Calpain sensitive regions in the N-terminal cytoplasmic domains of glycine transporters GlyT1A and GlyT1B.
Glycine transporters are members of the Na+/Cl- dependent transporter gene family and play crucial roles in regulating inhibitory as well as excitatory neurotransmission. In this report we show that calcium elevation in spinal cord synaptosomes decreases the levels of glycine transporter, GlyT1, N-terminal immunoreactivity, and that this decrease can be blocked by calpain inhibitor. Sequencing of GST fusion proteins containing the N-terminal domains of GlyT1A and B splice variants cleaved with rat recombinant calpain identified calpain cleavage sites after glycine 17 in GlyT1B and N-terminally of the first conserved arginine residue in both GlyT1A and GlyT1B. Expression in HEK293 cells revealed that truncation of the N-terminus of GlyT1 results in significant inhibition of glycine uptake. A syntaxin1A GST fusion protein was able to pull-down N-terminally deleted GlyT1, indicating that calpain cleavage does not eliminate syntaxin1A binding. These results suggest that calpain cleavage may regulate the transport activity/turnover of GlyT1 in vivo by cleaving its N-terminal domain.
Baliova
Martina
M
Department of Neurobiology, Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia. Frantisek.Jursky@savba.sk
Jursky
Frantisek
F
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D002460:Cell Line; D050487:Glycine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013574:Synaptosomes / Q000378:metabolism; D050827:Syntaxin 1 / Q000235:genetics / Q000378:metabolism
D050487:Glycine Plasma Membrane Transport Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C494213:Stx1a protein, rat; D050827:Syntaxin 1; D002154:Calpain; D002118:Calcium