Characterization of a human alternatively spliced truncated reduced folatecarrier increasing folate accumulation in parental leukemia cells

Human CEM-7A cells established by gradual deprivation of leucovorin from th e growth medium, display 100-fold, overexpression of methotrexate transport activity. We found that this was associated with 10-fold reduced folate ca rrier gene amplification and 50-fold overexpression of both the principal 3 kb reduced folate carrier transcript and, surprisingly, a novel truncated 2 kb reduced folate carrier mRNA poorly expressed in parental CEM cells. Th e molecular basis for the generation of this truncated reduced folate carri er transcript and its potential functional role in folate accumulation were studied. Reduced folate carrier genomic and cDNA sequencing revealed that the truncated transcript had an internal deletion of 987 nucleotides which was a result of an alternative splicing utilizing a cryptic accepter splice site within exon 6. This deletion consisted of the 3'-most 480 nucleotides of the reduced folate carrier ORF and the following 507 nucleotides of the 3'-UTR. These resulted in a truncated reduced folate carrier protein, whic h lacks the C-terminal 160 amino acids, but instead contains 58 new C-termi nal amino acids obtained from reading through the 3'-UTR, Consequently, a t runcated reduced folate carrier protein is generated that lacks the 12th tr ansmembrane domain and contains a new and much shorter C-terminus predicted to reside at the extracellular face. Western analysis with plasma-membrane fraction from CEM-7A cells revealed marked overexpression of both a broadl y migrating approximate to 65-90 kDa native reduced folate carrier and a ap proximate to 40-45 kDa truncated reduced folate carrier, the core molecular masses of which were confirmed by in vitro translation. However, unlike th e native reduced folate carrier, the truncated reduced folate carrier prote in failed to bind the affinity labels NHS-[H-3]MTX and NHS-[H-3]folic acid. Stable transfection of the truncated reduced folate carrier cDNA into mous e L1210 leukemia cells: increased folate accumulation, decreased their leuc ovorin and folie acid growth requirements, and increased their sensitivity to methotrexate. This constitutes the first documentation of an expressed a lternatively spliced truncated reduced folate carrier that, when coexpresse d along with the native carrier, augments folate accumulation and consequen tly decreases the cellular folate growth requirement. The possible mechanis ms by which the truncated reduced folate carrier may increase Folate accumu lation and/or metabolism in cells coexpressing the truncated and native red uced folate carrier are discussed.