Cloning and characterization of a pH-sensing regulatory factor that modulates transport activity of the human H+/peptide cotransporter, PEPT1.
We have isolated a cDNA encoding a pH-sensing regulatory factor protein that modulates transport activity of the human H+/peptide cotransporter, hPEPT1, from the human duodenum cDNA library. The cDNA (1,724 bp) for the regulatory factor (hPEPT1-RF) had an open reading frame encoding a 208-amino acid. The 18-195 amino acid residues of hPEPT1-RF were completely consistent with the 8-185 amino acid residues of hPEPT1, whereas the 1-17 and 196-208 residues were unique sequences. Using a reticulocyte lysate, the in vitro synthesized hPEPT1-RF RNA translated a product of approximately 23 kDa. Northern blot analysis and reverse transcription-coupled PCR revealed that both hPEPT1 and hPEPT1-RF mRNA transcripts are expressed in Caco-2 cells. When expressed in Xenopus oocytes, hPEPT1-RF showed no transport activity of glycylsarcosine, but shifted pH profile of the dipeptide transport mediated by the coexpressed hPEPT1. The pH profile of glycylsarcosine uptake in oocytes coexpressing hPEPT1 and hPEPT1-RF was almost similar to that in the Caco-2 cells. This is the first demonstration of cDNA isolation of a regulatory factor which modulates hPEPT1 activity.
Saito
H
H
Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Japan.
Motohashi
H
H
Mukai
M
M
Inui
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology; D000074011:Peptide Transporter 1; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D027981:Symporters; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014982:Xenopus laevis
D002352:Carrier Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D000074011:Peptide Transporter 1; D012333:RNA, Messenger; D011994:Recombinant Proteins; C082186:SLC15A1 protein, human; D027981:Symporters