Neuron,

Vol. 5, 583-595, November,

1990, Copyright

0 1990 by Cell Press

Cloning of a Novel Glutamate Receptor Subunit, GluR5: Expression in the Nervous System during Dkelopment Bernhard Bettler: Jim Boulter: Irm Hermans-Borgmeyer: Anne O’Shea-Greenfield: Evan S. DenerisFt Carl Mall: Uwe Borgmeyer,* Michael Hollmann: and Stephen Heinemann* *Molecular Neurobiology Laboratory *Gene Expression Laboratory The Salk Institute for Biological Studies San Diego, California 92138

Summary We have isolated cDNAs encoding a glutamate receptor subunit, designated CIuR5, displaying 40%-41% amino acid identity with the kainate/AMPA receptor subunits CIuRl, GIuR2, CIuR3, and GIuR4. This level of sequence similarity is significantly below the approximately 70% intersubunit identity characteristic of kainate/AMPA receptors. The GIuR5 protein forms homomeric ion channels in Xenopus oocytes that are weakly responsive to L-glutamate. The GluR5 gene is expressed in subsets of neurons throughout the developing and adult central and peripheral nervous systems. During embryogenesis, GluR5 transcripts are detected in areas of neuronal differentiation and synapse formation. introduction The amino acid L-glutamate is a major excitatory neurotransmitter in the mammalian CNS (Monaghan et al., 1989). Receptors for L-glutamate are important for fast synaptic transmission (Tang et al., 1989) and synaptic plasticity (Collingridge and Bliss, 1987). Most likely, synaptic plasticity is achieved by modification of synaptic efficiency, a process triggered by the flow of action potentials through neuronal circuits. Similar or identical mechanisms are expected to generate flexibility in the nervous system both during development and in memory formation processes. Support for this idea derives from the study of glutamate receptors, specifically from the observations that they are involved in developmental plasticity processes (Bear and Singer, 1986; Kleinschmidt et al., 1987; Woo et al., 1987; Lipton and Kater, 1989; Constantine-Paton et al., 1990) and play a role in long-term potentiation, the postulated electrophysiological correlate of memory formation (Collingridge and Bliss, 1987; Kennedy, 1989). Furthermore, the continuous activation of glutamate receptors may contribute to the pathogenesis of diseases such as epilepsy, schizophrenia, Huntington’s disease, and Alzheimer’s disease (Olney, 1989). Molecular genetic studies have demonstrated that the subunits for neurotransmitter receptors are en+ Present address: Center for Neurosciences, serve University, Cleveland, Ohio 44106.

Case Western

Re-

coded by two gene “superfamilies”: the G-proteincoupled receptor superfamily and the ligand-gated ion channel superfamily (Hall, 1987; Barnard et al., 1987; Schofield et al., 1990). The effects of L-glutamate are mediated by both types of receptors (Foster and Fagg, 1984; Sugiyama et al., 1987; Strange, 1988). Clutamate receptors that function through G-proteins and the inositol phosphate turnover are characterized by their selective activation by l-amino-cyclopentyll$dicarboxylate (ACPD). Presently, glutamate receptors mediating their effects through cation-selective ion channels are classified into four pharmacological subtypes based on differences in their agonist selectivity: N-methyl-o-aspartate (NMDA), AMPA (formerly called the quisqualate receptor), kainate (KA), and APB receptors. Our laboratory has used expression cloning and low stringency hybridization techniques to isolate cDNAs encoding four glutamate receptor subunits, GIuRl, GIuR2, GIuR3, and GIuR4 (Hollmann et al., 1989; Boulter et al., 1990; this work). The homologous subunit genes have been isolated by KeinPnen and coworkers (1990). Single protein subunits are sufficient to form functional receptor ion channel complexes that are activated by KA, AMPA, or quisqualate but not by NMDA or APB. These experiments provide direct evidence that KA, AMPA, and quisqualate can activate the same ion channels. Expression studies with the nicotinic acetylcholine (nAChR) and y-aminobutyrate (GABAAR) receptors have also demonstrated that single subunits can form functional ion channels, but that receptor and channel diversity is probably generated by using different subunit combinations (Duvoisin et al., 1989; Papke et al., 1989; Schofield et al., 1990). In line with this concept, combinations of GluRl, R2, R3, and R4 subunits may assemble more efficiently or differ in their channel properties from homomeric receptor-ion channel complexes (Boulter et al., 1990; Kein;inen et al., 1990). Here, we report the isolation and characterization of GIuR5, a polypeptide indicating that there is large sequence diversity among glutamate receptors. We have undertaken an initial in situ hybridization study that compares the subunit GIuR5, representing a new class of glutamate receptor subunits, with GIuR4, a subunit encoding a KAIAMPA receptor. Results GIuR5 Is a Member of the Glutamate Receptor Subunit Gene Family cDNA clones encoding the GluR4, GIuR5-I (Figure I), and GluR5-2 proteins were isolated as described in Experimental Procedures. The KA/AMPA subunit GIuR4 reported here (Figure 2) is virtually identical to the CIuR-D subunit described by KeinHnen et al. (1990). Differences in the amino acid sequences between the

