Functional interactions between arginine-133 and aspartate-88 in the human reduced folate carrier: evidence for a charge-pair association.
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.
Liu
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Cancer Biology Graduate Program, Wayne State University School of Medicine, 110 E. Warren Avenue, Detroit, MI 48201, USA.
Matherly
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D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000502:physiology; D001224:Aspartic Acid / Q000502:physiology; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D006224:Cricetinae; D006801:Humans; D020014:K562 Cells; D002955:Leucovorin / Q000378:metabolism; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D051381:Rats; D058978:Reduced Folate Carrier Protein; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014162:Transfection
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; C548700:Slc19a1 protein, mouse; D001224:Aspartic Acid; D001120:Arginine; D002955:Leucovorin; D008727:Methotrexate