VARIANT GPI STRUCTURE IN RELATION TO MEMBRANE-ASSOCIATED FUNCTIONS OFA MURINE FOLATE RECEPTOR

Two variant sublines of murine L1210 leukemia cells (L1210A and L1210J F) overexpress the cell surface folate receptor (FR). The membrane bou nd FR in L1210A cells exhibited significantly (up to 17-fold) greater relative affinities for (6S)-N-5-methyltetrahydrofolate, (6S)-N-5-form yltetrahydrofolate and methotrexate compared to the FR in L1210JF cell s, Furthermore, receptor-mediated transport of [H-3]-(6S)-N-5-methylte trahydrofolate was much more efficient in L1210A cells compared to L12 10JF cells. When solubilized with Triton X-100, the ligand binding cha racteristics of FR from both sublines resembled those of the receptor associated with L1210JF cell membranes. N-terminal amino acid sequence analysis as well as RT-PCR analysis of the entire coding region revea led a single species of FR in both cells, identical to murine FR-alpha . The FR in L1210JF cells was sensitive to phosphatidylinositol specif ic phospholipase C (PI-PLC) indicating the presence of a glycosyl-phos phatidylinositol (GPI) membrane anchor while the FR in L1210A cells wa s resistant to PI-PLC; however, the FR in L1210A cells was released fr om plasma membranes by nitrous acid, as expected for GPI and its PI-PL C resistant structural variants. Treatment of L1210A cell membranes wi th mild base rendered the protein PI-PLC sensitive as expected for GPI anchors acylated in the inositol ring and also decreased the affiniti es of the membrane associated FR for reduced folates, When the cDNA fo r murine FR-alpha was expressed in parental L1210 cells the protein wa s PI-PLC resistant but was sensitive to PI-PLC when the cDNA was expre ssed in human 293 fibroblasts. In L1210JF, L1210A, and parental L1210 cells, several cell surface proteins, including FR, incorporated [H-3] ethanolamine, a component of the GPI membrane anchor; however, the lab eled proteins were released by PI-PLC only in L1210JF cells, The above results preclude any peculiarity of the FR polypeptide in either L121 0 subline as the basis for the observed differences in PI-PLC sensitiv ity and membrane-associated functions of FR. Partial deglycosylation o f membrane associated FR from either cell with N-glycanase did not inf luence its ligand binding characteristics. The results of this study l ead to the hypothesis that variant GPI structures may modulate the fun ction of a protein by influencing its conformation/topography in the m embrane, Such effects may be identified by their disappearance/reducti on upon detergent solubilization or mild base treatment of the membran e.