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Regulation of Human Organic Cation Transporter 3 (hOCT3) by the Dual‐Specifity Tyrosine‐(Y)‐Phosphorylation Regulated Kinase (DYRK1A)
Author(s) -
Ciarimboli Giuliano,
Stapel Mira,
Barz Vivien,
Pavenstädt Hermann,
Rinschen Markus M.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.03245
Subject(s) - phosphorylation , chemistry , microbiology and biotechnology , monoamine neurotransmitter , harmine , biochemistry , kinase , dyrk1a , tyrosine phosphorylation , biology , serotonin , receptor , pharmacology
hOCT3, a transmembrane protein with broad tissue distribution, is involved in the low‐affinity, high‐capacity transport of monoamine substrates across the cell membrane. In mice OCT3 is important for cerebral monoaminergic neurotransmission and for the effects of monoamines on behavior. The molecular regulation of hOCT3, however, is unknown. In this study, we have identified the dual‐specificity tyrosine‐(Y)‐phosphorylation regulated kinase 1A (DYRK1A) as a key interaction partner of hOCT3 using a membrane‐based Split‐Ubiquitin‐Yeast‐Two‐Hybrid technique, and pull‐down experiments in hOCT3 and DYRK1A transfected human embryonic kidney (HEK) cells. In these cells, DYRK1A overexpression resulted in hOCT3 phosphorylation at the serines 3 and 537 as determined by phosphoproteomic analysis, and in a significant inhibition of the transport of 4‐4‐dimethylamino‐styryl‐N‐methylpyridinium (ASP + ), a fluorescent hOCT3 substrate. DYRK1A overexpression reduced the maximum transport velocity (V max ) of hOCT3, without changing its affinity for ASP + . The hOCT3 transporter function was rescued by inhibition of DYRK1A with 100 nM harmine. In the seminoma cell line Tcam‐2, which endogenously expresses hOCT3 and DYRK1A, harmine treatment resulted in 1) a significant increased hOCT3 expression in the plasma membrane, as determined by biotinylation experiments; 2) an increase of ASP + uptake; and 3) a decrease of hOCT3/DYRK1A co‐localization as determined by confocal microscopy. Moreover, in Tcam2 cells, phosphatase activation by 4 h incubation with 10 μM ceramide induced a significant increase of ASP + uptake. Taken together, these data show that hOCT3 is regulated by DYRK1A via interaction, phosphorylation and change of intracellular distribution. The gene coding for the DYRK1A is localized in the Down syndrome (DS) critical region of chromosome 21 and is a strong candidate gene for DS‐associated mental retardation. Since hOCT3 functions as neurotransmitter reuptake transporter in the brain, we speculate that its inhibition by DYRK1A results in an impaired reuptake of neurotransmitters, and therefore an impoverishment of brain neurotransmitters content. This mechanism may be one of the molecular causes of mental retardation and susceptibility to Alzheimer diseases in DS patients. Support or Funding Information Deutsche Forschungsgemeinschaft CI 107/11.1