Truncation of human dopamine transporter by protease calpain.
It has been shown recently that the N-terminal domain of the dopamine transporter (DAT) plays a role in several transporter functions. Here we provide evidence for a possible cellular mechanism of how the N-terminus of dopamine transporter might be removed in vivo. We isolated a recombinant N-terminal protein region of human dopamine transporter and cleaved it with calpain protease. Peptide fragment analysis revealed the existence of two calpain cleavage sites at positions Thr43/Ser44 and Leu71/Ser72 of the DATN-terminus. We show that calpain activation in rat striatal synaptosomes leads to a rapid decrease of dopamine transporter N-terminal epitopes corresponding to the protein sequences removed by a calpain cleavage at Thr43/Ser44 and that the process is totally blocked by a calpain inhibitor. Calpain truncation of the DATN-terminus abolishes its interaction with the receptor of activated protein kinase C, RACK1 and removes protein sequences previously implicated in amphetamine-induced dopamine release, PKC-dependent endocytosis and the interaction of DAT with the dopamine D2 receptor. The above suggests that cleavage of DAT by calpain may significantly modify dopamine homeostasis under pathological or physiological conditions.
Franekova
Veronika
V
Laboratory of Neurobiology, Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, Bratislava, Slovakia.
Baliova
Martina
M
Jursky
Frantisek
F
D000595:Amino Acid Sequence / Q000502:physiology; D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003342:Corpus Striatum / Q000378:metabolism*; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000378:metabolism; D017448:Receptors, Dopamine D2 / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
D050483:Dopamine Plasma Membrane Transport Proteins; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D017448:Receptors, Dopamine D2; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D011493:Protein Kinase C; D002154:Calpain; D004298:Dopamine