Roles of the N-terminal domain on the function and quaternary structure of the ionotropic glutamate receptor.
The alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of ionotropic glutamate receptors (iGluRs) mediates fast excitatory neurotransmission in the mammalian brain. Although the most N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain is suggested to play a role in the initial assembly of iGluR subunits, it is unclear how this domain is arranged and functions in intact iGluRs. Similarly, although recent crystallographic analyses indicate that the isolated ligand-binding lysine/arginine/ornithine-binding protein domain forms a 2-fold symmetric dimer, the subunit stoichiometry of intact iGluRs remains elusive. Here, we developed a new approach to address these issues. The LIVBP domain of the GluR1 subunit of AMPA receptors was replaced by leucine-zipper peptides designed to form stable symmetric dimers, trimers, tetramers, or pentamers. All these mutant GluR1s were expressed in human embryonic kidney 293 cells and were transported to the cell surface as well as wild type GluR1. Functional and biochemical analyses indicated that these oligomerizing peptides specifically controlled the formation of the expected number of subunits in a channel complex. However, the channel function was only restored by the tetramer-forming peptide. Although the purified LIVBP domain of GluR1 formed a dimmer in solution, a dimer-forming peptide could not restore the function of GluR1. Moreover, a cross-linking assay indicated that four LIVBP domains are located in proximity to each other. These results suggest that the function of the LIVBP domain is not simply to form initial dimers but to adopt a conformation compatible with the overall tetrameric arrangement of subunits in intact AMPA receptors.
Matsuda
Shinji
S
Department of Physiology, Keio University School of Medicine, Tokyo, Japan.
Kamiya
Yoshinori
Y
Yuzaki
Michisuke
M
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003432:Cross-Linking Reagents; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D051381:Rats; D018091:Receptors, AMPA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D021122:Protein Subunits; D018091:Receptors, AMPA