impact levels of gene expression, and to control the genetics of the mice in order to assess the effects of hRFC gene alterations on tissue folate uptake and distribution, none of which can be easily achieved in human populations.

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Functional interactions between arginine-133 and aspartate-88 in the human reduced folate carrier: evidence for a charge-pair association.

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