Neuron 584

Expression of Glutamate Receptors in the PNS and CNS 585

Figure 2. Alignment

of Deduced

Amino Acid Sequences

for the Rat Glutamate

Receptor

Subunits

Identical residues in all compared sequences are presented as white letters on a black background. Spaces were introduced to maximize homology. Predicted signal peptides and the four proposed (Hollman et al., 1989) membrane-spanning regions (MSRs I-IV) are indicated. Two arrowheads delimit the region proposed in CluRl (Hollmann et al., 1989) to correspond to the Cys-Cys loop found in other ligand-gated ion channel subunits (Barnard et al., 1987). The hatched line denotes the insertion of 15 amino acid residues found in the GIuR5-1 but not in the GluR5-2 protein. Differences between the amino acid sequence of the GIuR4 subunit reported here and that of GIuR-D (Keininen et al., 1990) occur at GluR4 amino acid positions 433 (S to I) and 713 (I to T), amino acid 1 being the predicted N-terminal residue of the mature GIuR4 protein and amino acids encoding the signal peptide having assigned negative numbers.

GIuR-D and the GluR4 subunits are pointed out in the legend to Figure 2. Translation of the cDNA nucleotide sequence for GIuR5-I predicts a single long open reading frame of 920 amino acid residues (Figure 1). The GIuR5 sequence has overall amino acid sequence identitywith each of the KA/AMPA subunits (Figure 2; Table 1). The 15 amino acid insertion in GIuRS-I is unique among the proteins listed; thus the shorter GluR5-2 variant is the counterpart to the KAiAMPA subunits character-

Figure 1. Nucleotide

and Amino Acid Sequence

ized. Table 1 shows that GIuR5 is thus far the most dissimilar glutamate receptor subunit identified, and the comparison of GIuR5 with the KA/AMPA subunits highlights the most conserved sequence elements (Figure2). Within other ligand-gated ion channel families, the nAChR, the GAE&,R, and the glycine receptor (GlyR) families, the N-terminal extracellular domain is most conserved and the C-terminal sequences diverge between membrane-spanning regions (MSRs) III and IV. In theglutamate receptor subunit genefam-

of a Single cDNA Clone (IRBZO) Encoding the Rat Glutamate

Receptor Subunit CluR5-1

The deduced amino acid sequence is shown above the nucleotide sequence. Cleavage of the assumed signal peptide is predicted at amino acid position 1 (van Heijne, 1986). This cleavage site is after a proline residue, which is atypical. Amino acids encoding the signal peptide were assigned negative numbers. Nucleotides are numbered in the 5’ to 3’ direction, beginning with the first residue of the codon for the putative N-terminal residue of the mature protein. The nucleotides on the 5’side of amino acid residue 1 are indicated by negative numbers. Stop codons flanking the open reading frame are denoted END. The polyadenylation signal is underlined. Sites of potential N-linked glycosylation are indicated by asterisks. Two arrowheads delimit the insertion of 45 nucleotides between nucleotides 1113 and 1159 found in LRB20, but not in 12 of the 29 cDNA clones isolated.

Neuron 586

Table 1. The Percent Amino Acid Sequence Identity among Pairwise Combinations of Members of the Glutamate Receptor Subunit Gene Family.

CluRl GIuR2 GIuR3 CIuR4 CIuR5

GluRl

GluR2

GIuR3

GIuR4

GIuR5

100

70 100

69 73 100

68 72 73 100

40 40 41 41 100

The sequences were compared and the percent identity between paired sequences was calculated as described in Experimental Procedures.

Figure 3. Hydropathy

ily, in contrast, the regions N-terminal of the proposed MSR I (Hollmann et al., 1989) have only 17% identity and are less similar than the regions C-terminal of MSR I, which have 45% identity. The Cys-Cys loop, a signature for ligand-gated neurotransmitter receptor channel complexes (Barnard et al., 1987), is not conserved in the glutamate receptor subunit gene family (Figure 2). The C-terminal halves of glutamate receptor subunits are suggested to be involved in channel formation and contain the postulated membranespanning helices (MSRs I-IV; Figure 2). The presumed MSR III is the most conserved continuous sequence, with only one conservative amino acid exchange (Val to Ile) in the GIuR5 protein (Figure 2). As mentioned above, in other ligand-gated channel families the segment between MSRs III and IV is divergent in length and sequence. In the glutamate receptor subunit family the similarity in this postulated segment is high (48%) and only GIuR5 exhibits a sequence length variation. The KAIAMPA receptors and the GIuR5 protein are generally divergent C-terminal of the proposed MSR IV. The hydrophobicity plot for GIuR5 (Figure 3) is similar to those for the KA/AMPA receptors, suggesting a conserved secondary structure in the proposed ion channel forming portion of the protein. However, the N-terminal half of the GIuR5 hydrophobicity plot is unusual. In this region, GIuR5, compared with the KAIAMPA subunits, is more hydrophobic and contains several segments that could span the membrane. Based on algorithms that search for membrane-associated helices, four (Rao and Argos, 1986) or seven (Eisenberg et al., 1984) putative transmembrane regions can be assigned to GIuR5 (Figure 3, bars). In GIuR5, the region homologous to the proposed MSR I in GluRl contains amino acid substitutions (Gly to His, Leu to Tyr; Figure 2) that decrease its average hydrophobicity, and the assignment of MSR properties to this segment is questionable, as it is not predicted by either of the algorithms used. However, a proline residue that is conserved in the MSR I (Barnard et al., 1987) of the nAChR (position 12), the GABAAR (position 8), and the GlyR (position 8) is maintained in the putative MSR I of all the glutamate receptor subunits (position 12, corresponding to amino acid residue 501 in GluRS-1). Finally, a comparison of the C-ter-

Plot of GIuR4 and GIuR5-1

The algorithm of Kyte and Dolittle (1982) was used with a window setting of 19. The hydropathy plot of GIuR4 is typical for the KA/AMPA subunits characterized thus far. The calculation was done using sequence analysis programs from the University of Wisconsin Genetics Computer Group (Devereux et al., 1984). The black bars mark predicted transmembrane regions (Eisenberg et al., 1984; Rao and Argos, 1986) corresponding to the proposed MSRs I-IV of GluRl (Hollmann et al., 1989). Open bars indicate additional tentative MSRs in the GIuR5 protein. The program of Rao and Argos (1986) identifies four MSRs encoded at amino acid positions Gly Zss_Ser30s,,te548_Thr570,Thr6’8..Vat636, and Asr?-Glyg5. The program of Eisenberg et al. (1984) predicts seven membrane-associated helices encoded at the amino acid positions Leu70-Gln90, Ilez13-Phez33, Gly285-Ser305, Asps4’-Ala 567, Va1586-Gln606, llebz4-LeuM4, and Ile~s-Gly825. The segment specified by a hatched bar, aligning with the proposed MSR I of GIuRl, is not predicted to span the membrane by those programs.

minal regions of all five glutamate receptor subunits with the frog (Gregor et al., 1989) and chicken (Wada et al., 1989) KA binding proteins demonstrates a similar extent of sequence conservation (35%-40% amino acid identity). A FASTA search (Pearson and Lipman, 1988) of the GenBank, EMBL, and SWISS-PROT databases with the GluR5 sequence uncovered no significant similarities to other proteins. Oocytes Injected with CIuR5 cRNA Respond to L-Glutamate Application In vitro synthesized GIuR5-1 and GIuR5-2 cRNAs were individually expressed in Xenopus oocytes. With either cRNA, the glutamate receptor agonists aspartate (100 PM), AMPA(50 PM), quisqualate (IO PM), APB (100 PM), and NMDA (100 uM, applied with 10 KM glycine) did not elicit membrane depolarizations (data not shown). However, we have recorded weak membrane depolarizations induced by L-glutamate (100 uM) in oocytes injected with GIuR5-I cRNA (maximal depolarization 3.5 mV) and GluR5-2 cRNA (maximal depolarization 4.5 mV; Figure 4). We were not able to find significantly stronger membrane depolarizations in response to L-glutamate in oocytes coinjected with GIuR5-1 and GluR5-2 cRNA as compared with oocytes injected with GIuR5-2 cRNA alone (data not shown). For any particular oocyte injected with GIuR5-I or GluR5-2 cRNA, the depolarizations were reproduc-

Expression of Glutamate Receptors in the PNS and CNS 587

1 GIuRS-1

1 GluRS-2

-

$

G---1Mh Figure 4. Voltage Recording Traces Obtained

in Xenopus Oocytes

The best responses induced by 100 uM L-glutamate measured 2-3 days after injection of 2 ng of cRNA are shown. The time point of L-glutamate application is indicated by an arrow. Potential measurements were monitored on a digital voltmeter and recorded on a Gould pen recorder. The resting membrane potentials of the oocytes from which recordings are shown were -72 (CIuRS-1) and -77 mV (GIuRS-2). Voltage traces were scanned for publication on a Macintosh Computer.

ible, showed fast onset, and were slowly (within 5 min) reversed when agonist superfusion was switched to buffer super-fusion. Neither uninjected oocytes nor water-injected oocytes showed a response to the glutamate receptor agonists tested. Responses to L-glutamate were recorded in 7 (out of 7) oocytes for GluR5-1 (membrane depolarization 2.29 f 0.26 mV SEM) and 29 (out of 33) oocytes for GIuR5-2 (2.27 f 0.19 mV SEM). The Genes for CIuR5 and the KAIAMPA Subunit GIuR4 Are Expressed in the Developing Central and Peripheral Nervous Systems For the developmental study of GIuR4and GluR5 gene expression, we have analyzed sections of mice from embryonic day IO (EIO) through postnatal day21 (P21). Autoradiographs from E12, E14, E16, P3, and PI2 are shown in Figure 5, and the complete analysis is presented in Figure 6. In the entire CNS, a diffuse expression of the GIuR4 and GIuR5 genes is detected at EIO. These first hybridization signals originate from postmitotic neurons. This is best demonstrated in the myelencephalon at El2 (Figures 5a and 5b). The ependymal layer is facing the neural canal and contains dividing neuroblasts. No hybridization is detectable in these cells. The postmitotic cells are located in the exterior part of the neural tube and express both genes. later in development, transcripts for CluR5 and, to a lesser extent, GIuR4 are particularly pronounced in areas where neuronsdifferentiateand assemble into nuclei. These temporal changes in the hybridization pattern are best observed for CIuR5 in the primary sensory nuclei of the medulla oblongata and the nuclei of the pons, which hybridize more intensely than surrounding structures (Figures 5c-5f). At E14, GIuR5 gene expression is particularly intense in several discrete brain nuclei, whereas GIuR4 gene expression is detectable over the entire rostra1 and caudal parts of the brain. During postnatal development, the spatial distribution of GIuR4 gene transcripts does not change, but

