Increased expression and characterization of two distinct folate binding proteins in murine erythroleukemia cells.
We previously identified two membrane-bound folate binding proteins, FBP1 and FBP2, in murine L1210 leukemia cells. We now report on the development of two variant murine erythroleukemia cell lines that were used for direct comparison and biochemical characterization of the two murine folate binding proteins. Based on the results of northern analysis and the mobilities of affinity-labeled proteins on polyacrylamide gels, these cell lines exhibit specific up-regulated expression of FBP1 or FBP2. The affinities of the folate binding proteins for various (anti)folates were determined based upon the ability of the compounds to inhibiting of [3H]folic acid. The two proteins exhibited considerably different affinities and stereospecificities and, in general, FBP2 consistently bound each test compound with lesser affinity than FBP1. Both proteins displayed greatest affinity for folic acid, 5-methyltetrahydrofolate, and the antifolates CB3717 and 5,10-dideazatetrahydrofolate (DDATHF). Conversely, the proteins exhibited poor affinity for the dihydrofolate reductase inhibitors methotrexate and aminopterin. For 5-formyltetrahydrofolate, FBP1 had high affinity for the (6S) diastereoisomer, whereas FBP2 showed preference for the non-physiologic (6R) diasterceoisomer. The binding properties of FBP1 and FBP2 overexpressed in these cell lines closely paralleled those of their respective human homologs. These lines provide a model system in which to examine the biochemical characteristics of the individual folate binding proteins without the potential problems associated with expression of proteins in dissimilar cell lines.
Brigle
K E
KE
Department of Medicine, Virginia Commonwealth University, Medical College of Virginia, Richmond 23298.
Spinella
M J
MJ
Westin
E H
EH
Goldman
I D
ID
D000581:Amidohydrolases; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002999:Clone Cells; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D002955:Leucovorin / Q000494:pharmacology; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011956:Receptors, Cell Surface; D013237:Stereoisomerism; D013763:Tetrahydrofolates / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D013763:Tetrahydrofolates; C045894:lometrexol; D005492:Folic Acid; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D002955:Leucovorin