Identification of amino acids required for the functional up-regulation of human dihydrofolate reductase protein in response to antifolate Treatment.
Human dihydrofolate reductase (DHFR) protein levels rapidly increase upon exposure to methotrexate, a potent inhibitor of this enzyme. A model to explain this increase proposes that DHFR inhibits its own translation by binding to its cognate mRNA and that methotrexate disrupts the DHFR protein-mRNA complex allowing its translation to resume. In the present study, Chinese hamster ovary cells lacking DHFR were transfected with wild type and mutants of human DHFR to identify amino acids that are essential for increases in DHFR in response to methotrexate. Glu-30, Leu-22, and Ser-118 were involved in the up-regulation of DHFR protein levels by methotrexate and certain other antifolates. Cells transfected with E30A, L22R, and S118A mutants that did not respond to methotrexate up-regulation had higher basal levels of DHFR, consistent with the model, i.e. lack of feedback regulation of these enzymes. Although cells containing the S118A mutant enzyme had higher levels of DHFR and had catalytic activity similar to that of wild type DHFR, they had the same sensitivity to the cytotoxicity of methotrexate, as were cells with wild type DHFR. This finding provides evidence that the adaptive up-regulation of DHFR by methotrexate contributes to the decreased sensitivity to this drug. Based on these observations, a new model is proposed whereby DHFR exists in two conformations, one bound to DHFR mRNA and the other bound to NADPH. The mutants that are not up-regulated by methotrexate are unable to bind their cognate mRNA.
Skacel
Nancy
N
Weill Cornell Graduate School of Medical Sciences, New York, New York, USA.
Menon
Lata G
LG
Mishra
Prasunkumar J
PJ
Peters
Rowayda
R
Banerjee
Debabrata
D
Bertino
Joseph R
JR
Abali
Emine Ercikan
EE
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D002384:Catalysis; D002470:Cell Survival; D003001:Cloning, Molecular; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D005493:Folic Acid Antagonists / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D018698:Glutamic Acid / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008727:Methotrexate / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000737:chemistry; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012694:Serine / Q000737:chemistry; D013053:Spectrophotometry; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis* / Q000737:chemistry; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D015854:Up-Regulation
D000596:Amino Acids; D017931:DNA Primers; D005493:Folic Acid Antagonists; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid; D012694:Serine; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase; D005982:Glutathione Transferase; D007930:Leucine; D003545:Cysteine; D008727:Methotrexate