A P425R mutation of the proton-coupled folate transporter causing hereditary folate malabsorption produces a highly selective alteration in folate binding

Daniel Sanghoon Shin, Rongbao Zhao, Enghui H. Yap, Andras Fiser, I. David Goldman

Research output: Contribution to journalArticle

24 Scopus citations

Abstract

Proton-coupled folate transporter (PCFT) mediates folate intestinal absorption and transport across the choroid plexus, processes defective in subjects with hereditary folate malabsorption (HFM). PCFT is also widely expressed in human solid tumors where it contributes to the transport of pemetrexed and other antifolates. This study defines the basis for the functional changes due to a P425R mutation detected in a subject with HFM. Among various substitutions, only positively charged mutants (P425R and P425K) lost function but in a highly selective manner. Transport of reduced folates mediated by P425R-PCFT was virtually abolished; the methotrexate influx Kt was increased fivefold (from 2 to 10 μM). In contrast, the pemetrexed influx Kt mediated by P425R-PCFT was decreased 30% compared with wild-type (WT)-PCFT. Methotrexate inhibition of pemetrexed influx was competitive with a Ki for WT-PCFT comparable to its influx Kt. However, the methotrexate influx Ki for P425R-PCFT was ~ 15-fold higher than the WT-PCFT influx Kt and threefold higher than the methotrexate influx Kt for the P425R-PCFT mutant. The confirmed secondary structure and homology modeling place the P425 residue at the junction of the 6th external loop and 12th transmembrane domain, remote from the aqueous translocation pathway, a prediction confirmed by the failure to label P425C-PCFT with N-biotinylaminoethyl methanethiosulfonate-biotin and the absence of inhibition of P425C-PCFT function by water-soluble sulfhydryl reagents. Hence, despite its location, the P425R-PCFT mutation produces a conformational change that fully preserves pemetrexed binding but markedly impairs binding of methotrexate and other folates to the carrier.

Original languageEnglish (US)
Pages (from-to)C1405-C1412
JournalAmerican Journal of Physiology - Cell Physiology
Volume302
Issue number9
DOIs
StatePublished - May 1 2012

Keywords

  • Heme carrier protein 1
  • Intestinal folate transport

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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