Role of the amino acid 45 residue in reduced folate carrier function and ion-dependent transport as characterized by site-directed mutagenesis.
In previous reports, an E45K mutation in reduced folate carrier (RFC1) resulted in marked substrate-specific changes in folate binding and the induction of an obligatory inorganic anion requirement for carrier function. In this study, site-directed mutagenesis was employed to further characterize the role of glutamate-45 in carrier function by replacement with glutamine, arginine, aspartate, leucine, or tryptophan followed by tranfection of the mutated cDNAs into the MTX(r)A line, which lacks a functional endogenous carrier. Alterations in transport function with amino acid substitutions at this residue were not charge related. Hence, E45Q, E45R, and E45K all 1) increased carrier affinity for 5-formyltetrahydrofolate approximately 4-fold, 2) increased affinity for folic acid approximately 6- to 10-fold, 3) did not change affinity for 5-methyltetrahydrofolate, and 4) except for E45R decreased affinity for methotrexate (2- to 3-fold). In contrast, mutations E45D, E45L, and E45W generally reduced affinity for all these folates except for folic acid. Finally, chloride-dependent influx was only noted in the E45R mutant. These data further substantiate the important role that glutamate-45 plays in the selectivity of binding of folates to RFC1 and establish that it is the addition of a positive charge at this site and not the loss of a negative charge that results in the induced anion dependence. These and other studies indicate that mutations in the first transmembrane domain can have a markedly selective impact on the affinity of RFC1 for folate compounds and in particularly a highly salutary effect on binding of the oxidized folate, folic acid.
Zhao
R
R
Department of Medicine, Albert Einstein Comprehensive Cancer Center, Albert Einstein College of Medicine, Bronx, New York, USA.
Gao
F
F
Wang
P J
PJ
Goldman
I D
ID
D019943:Amino Acid Substitution; D000818:Animals; D000838:Anions / Q000378:metabolism; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D005492:Folic Acid / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D002955:Leucovorin / Q000378:metabolism; D007939:Leukemia L1210; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D058978:Reduced Folate Carrier Protein; D013763:Tetrahydrofolates / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D000838:Anions; D000964:Antimetabolites, Antineoplastic; D002352:Carrier Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; C548700:Slc19a1 protein, mouse; C492292:Slc19a2 protein, mouse; D013763:Tetrahydrofolates; D018698:Glutamic Acid; D005492:Folic Acid; D002955:Leucovorin; C005984:5-methyltetrahydrofolate; D008727:Methotrexate