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, corre sponding to the bile acid-sensitive methotrexate carrier from rat liver by direct full-length rapid amplification of cDNA ends polymerase chain reacti on (RACE-PCR) using degenerated primers that were deduced from published se quences of tumor cell methotrexate transporters. When expressed in Xenopus lat-vis oocytes and cosM6 cells, both clones mediate methotrexate and bumet anide transport. RL-Mtx-1 consists of 2,445 bp with an open reading frame o f 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 splicin g. The insert contains a stop codon that shortens the RL-Mtx-2 protein to 3 30 amino acids (38 kd). Both cDNAs contain the binding site sequence for th e dioxin/ nuclear translocator responsive element (Ah/Arnt-receptor) in con junction 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. Expres sion of the mRNA for the methotrexate carrier is found in liver, kidney, he art, brain, spleen, lung, and skeletal muscle, but not in the testis as rev ealed by Northern blot analysis. The highest abundance of the mRNA is found in the kidney.