usually smaller amounts of mRNA than those seen at later embryonic stages are detectable. In contrast, GIuR5 gene expression appears to become more restricted spatially during development, and transcript levels are down-regulated. Extreme changes in the temporal GIuR5 hybridization pattern are apparent in the cerebellar cortex. Until P12, high GIuR5 transcript levels are detected in the granular and Purkinje cell layer. Later, the intensity of hybridization signals in the granular cell layer is reduced relative to the Purkinje cell layer, and starting at P14, only a faint hybridization signal is detected in the granular cell layer (see Figure 6b). In general, those regions of the brain that exhibit a dense labeling during embryonic development also have detectable transcript levels in adults. In P21 animals the highest GIuR4 transcript levels are observed in the cell layers of the olfactory bulb, the hippocampus, the cerebellum, and the retina. In the retina, strong hybridization is found in the ganglion cell layer and in the amacrine cells of the inner nuclear layer. No expression is detected in Muller cells (Figure 5). For GluR5, the strongest hybridization signals at P21 are found in the olfactory bulb, the amygdala, the colliculi, and some hypothalamic nuclei. In the developing PNS, the GIuR4 and GIuR5 genes are expressed to varying degrees in the cranial ganglia (e.g., trigeminal ganglion and acoustic ganglia), the dorsal root ganglia, and the mural ganglia of the intestinal organs (Figure 5). Comparable to the CNS, transcripts in the PNS are detected by El0 for GIuR4 and by El1 for GIuRL During development, hybridization signals for GIuR4 continuously increase until early postnatal stages and then persist with similar intensity in adults. Hybridization signals for GluR5 increase up to El6 and remain with comparable intensity in later developmental stages. In postnatal animals, the dorsal root ganglia (GIuR5) and the mural ganglia of the intestinal organs (GluR4and R5) exhibit higher levels of hybridization than those seen in the CNS. High resolution autoradiography in the dorsal root ganglia demonstrates hybridization of the GIuR5 probe over neuronal cells, whereas satellite cells are unlabeled (Figure 5). Expression of the Genes for GIuR4 and GIuR5 in Adult Rat Brain The distribution of the GIuR4 and GIuR5 transcripts in the adult CNS was studied by in situ hybridization. Coronal sections through the hippocampal region and the cerebellum are shown in Figure 7. In the forebrain region, high levels of GIuR4 transcripts are detected in the CA1 and the dentate gyrus of the hippocampus, in the medial habenula, and particularly in the reticular thalamic nucleus. The hippocampus shows only weak expression of the GIuR5 gene, and no transcripts are detected in the medial habenula. The GIuR5 hybridization signal is intense in the cingulate and piriform cortex, several hypothalamic nuclei, the amygdala, and the lateral septum. In the cerebellum, the hybridization patterns for GIuR4 and GIuR5

b

M

SC

E12; GluR5

E12; GluR4

d

SC

E14; GluR4

e

c dTh

E14: GluR5

Th M Cb

0 /

E16; GluR4

--?

E16; GluR5

.__ -

Figure 5. Localization of the GIuR4 and GIuR5 Gene Transcripts during Embryonic and Postnatal Development ization

Using In Situ Hybrid-

(a-f) Embryonic development; (g-m) postnatal development. Ventral surface to the left; dorsal surface to the right. The schematic drawings (60% of original size) indicate the plane of the sections. Sections shown were exposed for 10 days. Photoemulsion-dipped sections at higher magnification (I and m) demonstrate the neuronal distribution of GIuR4 mRNA in the retina (I) and GIuR5 mRNA in the dorsal root ganglia(m); top is bright-field; bottom is dark-field. Abbreviations: C, cortex; Cb, cerebellum; CbC, cerebellar cortex; CbN, cerebellar nuclei; CNG, cranial nerve ganglion; D, diencephalon; DRC, dorsal root ganglia; dTh, dorsal thalamus; ETh, epithalamus; CCL, ganglion cell layer; H, hippocampus; INL, inner nuclear layer; M, mesencephalon; Me, metencephalon; MC, mural ganglia of internal organs; MN, mesencephalic nuclei; MO, medulla oblongata; My, myelencephalon; 0, olfactory bulb; P, pons; R, retina; S, anterior septal area; SC, spinal cord; SN, primary sensory nuclei; St, striatum; T, telencephalon; Th, thalamus, V, vertebral bone tissue.

Expression of Glutamate Receptors in the PNS and CNS 509

P3; GluR4

-

k

i

P12: GluR4

P3; GluR5

P12; GluR5

m

.

._

t

“-;-P~

NeUrOn

590

GluR4

a El0

El1

El2

El4

E 1 fj

El8

1’0

1’3

1’7

I’12

I’ I .1

I’.) I

CNS Retina Telencephalon Olfactory bulb Isocortex I’iriform Cortex Hippocampus CA1 CA2 Dentate Cyrus Subiculum Amygdala %ialum Septum Dlenrephalon Epithalamus Medial Habenula Lateral Habenula Thalamlr Nuclei Dorsal Thai. Nurlel Ventral Thal. Nuclei Hypothalamus Mesenc-ephalon Tertum Mesenc. Nurlel !Gtenrephalon Cerebellum Cerebellar Cortex Molecular Layer I’urkinje Cell Layer Granular Cell Layer Cerebellar Nuclei I’ontine Nuclei Myelencephalon Medulla oblongata Medulla spmalis PNS

