Abstract
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 MTXrA 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 ∼4-fold, 2) increased affinity for folic acid ∼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.
Footnotes
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Send reprint requests to: Dr. I. David Goldman, Albert Einstein Comprehensive Cancer Center, Albert Einstein College of Medicine, Chanin Two, 1300 Morris Park Ave., Bronx, NY 10461. E-mail:igoldman{at}aecom.yu.edu
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1 This work was supported by Grant CA-39807 from the National Cancer Institute.
- Abbreviations:
- RFC1
- reduced folate carrier
- MTX
- methotrexate
- 5-CHO-THF
- 5-formyltetrahydrofolate
- 5-CH3-THF
- 5-methyltetrahydrofolate
- HBS
- HEPES-buffered saline
- Received July 14, 1999.
- Accepted October 21, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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