| Previous studies have shown that human dihydrofolate reductase (DHFR) protein levels increase upon exposure to methotrexate (MTX), a potent inhibitor of this enzyme. A model to explain this increase proposes that DHFR protein inhibits its own translation by binding to its cognate mRNA. Addition of MTX disrupts the DHFR protein-mRNA complex, allowing the translation to resume. In this study, Chinese hamster ovary DHFR null cells were transfected with wild type and mutants of human DHFR fused to enhanced green fluorescent protein (EGFP) to identify amino acids that are essential for increases in DHFR protein levels in response to MTX and other antifolates. Notably, when cells transfected with E30A, S118A, and L22R DHFR-EGFP were exposed to methotrexate, trimetrexate, raltitrexed, and pemetrexed there was no change in DHFR protein levels, indicating that these amino acids were essential for the up-regulation response. Substitutions at the sole cysteine 6 position did not affect the up-regulation response to MTX but modulated the response to raltitrexed and pemetrexed. This is the first study to identify residues required for the functional translational up-regulation response to antifolates. A new model for translational regulation is proposed in which DHFR can exist in two conformations, one bound to mRNA and one bound to NADPH. It is proposed that mutants that are not upregulated in response to MTX are fixed in a non-binding RNA conformation and cannot be induced.;We also present a comparative analysis of the translational regulation of DHFR amongst species. We demonstrate that there is a positive induction in DHFR protein levels in response to MTX in human, monkey, and dog cell lines, while there is no change in DHFR protein levels in either mouse or hamster cells. We created species chimeras of the putative mRNA binding site and tested the functionality of these structures in a mammalian cell culture system. We further demonstrate that human DHFR protein is promiscuous in that it can bind to more than one mRNA species. |
