Excitatory synaptic transmission in the brain of vertebrates is mediated by a family of 18 glutamate receptor ion channel genes (iGluRs) which exhibit subtype selective assembly, forming three major classes named AMPA, kainate and NMDA receptors1,2
. G-protein coupled receptors (mGluRs) also play key roles in the response to glutamate3
. Our first insights into the structure of glutamate receptors came from cDNA cloning and bioinformatic analysis4-6
. This revealed that iGluRs and mGluRs were multi-domain proteins, which likely evolved by fusion of bacterial periplasmic binding proteins with prokaryotic ion channels or signaling domains. A prototypical iGluR subunit contains an extracellular N-terminal assembly domain with a leucine/isoleucine/valine binding protein-like fold (LIVBP), and a glutamate or glycine binding domain with a glutamine binding protein-like fold (); by contrast mGluRs contain only a single LIVBP like domain and paradoxically this forms the binding site for glutamate. The assembly of mGluRs as dimers is well established7
, and although iGluRs are tetrameric proteins, their extracellular domains are also believed to assemble as pairs of dimers. The 1st
crystal structures of iGluR ligand binding domains were solved more than 10 years ago8
, followed by structures for mGluR ligand binding domains7
, but no high resolution structures for the 45 kDa amino terminal domain of an iGluR have been solved.
Figure 1 Expression and purification of the GluR6 amino terminal domain. (a) Topology diagram for a glutamate receptor subunit showing the R1 and R2 amino terminal domain clam shells, the S1S2 glutamate binding domain, and the ion channel. (b) SDS PAGE for GluR6 (more ...)
The ATDs of iGluRs are generally assumed to show ligand induced domain closure like that observed in LIVBP and mGluR1, and which is a key component in models developed to explain the allosteric modulation of NMDA receptors by Zn2+
and ifenprodil which bind to the ATD9-12
. However, the generation of homology models based on crystal structures for LIVBP and mGluR1 is complicated by both insertions in the ATD of iGluRs compared to LIVBP, and insertions in the ligand binding domain of mGluRs compared to the ATD of iGluRs. These homology models have focused on NMDA receptor allosteric modulators, have not addressed the mechanisms underlying subtype selective assembly, and assume that the ATDs of different classes of glutamate receptors are functionally similar9-12
To address these issues we performed crystallographic and sedimentation experiments. Our results reveal that the ATD of the rat kainate receptor GluR6 can be expressed as a soluble glycoprotein which forms dimers in solution at micromolar concentrations; that it crystallizes as a dimer with a much larger buried surface than found for mGluRs; and that both subunits in the dimer assembly have a partially closed conformation distinct from that observed for mGluRs and LIVBP. The structures reveal novel loop regions, different from those found in mGluRs, which likely play roles in the mechanism for subtype selective assembly. We also find striking amino acid sequence differences in the dimer interface of NMDA receptors versus AMPA and kainate receptors, which suggests unexpectedly that these families are functionally distinct.