The mutation of two amino acid residues in the N-terminus of tyrosine hydroxylase (TH) dramatically enhances the catalytic activity in neuroendocrine AtT-20 cells.
The sequence Arg37-Arg38 of tyrosine hydroxylase (TH) is known to play a significant role in the feedback inhibition by the end product DA. To clarify how deeply the sequence Arg37-Arg38 and the phosphorylated Ser40 of human TH type 1 (hTH1) are involved in the regulation of this feedback inhibition in mammalian cells, we generated the following mutants: (i) RR-GG, Arg37-Arg38 replaced by Gly37-Gly38; (ii) RR-EE, Arg37-Arg38 replaced by Glu37-Glu38; (iii) S40D, Ser40 replaced by Asp40; and (iv) S40A, Ser40 replaced by Ala40. In a cell-free system, the level of the DA inhibition of the RR-EE mutant enzyme was to the same or smaller degree than that of the phosphorylation-mimicking S40D. Next, AtT-20 neuroendocrine cells were transfected with wild-type and mutated TH genes because these cells were earlier shown to be capable of fully converting L-3,4-dihydroxyphenylalanine into DA, whereby the catalytic activity of TH would be expected to be inhibited by the end product DA accumulating in the cells. The level of DA accumulation in AtT-20 cells expressing the TH gene was in the order: RR-EE > S40D > S40A = RR-GG > wild-type, which was in accordance with the observations for the cell-free system. These results suggest that the sequence Arg37-Arg38 of hTH1 is a more potent determinant of the efficient production of DA in mammalian cells than is the phosphorylated Ser40-hTH1.
Nakashima
Akira
A
Department of Physiology, School of Medicine, Fujita Health University, Toyoake, Aichi 470-1192, Japan.
Kaneko
Yoko S
YS
Mori
Keiji
K
Fujiwara
Kentaro
K
Tsugu
Toshimitsu
T
Suzuki
Takahiro
T
Nagatsu
Toshiharu
T
Ota
Akira
A
D015102:3,4-Dihydroxyphenylacetic Acid / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D002384:Catalysis; D002460:Cell Line; D002474:Cell-Free System / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism / Q000493:pharmacokinetics; D025461:Feedback, Physiological / Q000502:physiology; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009490:Neurosecretory Systems / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D013329:Structure-Activity Relationship; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics* / Q000378:metabolism*
D015102:3,4-Dihydroxyphenylacetic Acid; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine