CELLULAR CONTROL OF FOLATE UPTAKE--CAVEOLAE V COATED PIT

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DESCRIPTION: The major hypothesis of this proposal is that folate receptors are expressed on the placenta and neural tube and that folate uptake to the developing fetus is regulated by potocytosis, a process thought to involve glycosol-phosphatidylinositol (GPI)-anchored proteins and caveolae. In addition renal reabsorption of folate from the glomerular filtrate is dependent upon the same process. The specific aims of this study are: (1) to develop a series of autofluorescent folate receptors with differing anchoring schemes or targeting sequences to direct them to caveolae and coated pits. These chimeric receptors will be used to precisely correlate pathway-specific folate biology with what may be artifact-free visualization of folate receptors in live cells; and (2) to use conditional lethal mutant cell lines with inducible defects in coated pit endocytosis to determine how specific molecules accomplish compartmentation and regulation of folate receptors in caveolae rather than coated pits. The experimental design employs cDNA constructs that when transfected into CHO cells produce chimeric proteins containing anchoring domains or targeting sequences, an autofluorescent reporter protein and the alpha-folate receptor. In addition temperature-sensitive mutant cell lines that are defective in coated-pit endocytosis at non-permissive temperatures will be used to study chimeric protein segregation and function. This highly innovative approach to the study of folate receptor biology is likely to provide important new information on receptor localization, recycling and functional mechanism.