Role of lysine 411 in substrate carboxyl group binding to the human reduced folate carrier, as determined by site-directed mutagenesis and affinity inhibition.

Role of lysine 411 in substrate carboxyl group binding to the human reduced folate carrier, as determined by site-directed mutagenesis and affinity inhibition. Research Abstract Details 

Research Abstract Table of Contents

Jump to the:

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 Text:

Yijun Deng,Zhanjun Hou,Lei Wang,Christina Cherian,Jianmei Wu,Aleem Gangjee,Larry H Matherly,

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.

Role of lysine 411 in substrate carboxyl group binding to the human reduced folate carrier, as determined by site-directed mutagenesis and affinity inhibition. Publishing Authors By Initials

Y Deng,Z Hou,L Wang,C Cherian,J Wu,A Gangjee,LH Matherly,

For similar abstracts research abstracts see: abstracts research

PUBMED ID PMID:

MEDLINE DATE:

Role of lysine 411 in substrate carboxyl group binding to the human reduced folate carrier, as determined by site-directed mutagenesis and affinity inhibition. Journal Published:

PUBLICATION TYPE: Journal Article

Journal: Molecular pharmacology

VOLUME: 73

Page Numbers: 1274-81

Journal Abbreviation: Mol. Pharmacol.

ISSN: 1521-0111

DAY: 8

MONTH: 01

YEAR: 2008

Role of lysine 411 in substrate carboxyl group binding to the human reduced folate carrier, as determined by site-directed mutagenesis and affinity inhibition. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 35623

Role of lysine 411 in substrate carboxyl group binding to the human reduced folate carrier, as determined by site-directed mutagenesis and affinity inhibition. Keywords Mesh Terms:

KEYWORDS:

MESH TERMS:

Chemical & Substance for Abstract: Role of lysine 411 in substrate carboxyl group binding to the human reduced folate carrier, as determined by site-directed mutagenesis and affinity inhibition. Information

Substance Name:

Registry Number:

Grant and Affiliation Information for Role of lysine 411 in substrate carboxyl group binding to the human reduced folate carrier, as determined by site-directed mutagenesis and affinity inhibition.

AFFILIATION: Developmental Therapeutics Program, Barbara Ann Karmanos Cancer Institute, 110 E. Warren Avenue, Detroit, MI 48201. matherly@kci.wayne.edu.

Country: United States

AGENCY:

GRANT:

ACRONYM:

MEDLINETA: Mol Pharmacol

REFSOURCE:

DATABASENAME:

ACCESSION NUMBER:

Number Hits: 0

Role of lysine 411 in substrate carboxyl group binding to the human reduced folate carrier, as determined by site-directed mutagenesis and affinity inhibition Related Publications

• A humanized mouse model for the reduced folate carrier.
• Effects of 5' untranslated region diversity on the posttranscriptional regulation of the human reduced folate carrier.
• Role of lysine 411 in substrate carboxyl group binding to the human reduced folate carrier, as determined by site-directed mutagenesis and affinity inhibition.
• Role of rat organic anion transporter 3 (Oat3) in the renal basolateral transport of glutathione.
• Transmembrane domains 4, 5, 7, 8, and 10 of the human reduced folate carrier are important structural or functional components of the transmembrane channel for folate substrates.