REVIEWNew insights into the structure and function of glutamate
receptors: the orphan receptor d2 reveals its family’s secrets
Michisuke Yuzaki
Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, TN, USA(Received for publication on December 27, 2002)

Abstract. The ionotropic glutamate receptors (iGluRs) form ligand-gated ion channels that mediate
the vast majority of excitatory neurotransmission in the mammalian brain. These receptors play central
roles not only in normal neurodevelopmental and neurophysiological processes but also in certain
neuropathological processes. Molecular cloning of genes for iGluRs in the past decade has advanced
our understanding of the basic properties of iGluRs, such as ion selectivity, ligand binding, andanchoring at synapses. Although the gene for the d2 glutamate receptor (GluRd2) was cloned on thebasis of homology screening, GluRd2 has been referred to as an ‘‘orphan’’ receptor because it does notform functional glutamate-gated ion channels. However, ataxia in many types of mice is caused byspontaneous mutation of GluRd2. Analysis of two such mutants, lurcher and hotfoot, has provided key
insights into the GluRd2 signaling in neurons. Furthermore, characterization of mutant GluRd2 hasrevealed unexpected clues to two fundamental features regarding the structure and function of iGluRs– gating and assembly. Studies have recently shown that the transmembrane region where the lurcher
mutation is located probably plays a crucial role in channel gating. The mechanism that controls iGluRsubunit assembly seems to involve the extracellular N-terminal domain where the hotfoot mutation is
located. An understanding of mechanisms responsible for gating and assembly is essential for thecomprehension of neuronal function and dysfunction. Although reverse genetics is useful in deciphering glutamate signaling, these ﬁndings demonstrate the power of classic approaches to forward genetics
on mutant mice. (Keio J Med 52 (2): 92–99, June 2003)
Key words: assembly, gating, Purkinje cell, lurcher, hotfootand-Hyde’’ type of signaling – it is crucial for normal
synaptic functions but has the potential to cause severe neuronal destruction. In other words, neurons areequipped with mechanisms to precisely control glutamate signaling; when these mechanisms are disrupted,
then the neurons undergo destruction. An understanding of such mechanisms is essential for an appreciationof neuronal function and dysfunction.
In the past decade, molecular cloning efforts
have identiﬁed genes for four AMPA receptor subunits (GluR1–GluR4), ﬁve kainate receptor subunits(GluR5–GluR7, KA1, and KA2), and seven NMDA
receptor subunits (NR1, NR2A–NR2D, NR3A, andNR3B).1,4–6 These ﬁndings have greatly advanced ourunderstanding of the basic properties of iGluRs, such

Introduction
Fast excitatory neurotransmission in the mammalian central nervous system is mediated by ionotropic glutamate-gated receptors (iGluRs): a-amino3-hydroxy-5-methyl-4-isoxazolepropionate(AMPA),
kainate, and N-methyl-d-aspartate (NMDA) receptors.1 Glutamate signaling is essential for normal neurodevelopment and synaptic plasticity associated withlearning and memory. However, when iGluRs are overstimulated, glutamate signaling causes ‘‘excitotoxicity,’’which plays a role in neuronal cell death in manyneuropathological processes, such as ischemia, Alzheimer’s disease, Parkinson’s disease, and Huntington’sdisease.2,3 Therefore, glutamate signaling is a ‘‘Jekyll-

Presented at the 1280th Meeting of the Keio Medical Society in Tokyo, October 16, 2002.
Present afﬁliation: Department of Physiology, School of Medicine, Keio University, Tokyo, Japan
Reprint requests to: Dr. Michisuke Yuzaki, Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, 332 N. Lauderdale St., Memphis, TN 38105-2794, USA, e-mail: michi.yuzaki@stjude.org
92

