Two regions in the N-terminal domain of ionotropic glutamate receptor 3 form the subunit oligomerization interfaces that control subtype-specific receptor assembly.
The N-terminal domain (NTD) of alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) and kainate glutamate receptors plays an important role in controlling subtype specific receptor assembly. To identify NTD subdomains involved in this process we generated AMPA glutamate receptor 3 (GluR3) mutants having intra-NTD substitutions with the corresponding regions of the kainate receptor GluR6 and tested their ability to form functional heteromers with wild-type subunits. The chimeric design was based on the homology of the NTD to the NTD of the metabotropic GluR1, shown to form two globular lobes and to assemble in dimers. Accordingly, the NTD was divided into four regions, termed here N1-N4, of which N1 and N3 correspond to the regions forming lobe-1 and N2 and N4 to those forming lobe-2. Substituting N1 or N3 impaired functional heteromerization but allowed protein-protein interactions. Conversely, exchanging N2 or N4 preserved functional heteromerization, although it significantly decreased homomeric activity, indicating a role in subunit folding. Moreover, a deletion in GluR3 corresponding to the hotfoot mouse mutation of the glutamate receptor delta2, covering part of N2, N3, and N4, impaired both homomeric and heteromeric oligomerization, thus explaining the null-like mouse phenotype. Finally, computer modeling suggested that the dimer interface, largely formed by N1, is highly hydrophobic in GluR3, whereas in GluR6 it contains electrostatic interactions, hence offering an explanation for the subtype assembly specificity conferred by this region. N3, however, is positioned perpendicular to the dimer interface and therefore may be involved in secondary interactions between dimers in the assembled tetrameric receptor.
Ayalon
Gai
G
Department of Anatomy and Cell Biology, Institute of Basic Dental Sciences, Hadassah Dental School, The Hebrew University, Jerusalem 91120, Israel.
Segev
Eitan
E
Elgavish
Sharona
S
Stern-Bach
Yael
Y
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D017353:Gene Deletion; D047468:Immunoprecipitation; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010641:Phenotype; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018075:RNA, Complementary / Q000378:metabolism; D018091:Receptors, AMPA / Q000737:chemistry* / Q000378:metabolism; D017470:Receptors, Glutamate / Q000737:chemistry; D018092:Kainic Acid Receptors / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012984:Software; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000737:chemistry; D000097806:GluK2 Kainate Receptor
D000409:Alanine; D003545:Cysteine; D018075:RNA, Complementary; D018091:Receptors, AMPA; D017470:Receptors, Glutamate; D018092:Kainic Acid Receptors; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D000097806:GluK2 Kainate Receptor; C104723:glutamate receptor ionotropic, AMPA 3