Calpain-mediated proteolytic cleavage of the neuronal glycine transporter, GlyT2.
The glycine transporter 2 (GlyT2) belongs to the family of Na+/CL--dependent plasma membrane transporters and is localized on the presynaptic terminals of glycinergic neurons. GlyT2 differs from other family members by its extended N-terminal cytoplasmic region. We report that activation of a Ca2+-dependent protease, most likely calpain, in spinal cord synaptosomes or cultured spinal cord neurons, results in partial proteolysis of GlyT2. Regions sensitive to calpain cleavage in vivo are located in the N-terminal and, to a lesser extent, C-terminal regions of the transporter protein. Incubation of a GlyT2 N-terminal fusion protein with spinal cord extract in the presence of calcium followed by protein sequence analysis localized the major N-terminal cleavage site after methionine 156, with a second cleavage site being situated after glycine 164. Interestingly, the size of the N-terminally truncated GlyT2 protein (70 kDa) is similar to that of most other transporter family members, and truncated GlyT2 displayed full transport activity upon expression in HEK293 cells. Our data suggest that Ca2+-triggered proteolysis may contribute to the regulation of GlyT2 trafficking and/or function in the neuronal plasma membrane.
Baliova
Martina
M
Department of Neurobiology, Institute of Molecular Biology, SAS, Bratislava, Slovakia.
Betz
Heinrich
H
Jursky
Frantisek
F
D000595:Amino Acid Sequence; D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002154:Calpain / Q000378:metabolism*; D002478:Cells, Cultured; D050487:Glycine Plasma Membrane Transport Proteins; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis / Q000096:biosynthesis; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013116:Spinal Cord / Q000737:chemistry / Q000166:cytology; D013574:Synaptosomes / Q000737:chemistry / Q000378:metabolism
D026921:Amino Acid Transport Systems, Neutral; D050487:Glycine Plasma Membrane Transport Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C493271:SLC6A5 protein, human; C493269:Slc6a5 protein, rat; D002154:Calpain; D002118:Calcium