title: Role of lysine 411 in substrate carboxyl group binding to the human reduced folate carrier, as determined by site-directed mutagenesis and affinity inhibition.
abstract: 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)-[(3)H]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-[(3)H]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 alpha- and gamma-carboxylates (1), hydrogen- or methyl-substituted alpha-(2,3) or gamma-(4,5) carboxylates, or substitutions of both alpha- and gamma-carboxylates (6-9) were used to inhibit [(3)H]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 gamma-carboxylate; however, this is not essential for transport. An unmodified alpha-carboxylate is required for high-affinity substrate binding to RFC, whereas the gamma-carboxyl is not essential.
authors.affiliation: Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, 110 E. Warren Avenue, Detroit, MI 48201, USA.
authors.forename: Yijun
authors.initials: Y
authors.lastname: Deng
authors.forename: Zhanjun
authors.initials: Z
authors.lastname: Hou
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Christina
authors.initials: C
authors.lastname: Cherian
authors.forename: Jianmei
authors.initials: J
authors.lastname: Wu
authors.forename: Aleem
authors.initials: A
authors.lastname: Gangjee
authors.forename: Larry H
authors.initials: LH
authors.lastname: Matherly
mesh_terms: D000345:Affinity Labels; D001692:Biological Transport / Q000187:drug effects; D004952:Esters / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008727:Methotrexate / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D050505:Mutant Proteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D058978:Reduced Folate Carrier Protein; D013329:Structure-Activity Relationship; D013388:Succinimides / Q000378:metabolism; D014162:Transfection
chemical_list: D000345:Affinity Labels; D004952:Esters; D005493:Folic Acid Antagonists; D026901:Membrane Transport Proteins; D050505:Mutant Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D013388:Succinimides; D008239:Lysine; C001426:N-hydroxysuccinimide; D008727:Methotrexate