Cloning and functional characterization of the bile acid-sensitive methotrexate carrier from rat liver cells.
We have cloned two complementary DNAs (cDNAs), RL-Mtx-1 and RL-Mtx-2, corresponding to the bile acid- sensitive methotrexate carrier from rat liver by direct full-length rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) using degenerated primers that were deduced from published sequences of tumor cell methotrexate transporters. When expressed in Xenopus laevis oocytes and cosM6 cells, both clones mediate methotrexate and bumetanide transport. RL-Mtx-1 consists of 2,445 bp with an open reading frame of 1,536 bp. The corresponding protein with 512 amino acids has a molecular weight of 58 kd. RL-Mtx-2 (2,654 bp) differs by an additional insert of 203 bp. This insert is located in frame at position 1,196 of the RL-Mtx-1 and contains the typical splice junction sites at the 5' and 3' end, indicating that the RL-Mtx-2 messenger RNA (mRNA) is generated by alternative splicing. The insert contains a stop codon that shortens the RL-Mtx-2 protein to 330 amino acids (38 kd). Both cDNAs contain the binding site sequence for the dioxin/nuclear translocator responsive element (Ah/Arnt-receptor) in conjunction with a barbiturate recognition sequence (Barbie box). Preliminary results show that the Barbie box acts as a negative regulatory element. The two liver cDNA clones show homologies to the published sequences of folate and the reduced folate carriers, but no homology is found to the transport systems for organic anions like the Ntcp1, oatp1, OAT-K1, and OAT1. Expression of the mRNA for the methotrexate carrier is found in liver, kidney, heart, brain, spleen, lung, and skeletal muscle, but not in the testis as revealed by Northern blot analysis. The highest abundance of the mRNA is found in the kidney.
Honscha
W
W
Institute of Pharmacology and Toxicology, Giessen, Germany. Walter.Honscha@vetmed.uni-giessen.de
Dötsch
K U
KU
Thomsen
N
N
Petzinger
E
E
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D001647:Bile Acids and Salts / Q000494:pharmacology*; D002034:Bumetanide / Q000378:metabolism; D019556:COS Cells / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000378:metabolism; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D009865:Oocytes / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D058978:Reduced Folate Carrier Protein; D014018:Tissue Distribution; D014982:Xenopus laevis
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D001647:Bile Acids and Salts; D002352:Carrier Proteins; D018076:DNA, Complementary; D026901:Membrane Transport Proteins; D015778:Minor Histocompatibility Antigens; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D058978:Reduced Folate Carrier Protein; C043408:Slc19a1 protein, rat; D002034:Bumetanide; D008727:Methotrexate