Variant GPI structure in relation to membrane-associated functions of a murine folate receptor.
Two variant sublines of murine L1210 leukemia cells (L1210A and L1210JF) overexpress the cell surface folate receptor (FR). The membrane bound FR in L1210A cells exhibited significantly (up to 17-fold) greater relative affinities for (6S)-N5-methyltetrahydrofolate, (6S)-N5-formyltetrahydrofolate and methotrexate compared to the FR in L1210JF cells. Furthermore, receptor-mediated transport of [3H]-(6S)-N5-methyltetrahydrofolate was much more efficient in L1210A cells compared to L1210JF cells. When solubilized with Triton X-100, the ligand binding characteristics of FR from both sublines resembled those of the receptor associated with L1210 JF cell membranes. N-terminal amino acid sequence analysis as well as RT-PCR analysis of the entire coding region revealed a single species of FR in both cells, identical to murine FR-alpha. The FR in L1210JF cells was sensitive to phosphatidylinositol specific phospholipase C (PI-PLC) indicating the presence of a glycosyl-phosphatidylinositol (GPI) membrane anchor while the FR in L1210A cells was resistant to PI-PLC; however, the FR in L1210A cells was released from plasma membranes by nitrous acid, as expected for GPI and its PI-PLC resistant structural variants. Treatment of L1210A cell membranes with mild base rendered the protein PI-PLC sensitive as expected for GPI anchors acylated in the inositol ring and also decreased the affinities of the membrane associated FR for reduced folates. When the cDNA for murine FR-alpha was expressed in parental L1210 cells the protein was PI-PLC resistant but was sensitive to PI-PLC when the cDNA was expressed in human 293 fibroblasts. In L1210JF, L1210A, and parental L1210 cells, several cell surface proteins, including FR, incorporated [3H]ethanolamine, a component of the GPI membrane anchor; however, the labeled proteins were released by PI-PLC only in L1210JF cells. The above results preclude any peculiarity of the FR polypeptide in either L1210 subline as the basis for the observed differences in PI-PLC sensitivity and membrane-associated functions of FR. Partial deglycosylation of membrane associated FR from either cell with N-glycanase did not influence its ligand binding characteristics. The results of this study lead to the hypothesis that variant GPI structures may modulate the function of a protein by influencing its conformation/topography in the membrane. Such effects may be identified by their disappearance/reduction upon detergent solubilization or mild base treatment of the membrane.
Wang
X
X
Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo 43699-0008, USA.
Jansen
G
G
Fan
J
J
Kohler
W J
WJ
Ross
J F
JF
Schornagel
J
J
Ratnam
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D017931:DNA Primers; D019856:Ethanolamine; D004983:Ethanolamines / Q000378:metabolism; D058973:Folate Receptors, GPI-Anchored; D017261:Glycosylphosphatidylinositols / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007668:Kidney; D002955:Leucovorin / Q000378:metabolism; D007939:Leukemia L1210 / Q000378:metabolism; D008727:Methotrexate / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011869:Radioligand Assay; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013763:Tetrahydrofolates / Q000378:metabolism; D014162:Transfection; D014316:Tritium; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D017931:DNA Primers; D004983:Ethanolamines; D058973:Folate Receptors, GPI-Anchored; D017261:Glycosylphosphatidylinositols; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D013763:Tetrahydrofolates; D014316:Tritium; D019856:Ethanolamine; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002955:Leucovorin; C005984:5-methyltetrahydrofolate; D008727:Methotrexate