Keio J Med 2003; 52 (2): 92–99

as ion selectivity, ligand binding, and anchoring at synapses. However, two fundamental issues remain unclear.
One is the mechanism of ‘‘gating,’’ i.e., the conformational changes associated with the opening and closing of ion channels induced by external signals. The
importance of the precise control of gating of ion channels is illustrated by several hereditary diseases attributable to a point mutation that causes sub-millisecond
to millisecond delays in the activation or inactivation ofgating.7,8 Another unclear issue is the mechanism that
controls subunit assembly; functional iGluRs are heteromers in vivo, and various combinations of the subunits are responsible for the functional heterogeneity ofiGluRs.9,10
Although the d2 glutamate receptor (GluRd2) isclassiﬁed as an iGluR, GluRd2 has been referred to asan ‘‘orphan’’ receptor because it does not form functional glutamate-gated ion channels when expressed,
either alone or with other iGluRs, in heterologous cells,
nor does it bind to glutamate analogs.11,12 Nevertheless, GluRd2, which is predominantly expressed in cerebellar Purkinje cells, plays a crucial role in cerebellar
functions: mice that lack the gene encoding GluRd2
display ataxia and impaired synaptic plasticity.13 The
mechanisms by which GluRd2 is involved in cerebellar
functions have not been fully elucidated, because of
the lack of speciﬁc pharmacological tools with which tomanipulate GluRd2. It is unlikely that GluRd2 directly
participates in normal parallel ﬁber – Purkinje cell synaptic transmission because the transmission was completely blocked by speciﬁc antagonists to conventional
AMPA receptors.14 Recently, studies of several mutant
mice, such as lurcher, hotfoot, and GluRd2 knock-out
mice, have provided the key to understanding GluRd2
signaling in neurons.14 GluRd2 has a channel pore
similar to that of other glutamate receptors; the channel
is functional at least when the lurcher mutation ispresent. GluRd2 must be transported to the Purkinje
cell surface to function; the absence of surface GluRd2
causes the ataxic phenotype of hotfoot mice. Furthermore, the characterization of GluRd2 has also revealed
unexpected clues to the two fundamental yet unclear
issues regarding the structure and function of iGluRs –gating and assembly. These two issues are addressedin this review, which is based on part of my presentation at the 1280th Meeting of the Keio Medical Society.
Here I will focus on ﬁndings from studies of lurcher andhotfoot mice performed in my laboratory.
The Lurcher Mutation and Gating Mechanisms ofiGluRsGeneral design of iGluRs
Recent studies have established that iGluRs have amodular structure (Fig. 1A): an amino-terminal leucine/93

isoleucine/valine-binding protein (LIVBP)-like domain
and a ligand-binding bipartite lysine/arginine/ornithinebinding protein (LAOBP)-like domain on the extracellular side of the plasma membrane, three transmembrane domains (TM1, TM3, and TM4), an ion
channel-forming re-entrant loop segment (P-loop or
TM2), and a cytoplasmic carboxyl-terminal region.3
Ligand binding is achieved by the LAOBP-like domain,which consists of S1 and S2 regions.15 Two lobes formedby these regions close upon ligand binding, which leads
to opening of the ion channel gate (Fig. 1A). The reentrant loop formed by TM2 determines the ion selectivity of the channel pore.16 The cytoplasmic tails of
iGluRs are important in synaptic clustering and in regulating receptor activity that involves intracellular signaling via interaction with anchoring proteins.17
The lurcher mutation
The ataxia of lurcher mice results from a point mutation at position 654 (Ala ! Thr) at the end of TM3 of
GluRd2 (Fig. 1B); this particular mutation causes constitutive activation of mutant channels (GluRd2 Lc ) in
the absence of ligand binding18 and eventually leads tothe death of Purkinje cells. Because the channel properties of GluRd2 Lc are similar to those of AMPA or
kainate receptors, it has been concluded that GluRd2
has a channel pore that can be functional at least when
this mutation is present at TM3.19
Interestingly, the lurcher mutation in GluRd2 is ina highly conserved motif (SYTANLAAF [the site of
mutation is underlined]), which is at the end of TM3 in
all iGluRs (Fig. 1B). Moreover, a similar yet rudimentary motif is found in TM2 of the bacterial glutamategated channel GluR020 and the bacterial pH-gated Kþchannel KcsA (Fig. 1B). Thus, this motif may play a
crucial role in the function of iGluRs and may have
evolved with Kþ channels from a common ancestor.21
Recently, the structure of TM2 in KcsA Kþ channels
during gating-associated movement was characterized:
the TM2 helix rotates and tilts away from the permeation pathway22,23 to open the channel gate. Therefore,
coworkers in my laboratory and I hypothesize that thegating of iGluRs is achieved by a similar mechanism
associated with the movement of this motif in TM3.
Indeed, this motif is ideally located, between theagonist-binding pocket and the channel pore, for participation in the transduction of agonist binding tochannel gating (Fig. 1A).
TM3 of iGluRs: to gate or not to gate
To test our hypothesis, we introduced the Ala ! Thr
mutation, i.e., the lurcher mutation, at the corresponding position in an AMPA receptor GluR1 (GluR1 Lc ).
Like GluRd2 Lc , GluR1 Lc formed channels that were94

