Multifunctional basic motif in the glycine receptor intracellular domain induces subunit-specific sorting.
The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated ion channel that mediates fast synaptic inhibition in the vertebrate central nervous system. As a member of the family of Cys-loop receptors, it assembles from five homologous subunits (GlyRalpha1-4 and -beta). Each subunit contains an extracellular ligand binding domain, four transmembrane domains (TM), and an intracellular domain, formed by the loop connecting TM3 and TM4 (TM3-4 loop). The TM3-4 loops of the subunits GlyRalpha1 and -alpha3 harbor a conserved basic motif, which is part of a potential nuclear localization signal. When tested for functionality by live cell imaging of green fluorescent protein and beta-galactosidase-tagged domain constructs, the TM3-4 loops of GlyRalpha1 and -alpha3, but not of GlyRalpha2 and -beta, exhibited nuclear sorting activity. Subunit specificity may be attributed to slight amino acid alterations in the basic motif. In yeast two-hybrid screening and GST pulldown assays, karyopherin alpha3 and alpha4 were found to interact with the TM3-4 loop, providing a molecular mechanism for the observed intracellular trafficking. These results indicate that the multifunctional basic motif of the TM3-4 loop is capable of mediating a karyopherin-dependent intracellular sorting of full-length GlyRs.
Melzer
Nima
N
From the Institut für Biochemie (Emil-Fischer-Zentrum), Universität Erlangen-Nürnberg, Erlangen 91054, Germany.
Villmann
Carmen
C
From the Institut für Biochemie (Emil-Fischer-Zentrum), Universität Erlangen-Nürnberg, Erlangen 91054, Germany.
Becker
Kristina
K
From the Institut für Biochemie (Emil-Fischer-Zentrum), Universität Erlangen-Nürnberg, Erlangen 91054, Germany.
Harvey
Kirsten
K
the Department of Pharmacology, The School of Pharmacy, London WC1N 1AX, United Kingdom, and.
Harvey
Robert J
RJ
the Department of Pharmacology, The School of Pharmacy, London WC1N 1AX, United Kingdom, and.
Vogel
Nico
N
From the Institut für Biochemie (Emil-Fischer-Zentrum), Universität Erlangen-Nürnberg, Erlangen 91054, Germany.
Kluck
Christoph J
CJ
From the Institut für Biochemie (Emil-Fischer-Zentrum), Universität Erlangen-Nürnberg, Erlangen 91054, Germany.
Kneussel
Matthias
M
the Zentrum für Molekulare Neurobiologie Hamburg, ZMNH, Universität Hamburg, Hamburg 20251, Germany.
Becker
Cord-Michael
CM
From the Institut für Biochemie (Emil-Fischer-Zentrum), Universität Erlangen-Nürnberg, Erlangen 91054, Germany. Electronic address: cmb@biochem.uni-erlangen.de.
D021581:Active Transport, Cell Nucleus; D000328:Adult; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D017208:Rats, Wistar; D018009:Receptors, Glycine / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques; D028901:alpha Karyopherins / Q000235:genetics / Q000378:metabolism
C106306:KPNA3 protein, human; C106259:KPNA4 protein, human; D021122:Protein Subunits; D018009:Receptors, Glycine; D028901:alpha Karyopherins; D049452:Green Fluorescent Proteins