TY - JOUR
T1 - A P425R mutation of the proton-coupled folate transporter causing hereditary folate malabsorption produces a highly selective alteration in folate binding
AU - Shin, Daniel Sanghoon
AU - Zhao, Rongbao
AU - Yap, Enghui H.
AU - Fiser, Andras
AU - David Goldman, I.
PY - 2012/5/1
Y1 - 2012/5/1
N2 - 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.
AB - 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.
KW - Heme carrier protein 1
KW - Intestinal folate transport
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UR - http://www.scopus.com/inward/citedby.url?scp=84860528730&partnerID=8YFLogxK
U2 - 10.1152/ajpcell.00435.2011
DO - 10.1152/ajpcell.00435.2011
M3 - Article
C2 - 22345511
AN - SCOPUS:84860528730
SN - 0363-6143
VL - 302
SP - C1405-C1412
JO - American Journal of Physiology - Cell Physiology
JF - American Journal of Physiology - Cell Physiology
IS - 9
ER -