CHARACTERIZATION OF A MUTATION IN THE REDUCED FOLATE CARRIER IN A TRANSPORT DEFECTIVE L1210 MURINE LEUKEMIA-CELL LINE

This laboratory previously described an L1210 leukemia cell line (MTX( r)A) selected for resistance to methotrexate by virtue of impaired tra nsport due to a functional defect in the translocation process. We now report on the sequence analysis of cDNAs encoding the reduced folate carrier from this line and identify a single mutation that results in the substitution of a proline for an alanine in a highly conserved tra nsmembrane region of the protein. Transfection of the parental reduced folate carrier into MTX(r)A cells resulted in a cell line which exhib ited a complete restoration of methotrexate uptake and an enhanced sen sitivity to methotrexate. Northern analysis and specific [H-3]MTX cell surface binding indicated that expression of the reduced folate carri er was elevated similar to 5-fold in the transfectant compared to pare ntal and MTX(r)A cells. The MTX influx properties of the transfectant cell line were identical to those of the well characterized reduced fo late carrier from parental L1210 cells in terms of: 1) patterns of sen sitivity to competing folates, 2) sensitivity to the organic anion sul fobromophthalein, 3) lack of energy dependence, and 4) capacity for tr ans-stimulation. We also provide new data which suggests that the nucl eotide sequence 5' of the predicted ATG initiation codon may encode ad ditional protein information in the form of a leader sequence. Finally , we demonstrate that the MTX(r)A line has both the mutant and the par ental reduced folate carrier alleles but that expression appears to be restricted to the mutant allele. Thus, the methotrexate transport phe notype and resultant drug resistance in this cell line result from gen etic/regulatory events at both alleles.