
<ArticleTitle>Functional interactions between arginine-133 and aspartate-88 in the human <prot>reduced folate carrier</prot>: evidence for a charge-pair association.</ArticleTitle>
<AbstractText>The human <prot>reduced folate carrier</prot> (<prot>hRFC</prot>) is an integral membrane protein that mediates cellular uptake of reduced folates and antifolates. <prot>hRFC</prot> 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 <prot>hRFC</prot> structure and function, this residue was systematically mutagenized to histidine, leucine, lysine and glutamate. When transfected into transport-impaired K562 cells, the mutant <prot>hRFC</prot> constructs were expressed at high levels; however, only lysine-133 <prot>hRFC</prot> was able to transport methotrexate and (6S)-5-formyl tetrahydrofolate. Substitution of aspartate-453 (in <prot>hRFC</prot> 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 <prot>hRFC</prot> tertiary structure, both charges were neutralized (by substituting leucine and valine, respectively) in the same construct. In contrast to the singly mutated <prot>hRFC</prot>s, 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 <prot>hRFC</prot> tertiary structure.</AbstractText>
