Cell surface receptors for folic acid (membrane folate receptors, MFRs)have generated recent excitement because of the recognition of their physiological importance in cellular folate uptake and as likely transporters of potent novel chemotherapeutic antifolates. MFRs generate soluble folate binding proteins of nutritional and pathological significance in extracellular fluids. MFRs may be evolutionarily related to other cell surface proteins and may also possess an additional role in signalling cell proliferation. Despite intensive recent investigations of MFR isoforms at the molecular level much needs to be learned about the molecular mechanisms governing tissue specificity, regulation by folates and upregulation in malignancy. Furthermore, although the primary structures of two MFR isoforms are known and a mechanistic model for folate transport has been proposed, little is known about the location of the ligand binding site and other functional domains in MFRs. This project aims to use molecular and biochemical methods to understand (a) the transcriptional regulation that most likely controls MFR expression in response to changes in folate concentrations, tissue specific .regulation and regulation in neoplastic situations; the approach will include isolation and characterization of the promoter for a novel MFR isoform expressed in leukemic cells, examining the promoter transcripts in various cells expressing this MFR and identifying regions of the promoter important in folate dependent and tissue specific regulation; transcription rates and promoter activities will be monitored by nuclear run on and CAT assays (b) certain important aspects of structure-function relationships in MFRs including the site of ligand interaction, structural domains necessary for the transport function in MFRs, the location and significance of a glycosyl phosphatidyl inositol membrane anchor that is present in two MFR isoforms but absent in a third and the significance of glycosylation of MFRs in folate transport; the approaches used here will involve (1) affinity labeling of MFR and identification of labeled residues and (2) the production of deletion, insertional and point mutations in a MFR cDNA followed by expression of the altered MFR in COS or CHO cells and examining the mutant protein for its ability to bind or transport folate. These studies will provide a molecular basis for understanding the regulation and transport function of MFRs and possibly other related proteins and will help in the exploitation of MFRs in medicine.