Yuzaki M: Structure and function of glutamate receptors

A

N

N

LIVBP-likedomainglutamateion flowS2

LAOBP-likedomain

S1

3 4

1
2

“Gating”
CC

C-terminal
tailchannel poreB

TM3lurcher (Lc)

GluR1 IVGGVWWFFTLIIISSYTANLAAFLTV
GluR6 IVGGIWWFFTLIIISSYTANLAAFLTV
NR1
ILGMVWAGFAMIIVASYTANLAAFLVL2
MMMGAWWLFALIVISSYTANLAAFLTI
GluR0 LVAGVWMLVALLSFSSITAGLASAFST
KcsA LVAVVVMVAGITSFGLVTAALATWFVG
gate
Fig. 1 Schematic illustration of the structure of ionotropic glutamate receptors and glutamate-induced conformational changes associated withchannel gating. (A) The LIVBP-like domain is at the N terminus. Two extracellular segments of the LAOBP-like domain (S1 and S2) form theligand-binding pocket of ionotropic glutamate receptors (left). Two lobes formed by S1 and S2 segments close upon ligand binding, which leads
to opening of the ion channel gate (right). Three short segments connect the structure to the transmembrane regions (numbered 1–4). The siteof the mutation in GluRd2 of lurcher mice is in the conserved motif SYTANLAAF in transmembrane domain 3 (TM3) and is indicated by P.
(B) Comparison of the amino acid sequence of GluRd2 with those of representative glutamate receptors and Kþ channels (AMPA receptor
GluR1, kainate receptor GluR6, NMDA receptor NR1, prokaryotic glutamate-gated channel GluR0, and bacterial Kþ channel KcsA). TM3 ofglutamate receptors and of GluR0 and TM2 of KcsA are shown. Letters are shaded according to the percentage of conservation of similaramino acids (100%, 80%, and 60%) in each position. A box indicates the residues in KcsA that rotate during channel opening24,25. The locationof a mutation in GluRd2 of lurcher mice is also indicated by P.

constitutively activated in the absence of ligand binding.19 When glutamate was applied to 293 cells expressing GluR1 (Fig. 2A), rapidly desensitizing currents wererecorded (Fig. 2B, left). Glutamate also activated currents through GluR1 Lc despite constitutive channel
activation (Fig. 2B, right), a ﬁnding suggesting that theconstitutively activated channels were not locked in the
open conﬁguration. However, the mutation severely
slowed the kinetics of ligand-gated channels. We also
observed similar effects of the lurcher mutation on kainate receptor GluR6 and NMDA receptors NR1 andNR2A.19 Furthermore, in the SYTANLAAF motif of
GluR1, we have recently identiﬁed additional residues
that cause lurcher mutation-like effects (Kamiya et al.,submitted) and whose periodicity is characteristic ofamino acids in a-helices (Fig. 3A). These results are
consistent with the view that the gating of iGluRs is
mediated by the structure at the end of TM3, including
the SYTANLAAF motif. According to this model, theend of TM3 forms a-helices that cross one another, andligand binding at the LAOBP-like domain (Fig. 1A)
induces tilting and rotation of the SYTANLAAF motif
in TM3 to open the ion permeation pathway (Fig. 3B).
When the lurcher mutation is introduced into this motif,
the larger side-chain volume of the substituted aminoacid may inhibit the complete closure of the gate evenin the absence of the ligand and may slow the closure of
the gate after the ligand is released (Fig. 3B).
An alternative to our hypothesis about the channelgating mechanisms of iGluRs is that the gating ofiGluRs is achieved by the conformational change in thechannel pore at TM2. These two hypotheses are notmutually exclusive, because the rotation at the end of
TM3 can be allosterically coupled with the conformational change at the channel pore. For example, in thefamily of cyclic nucleotide-gated Kþ channels, ligand
binding-induced rotation of the S6 helices, which correspond to TM3 of iGluRs, is thought to be associatedKeio J Med 2003; 52 (2): 92–99

