Folate is an essential nutrient that functions as a carrier of single carbon units for the biosynthesis of nucleic acids, the methylation of homocysteine for the biosynthesis of methionine, for the metabolism of histidine, and for the serine-glycine interconversion. This important role of folate in intracellular metabolism is reflected in the clinical disorder of folate deficiency and megaloblastic anemia that occurs with nutritional deficiency malabsorption (acquired and congenital), augmented consumption (pregnancy, hemolytic anemia) and disorders of metabolism (drugs and toxins). It also has been the basis for the development and therapeutic application of antifolate drugs to inhibit the growth of neoplastic cells. An essential component of folate homeostasis is the process by which folate is transported into cells and this occurs by two mechanisms: 1) an invariant pathway which utilizes a transmembrane anion channel that is selective for reduced anionic folates and the antifol, methotrexate, and excludes pteroyglutamic acid, an anionic but non-reduced folate; and 2) a variant mechanism which utilizes a folate binding protein (FBP) (or receptor) that is anchored to the external surface of the plasma membrane by a glycosylphosphatidylinositol (GPI) adduct. In contrast to the invariant transmembrane channel system, the GPI-anchored FBP is constitutively expressed on very few cells but may be induced by slow conditioning in a low folate environment. However, cells that constitutively express the GPI-FBP can grow under low folate conditions (approximately 1 nM) without such conditioning. The purification and structural and functional properties of a number of human FBP(s) including the GPI-FBP, the cloning of the complementary DNA (cDNA) encoding one GPI- FBP, the isolation of an unprocessed FBP pseudogene and the isolation and characterization of a gene encoding a FBP expressed in human placenta have been reported from this laboratory. This gene (assigned FBP/PL-1 gene) has an unusual cis promoter element comprised of a G-rich SP1 binding sequence linked to tandemly duplicated GGAAG pentameric sequences that are the motifs for the ets oncogene encoding GA binding nuclear transcription factor and which have been identified as regulatory elements in the genes encoding subunits of cytochrome c oxidase. We now propose to continue our studies with the following two objectives: 1) To isolate and characterize additional genes that constitute the multigene family that encode the membrane-associated and other cellular FBP(s); 2) To characterize cis promoter elements and enhancer sequences that regulate transcription, identify and purify trans-active nuclear factors that interact with these regulatory genomic elements and characterize cytoplasmic factors that regulate expression at the level of translation. These studies will enhance our understanding of the cellular transport and intracellular metabolism of folate cofactors and could provide a basis for up-modulation of these mechanisms to maintain normal folate homeostasis when environmental factors may induce folate deficiency or impaired metabolism.
