Two Regions in the N-terminal Domain of Ionotropic Glutamate Receptor 3 Form the Subunit Oligomerization Interfaces That Control Subtype-specific Receptor Assembly

Abstract

The N-terminal domain (NTD) of ␣-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 ␦2, 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.Glutamate, the major excitatory neurotransmitter, activates two receptor families: metabotropic glutamate receptors (mGluRs), 1 which are coupled to G-proteins, and ionotropic glutamate receptors (iGluRs), which form cation selective ion channels. The iGluRs are further divided into three subtypes: AMPA, kainate, and NMDA receptors, originally named after their most selective agonist (1). Each subtype, in turn, consists of several subunits that assemble as tetramers in various combinations to form channels with distinct properties (2). Of the cloned subunits, GluR1-4 form the AMPA receptors, GluR5-7 and KA1-2 form the kainate receptors, and NR1, NR2A-D, and NR3A-B form the NMDA receptors. The ␦1-2 subunits constitute an orphan group of iGluRs, and their relation to the three principal subtypes is unclear.Overall, all iGluR subunits have a common domain organization (3). Each subunit contains four membrane regions (M1-M4), of which M1, M3, and M4 traverse the plasma membrane and M2 is a reentrant loop aligning the channel pore. The N terminus is extracellular and comprises an ϳ400-residue Nterminal domain (NTD; also termed "X domain"), which is homologous to bacterial periplasmic amino acid-binding proteins and to the N-terminal domain of mGluRs (4). Following the NTD is S1, an ϳ150-residue segment, which ...

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