95A
glutamatetube

293 cells expressing GluR1

B

glutamate application (100 ms)

constitutive current

rapid

GluR1wt

slow kinetics

100 pA
50 ms

GluR1Lc

Fig. 2 Patch-clamp recordings of constitutive and glutamate-induced currents in 293 cells expressing glutamate receptors. (A) Schematic
illustration of the patch-clamp recording. Currents passing through the glutamate receptor under a ﬁxed voltage are monitored by a recording
electrode placed in the 293 cells expressing glutamate receptors. Control solution was gravity-fed into one of the two lumens of the y-shaped
glass tubing; glutamate-containing solution was fed into the other. The cell or a patch of cell membranes was positioned near the interface
formed between continuously ﬂowing control and drug solutions. Solution exchange was made by rapidly moving the y-shaped glass with aPiezo translator. With this system, all of the solution can be exchanged in 200 ms. (B) Typical current responses in 293 cells expressing wild-type
GluR1 (GluR1 wt ) or lurcher GluR1 (GluR1 Lc ). Application of 1 mM glutamate for 100 ms to 293 cells expressing GluR1 wt induced rapidly
inactivated current. In contrast, constitutive current was observed in GluR1 Lc -expressing cells in the absence of ligands. Moreover, the application of glutamate induced further currents with very slow desensitization and deactivation kinetics.

with conformational changes in the channel pore24,25;
these conformational changes open the channel gate.
Clinical implications
Studies of the lurcher mutation provided the ﬁrst
direct evidence that a mutation that affects the gating ofa neurotransmitter-gated channel causes the degeneration of the neurons that express it. Because the therapeutic potential of voltage-gated channel blockers isoften related to their effects on gating,26,27 a drug targeted against the gate-related structure at the end of
TM3 of iGluRs may hold therapeutic potential for diseases associated with excitotoxicity.
The Hotfoot Mutations and Assembly of iGluRs
The hotfoot mutations
Ataxia in the hotfoot mouse ho4J is caused by adeletion that occurs in the GluRd2 gene and resultsin the loss of 170 amino acids from the LIVBP-likedomain (approximately 400 amino acids) (Fig. 4A).28
Unlike the dominantly inherited lurcher allele, the hotfoot alleles carry recessive loss-of-function mutations

that cause ataxia in the absence of obvious Purkinje-celldeath. We found that the mutation in ho4J mice inhibited the function of GluRd2 by impairing its exit fromthe endoplasmic reticulum29 (ER), which was mainly
localized in the Purkinje cell soma (Fig. 4B). In addition
to ho4J, at least 16 independent hotfoot alleles havebeen identiﬁed.30 Interestingly, many of these hotfootalleles also have deletions of nucleotides that code for
40 to 95 amino acids in the LIVBP-like domain of
GluRd2; these deletions also affect the transport of
GluRd2 to the cell surface.31 Coimmunoprecipitation
assays indicated that all these deletions reduced theintermolecular interaction between individual GluRd2
molecules. Thus, the LIVBP-like domain seems to be
crucial for oligomerization of GluRd2. These results
indicate that GluRd2 must be transported to the surfaceof Purkinje cells to function and that the ataxic phenotype of hotfoot mutants is caused by the loss of GluRd2
from the cell surface.
LIVBP-like domain of iGluRs: some assembly required
Unlike the functions of other domains, the functionof the LIVBP-like domain in iGluRs has not been

96

Yuzaki M: Structure and function of glutamate receptors

A

Gating-related region ?
(at the end of TM3)

Channel pore
(TM2)

