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Trypsin disrupts the trafficking of the human dopamine transporter by alpha-synuclein and its A30P mutant.
Biochemistry. 2004 Feb 10; 43(5):1242-53.B

Abstract

Alpha-synuclein modulates dopamine homeostasis in dopamine-producing neurons of substantia nigra, partly through regulation of human dopamine transporter (hDAT) activity. To identify the underlying mechanisms, we disrupted the modulation of hDAT activity by wild-type (wt) alpha-synuclein, and its familial Parkinson's disease linked mutants A30P and A53T, by mild trypsinization (0.1%, 30 s) of Ltk(-) cotransfected cells. Trypsin completely reversed the attenuation of hDAT function mediated by wt and the A30P mutant. In A53T coexpressing cells, where DAT activity is not downregulated, trypsinization did not induce any changes. These effects of trypsin were mimicked by collagenase I and Dispase (0.1%, 1 min each) but not by chymotrypsin, Pronase, or papain (0.1%, up to 2 min each). Trypsin increased dopamine uptake in rat primary mesencephalic neurons, suggesting that DAT activity is also subjected to modulation by alpha-synuclein in these neurons that endogenously coexpress both proteins. In trypsinized cells, dopamine accelerated both production of reactive oxygen species and cell death in hDAT and wt or A30P, but not A53T, coexpressing cells, compared to nontrypsinized cells. Paradoxically, trypsin increased the protein-protein interactions between the synuclein variants and hDAT, without any noticeable proteolysis of these proteins. hDAT-alpha-synuclein protein-protein interactions occurred through residues 58-107 (NAC domain) of the alpha-synuclein variants and residues 598-620 of the carboxy-terminal tail of hDAT, in both trypsinized and nontrypsinized cells. Confocal microscopy and biotinylation studies show that, in cells expressing the wt or A30P variants, but not the A53T mutant, hDAT is sequestered away from the plasma membrane into the cytoplasm, an effect that is reversed by trypsin. These results show that alpha-synuclein modulates hDAT function through trafficking of the transporter in a process that can be disrupted by trypsin.

Authors+Show Affiliations

Department of Pediatrics, Georgetown University, Washington, D.C. 20007, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

14756560

Citation

Wersinger, Christophe, et al. "Trypsin Disrupts the Trafficking of the Human Dopamine Transporter By Alpha-synuclein and Its A30P Mutant." Biochemistry, vol. 43, no. 5, 2004, pp. 1242-53.
Wersinger C, Vernier P, Sidhu A. Trypsin disrupts the trafficking of the human dopamine transporter by alpha-synuclein and its A30P mutant. Biochemistry. 2004;43(5):1242-53.
Wersinger, C., Vernier, P., & Sidhu, A. (2004). Trypsin disrupts the trafficking of the human dopamine transporter by alpha-synuclein and its A30P mutant. Biochemistry, 43(5), 1242-53.
Wersinger C, Vernier P, Sidhu A. Trypsin Disrupts the Trafficking of the Human Dopamine Transporter By Alpha-synuclein and Its A30P Mutant. Biochemistry. 2004 Feb 10;43(5):1242-53. PubMed PMID: 14756560.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Trypsin disrupts the trafficking of the human dopamine transporter by alpha-synuclein and its A30P mutant. AU - Wersinger,Christophe, AU - Vernier,Philippe, AU - Sidhu,Anita, PY - 2004/2/6/pubmed PY - 2004/6/21/medline PY - 2004/2/6/entrez SP - 1242 EP - 53 JF - Biochemistry JO - Biochemistry VL - 43 IS - 5 N2 - Alpha-synuclein modulates dopamine homeostasis in dopamine-producing neurons of substantia nigra, partly through regulation of human dopamine transporter (hDAT) activity. To identify the underlying mechanisms, we disrupted the modulation of hDAT activity by wild-type (wt) alpha-synuclein, and its familial Parkinson's disease linked mutants A30P and A53T, by mild trypsinization (0.1%, 30 s) of Ltk(-) cotransfected cells. Trypsin completely reversed the attenuation of hDAT function mediated by wt and the A30P mutant. In A53T coexpressing cells, where DAT activity is not downregulated, trypsinization did not induce any changes. These effects of trypsin were mimicked by collagenase I and Dispase (0.1%, 1 min each) but not by chymotrypsin, Pronase, or papain (0.1%, up to 2 min each). Trypsin increased dopamine uptake in rat primary mesencephalic neurons, suggesting that DAT activity is also subjected to modulation by alpha-synuclein in these neurons that endogenously coexpress both proteins. In trypsinized cells, dopamine accelerated both production of reactive oxygen species and cell death in hDAT and wt or A30P, but not A53T, coexpressing cells, compared to nontrypsinized cells. Paradoxically, trypsin increased the protein-protein interactions between the synuclein variants and hDAT, without any noticeable proteolysis of these proteins. hDAT-alpha-synuclein protein-protein interactions occurred through residues 58-107 (NAC domain) of the alpha-synuclein variants and residues 598-620 of the carboxy-terminal tail of hDAT, in both trypsinized and nontrypsinized cells. Confocal microscopy and biotinylation studies show that, in cells expressing the wt or A30P variants, but not the A53T mutant, hDAT is sequestered away from the plasma membrane into the cytoplasm, an effect that is reversed by trypsin. These results show that alpha-synuclein modulates hDAT function through trafficking of the transporter in a process that can be disrupted by trypsin. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/14756560/Trypsin_disrupts_the_trafficking_of_the_human_dopamine_transporter_by_alpha_synuclein_and_its_A30P_mutant_ L2 - https://doi.org/10.1021/bi035308s DB - PRIME DP - Unbound Medicine ER -