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Role of Lysine 411 in Substrate Carboxyl Group Binding to the Human Reduced Folate Carrier, as Determined by Site-Directed Mutagenesis and Affinity Inhibition

Graduate Program in Cancer Biology (Y.D., L.H.M.) and Department of Pharmacology (L.H.M.), Wayne State University School of Medicine, Detroit, Michigan; Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan (Z.H., C.C., J.W., L.H.M.); and Division of Medicinal Chemistry, Graduate School of Pharmaceutical Science, Duquesne University, Pittsburgh, Pennsylvania (L.W., A.G.)

Reduced folate carrier (RFC) is the major membrane transporter for folates and antifolates in mammalian tissues. Recent studies used radioaffinity labeling with N-hydroxysuccinimide (NHS)-[³H]methotrexate (MTX) to localize substrate binding to residues in transmembrane domain (TMD) 11 of human RFC. To identify the modified residue(s), seven nucleophilic residues in TMD11 were mutated to Val or Ala and mutant constructs expressed in RFC-null HeLa cells. Only K411A RFC was not inhibited by NHS-MTX. By radioaffinity labeling with NHS-[³H]MTX, wild-type (wt) RFC was labeled; for K411A RFC, radiolabeling was abolished. When Lys411 was replaced with Ala, Arg, Gln, Glu, Leu, and Met, only K411E RFC showed substantially decreased transport. Nine classic diamino furo[2,3-d]pyrimidine antifolates with unsubstituted - and -carboxylates (1), hydrogen- or methyl-substituted -(2,3) or -(4,5) carboxylates, or substitutions of both - and -carboxylates (6-9) were used to inhibit [³H]MTX transport with RFC-null K562 cells expressing wt and K411A RFCs. For wt and K411A RFCs, inhibitory potencies were in the order 4 > 5 > 1 > 3 > 2; 6 to 9 were poor inhibitors. Inhibitions decreased in the presence of physiologic anions. When NHS esters of 1, 2, and 4 were used to covalently modify wt RFC, inhibitory potencies were in the order 2 > 1 > 4; inhibition was abolished for K411A RFC. These results establish that Lys411 participates in substrate binding via an ionic association with the substrate -carboxylate; however, this is not essential for transport. An unmodified -carboxylate is required for high-affinity substrate binding to RFC, whereas the -carboxyl is not essential.

Received for publication November 2, 2007.

Accepted for publication January 8, 2008.

Address correspondence to: Dr. Larry H. Matherly, Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, 110 E. Warren Avenue, Detroit, MI 48201. E-mail: matherly{at}kci.wayne.edu

This article has been cited by other articles:

				Z. Hou and L. H. Matherly Oligomeric Structure of the Human Reduced Folate Carrier: IDENTIFICATION OF HOMO-OLIGOMERS AND DOMINANT-NEGATIVE EFFECTS ON CARRIER EXPRESSION AND FUNCTION J. Biol. Chem., January 30, 2009; 284(5): 3285 - 3293. [Abstract] [Full Text] [PDF]