B

no ligand+ glutamate

washout

ion flow

GluR1wt

ion flow
Lc mutation
GluR1Lc

constitutive current

Slow inactivation

Fig. 3 A model of glutamate-receptor gating. (A) An oblique view of TM3 of GluR1. The position of TM3 is based on coordinates for KcsA
Kþ channels. In the region possessing the lurcher mutation, the red areas are the sites of substituted amino acids with larger side chains than theoriginal residues. A channel pore made by TM2 is also shown. (B) A potential arrangement of the TM3 helices in the closed and open conformation. This model is based on the KcsA Kþ gating model. Shown are top views of TM3 helices of glutamate receptors in the closed state
without ligand binding (left panels), the open state during glutamate application (middle panels), and the deactivation state after the ligand iswashed away (right panels). According to this model, the end of TM3 helices form bundles, the ends of the bundles cross one another, andligand binding at the LAOBP-like domain (Fig. 1A) induces tilting and rotation of the TM3 to open the ion permeation pathway. When thelurcher mutation is present (GluR1 Lc ), the larger side-chain volume of the substituted amino acid residue may inhibit the complete closure of
the gate, even in the absence of the ligand; thus, constitutive currents are produced. Although ligand binding can further rotate the TM3 toallow more ion ﬂow, TM3 cannot rapidly return to its original position because of the large side-chain volume; thus, current inactivation in
GluR1 Lc channels is relatively slow.

A

B
N
Deleted in
ho4J

LIVBP-likedomain

TM1

TM4
TM3
TM2

C

anti-calbindinanti-GluR 2

Keio J Med 2003; 52 (2): 92–99

well characterized. As the name suggests, LIVBP is abacterial periplasmic amino acid-binding protein, likeLAOBP. Indeed, in the glutamate receptors of themetabotropic family, the LIVBP-like domain serves asa ligand-binding domain.32 In contrast, the LIVBP-likedomain is important in the subtype-speciﬁc assemblyof the GluR1.33 This domain is also important in theassociation between NR1 and NR2A.34 Interestingly,
LIVBP-like domains of various receptors, including
those of GluR435 and metabotropic glutamate receptors,32 tend to dimerize with each other. These ﬁndings
and the reduced intermolecular interaction between
hotfoot GluRd2 suggest that a major function of thisdomain in iGluRs is oligomerization of subunits. We
hypothesize that deletions in the LIVBP-like domain
of GluRd2 in hotfoot mice correspond to the interface
for dimerization or the structure that underlies theinterface.
To examine whether this hypothesis is applicable toother iGluRs, we introduced a 170-amino acid deletion,i.e., the ho4J mutation, at the corresponding position in
the AMPA receptor GluR1 (GluR1 ho4J ). Glutamate

97

activated no current in 293 cells expressing GluR1 ho4J
(Fig. 5A). Like GluRd2 with a hotfoot mutation, thismutation inhibited the function of GluR1 by impairing its exit from the ER. Coimmunoprecipitation assays
further indicated that these deletions reduced the intermolecular interaction between individual GluR1 molecules. These results suggested that the hotfoot region
in the LIVBP-like domain of other iGluRs is involvedin subunit oligomerization and subsequent exit fromthe ER.
The mechanisms by which GluRd2 ho4J and GluR1 ho4J
are retained in the ER are unclear. Although Nglycosylation of the N-terminal region of glycine receptors and nicotinic acetylcholine receptors is critical for
their oligomerization and their suubsequent exit fromthe ER,36,37 it is not the case with iGluRs.29 It is possible that a quality control mechanism detects the conformation of the N-terminal region of the oligomer.
If the assembled subunits are unstable because of an
incompatible LIVBP-like domain, the oligomer may beretained in the ER (Fig. 5B). Alternatively, the LIVBPlike domain of iGluRs may possess a speciﬁc signal

Fig. 5 The effect of the ho4J mutation in GluR1 and a model of the mechanism by which the ho4J mutant receptor is retained in the endoplasmic reticulum (ER). (A) Electrophysiological analysis of wild-type GluR1 (GluR1 wt ) and ho4J GluR1 (GluR1 ho4J ) expressed in 293 cells.
Glutamate-induced currents were recorded by the method described in Fig. 2A. (B) Model of the retention of the ionotropic glutamate receptors (e.g., GluR1) in the ER and the expression of these receptors on the cell surface. Ionotropic glutamate receptors oligomerize in the ER, andthe oligomers are then transported to the cell surface. Oligomers that contain a ho4J subunit are retained in the ER, probably by the ER quality
control mechanism, and are eventually degraded.

