Human folate binding proteins (FBP), or folate receptor (hFR), bind and transport physiologic reduced folates and antifolates (e.g. methotrexate). In the preceding year, we have described and characterized the structure and expression of members of the human hFR gene family, determined the mechanism(s) of drug resistance in a set of methotrexate resistance KB cells, and demonstrated the role of the hFR in methotrexate sensitivity and growth under folate limiting culture conditions. In preliminary experiments of structure-function relationships, we have determined that Trp 142 is important in formation of the ligand binding domain or in ligand interaction; that membrane attachment of the hFR is unique and may consist of either a GPI tail or a transmembrane domain; that the membrane anchor is determined by cell type; the heterogeneous hFR cDNAs are translated at different efficiencies which is a function of their length and sequence; isolated, sequenced and characterized 2 additional hFR- KB cDNA isoforms; defined the structure of the hFR-KB cell hFR gene which is characterized by unique alternative splicing of the 5' exons; observed that alternative splicing appears to be tissue dependent; determined the multiple transcription start sites of the hFR-KB gene and delineated its potential cis and trans acting elements; demonstrated that the induction of hFR in cells culture in low folate concentrations is, at least partly, due to increased transcription; that the decreased expression of hFR by MTX-resistant clone C2 appears to result from decreased transcription and is associated with alterations in a trans acting factor; that a recombinant GST-hFR fusion protein can be highly expressed in E. coli; and that human salivary gland expresses cDNAs homologous to but distinct from the hFR cDNAs. In general, the Experimental Hematology Section is investigating the following: l) Structure, Function, and Molecular Biology of Human hFRs; 2) Role of the hFRs in Transport of Folates and in the development of Acquired Methotrexate Resistance; and 3) Expression of the hFRs.