Fig. 4 Schematic drawing of the hotfoot mutation ho4J and its effect on GluRd2 localization in Purkinje cells. (A) The ho4J mutation is locatedin the LIVBP-like domain of GluRd2. (B) Confocal microscopic analysis of the retention of GluRd2 in ho4J Purkinje cells. Cerebellar sections
from wild-type (wt) and ho4J heterozygous mice were prepared and stained with anti-GluRd2 antibody and anti-calbindin D28K antibody. In
wild-type mice (left), GluRd2 (green) is located predominantly in ﬁne dendritic spines of Purkinje cells (PC; red) in the molecular layer (ml).
However, in ho4J mice (right), GluRd2 remains in the somas of PCs, thus causing cerebellar ataxia.

98

Yuzaki M: Structure and function of glutamate receptors

recognized by unidentiﬁed protein trafﬁcking mechanisms, as reported recently for GluR1 and GluR2.38 In
this case, the hotfoot deletions may impair the proper
presentation of this motif to the trafﬁcking machinery.
Both views are consistent in that iGluRs are preassembled in the ER and only the oligomers that pass
the control mechanism can be transported to the cellsurface.

affect GluRd2, probably because it is encoded by alarge gene (approximately 1.4 Mb) and its mutation
causes a nonlethal ataxic phenotype. Therefore, we
expect that forward-genetics approaches in which thealkylating agent ethylnitrosurea is used as a mutagen
will result in the production of new types of ataxic
mutant mice and provide further clues to the functionof GluRd2 and iGluRs.

Clinical implications

Acknowledgements: The author thanks current and past members of
his laboratory for helpful discussions. Work in the author’s laboratory
was supported by the NIH grant NS36925, the Cancer Center Support
Grant CA21765, and the American Lebanese Syrian AssociatedCharities.

All iGluRs function as heteromers in vivo.9,10 For
example, AMPA receptors are heteromers that consistof GluR1 through GluR4, and each subunit has a speciﬁc function. A dynamic combination of these subunits
may control the functional heterogeneity of iGluRs invivo, but whether a mechanism prevents the coassemblyof iGluR subunits belonging to different families is still
unclear. Therefore, characterization of the LIVBP-likedomain will provide key insight into the developmental
and tissue-speciﬁc functions of iGluRs. As proposed for
G protein-coupled receptors,39 deﬁning the oligomerization interface and its molecular dynamics could yieldpreviously unidentiﬁed sites that can serve as targets fornovel therapeutic agents. Compounds that modulate
receptor assembly could represent a new class of noncompetitive drugs with distinct selectivity and activity
proﬁles.
It has become increasingly clear that glutamate signaling is mainly modulated in vivo by membrane trafﬁcking processes controlling the expression of receptors
on the cell surface.17 For example, AMPA receptor
trafﬁcking plays a critical role in two prominent examples of synaptic plasticity: long-term potentiation andlong-term depression. Indeed, studies of the hotfoot
mice have demonstrated the importance of the LIVBPlike domain in the function of GluRd2 in vivo. Therefore, further studies are warranted to better characterize the mechanisms of the LIVBP-like domains incontrolling the trafﬁcking of iGluRs.
Concluding Remarks
It is ironic that natural mutations affecting GluRd2,
an enigmatic member of the iGluR family, haverevealed very important clues to two fundamental
issues regarding the structure and function of iGluRs –gating and assembly. In the era in which the wholegenome sequence of several organisms and reverse
genetics are available, these fortuitous discoveries havemade us realize again the power of classic genetic approaches in naturally occurring mutant mice. Perhaps
the lesson we should learn from these mutant mice isthat we should be humble and listen to what nature is
telling us. A large number of spontaneous mutations

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