Figure 6. Distribution

of GIuR4 and GIuR5 mRNA Expression during Embryonic and Postnatal Development

in the Mouse

Nervous

System The GIuR4 (a) intensity scale is not comparable to the CIuR5 (b) scale. The strongest hybridization signal observed in a particular morphological structure defines the score assigned to this structure. The subdivisions into the morphological structures indicated were done only at developmental stages and in sections where they were clearly distinguishable. Relative density of in situ hybridization is indicated as follows: black, strong signal; dark gray, moderate signal; light gray, weak signal; white, not detected; hatched, not determined. Regions not listed should not be considered as nonexpressing

probes are overlapping but distinct. Both probes are detected at high levels in the Purkinje cell layer. In the granular cell layer the GIuR4 probe produces strong labeling, whereas GIuR5 probe labeling is weak. Discussion A Second Class of Glutamate Receptor Subunits CIuR5 has 40% sequence identity with the KAIAMPA subunits. Members within the KA/AMPA subunit class display approximately 70% sequence identity (Table 1). The GABAAR subunits a and B share with the y2 and 6 subunits 35% sequence conservation (Shivers et al., 1989). By analogy to the GABA* neurotransmitter system, the degree of sequence conservation between GluR5 and WVAMPA receptors defines a new

class of glutamate receptor subunits. The GIuR5 subunit is not a unique member of this class. Our data indicate additional subunits with sequences closely related to that of GIuR5 (Bettler et al., unpublished data). The highest amino acid similarity between GIuR5 and the UAIAMPA receptors is in the region postulated to participate in ion channel formation (Figure 2; Figure 3). However, the seven possible MSRs of GIuR5 (Figure 3) are also suggestive of a C-proteincoupled glutamate receptor. Electrophysiological Properties of the CIuR5 Protein Expressed in Xenopus Oocytes Weak L-glutamate-induced membrane depolarizations characteristic of an ion channel were observed in oocytes injected with GIuR5 cRNA (Figure 4). Quis-

Expression of Glutamate Receptors in the PNS and CNS 591

GluR5

b El0

El1

El2

El4

El6

El8

PO

P3

I’7

1’1 2

I’1 4

1’2 I

CNS Retina Telencephalon OlfactoIy bulb lsocortex Piriform Cortex Hippocampus CA1 CA3 Dentate Gyrus Sublculum Amygdala Striatum Septum Diencephalon Epithalamus Medial Habenula Lateral Habenula Thalamic Nuclei Dorsal Thai. Nuclei Ventral Thai. Nuclei Hypothalamus .Mesenrephalon Tertum Mcsenc. Nuclei

^___...

^

Metrnrephalon Cerebellum Cerebellar Cortex Molecular Layer Purkinje Cell Layer Granular Cell Layer Cercbellar Nuclei Pontine Nurlel .?Jyelencephalon Medulla oblongata Medulla spinalis PNS Cranial Nerve Gangha Spinal Nerve Ganglia Mural Ganglia

qualate, an agonist for the G-protein-coupled ACPD receptor (Sugiyama et al., 1987), does not evoke a response. Since the small responses recorded in oocytes injected with GIuR5-1 and GluR5-2 cRNA are elicited only by L-glutamate, our experiments do not allow us to assign them to an established pharmacological subtype. In initial experiments, we were not able to detect novel pharmacological properties in oocytes injected with pairwise combinations of cRNAs encoding GIuR5 and one of the KAIAMPA subunits, GIuRl, GIuR2, or GIuRA There are several possible explanations for the weak depolarizations recorded in oocytes injected with GIuR5 cRNA. First, the GIuR5 proteins might require the presence of additional subunits to assemble efficiently into receptor-ion channel complexes (Boulter et al., 1990; KeinPnen et al., 1990). These additional subunits might confer sensitivity to one of the typical glutamate-related agonists. Second, it is conceivable that the GIuR5 protein forms ion channels that desensitize very rapidly. Such glutamate receptors have, for example, been described in the C-fibers

of the dorsal root ganglia (Huettner, 1990). Third, the polypeptide presented here may also participate in the formation of a neurotransmitter-gated ion channel system that is only weakly activated by the L-glutamate-related agonists tested. Glutamate receptors with novel specificities for L-glutamate agonists have been described (Aas et al., 1989). Fourth, it is possible that a receptor expressed in the oocyte system does not undergo posttranslational modifications as it would in neuronal cells and is therefore not completely activated upon agonist application. We have found, for example, potential sites for protein kinase C in the putative intracellular domain of the GluR5-1 protein (amino acid residues 695 and 731), and there is evidence that phosphorylation can modulate the activity of glutamate receptors (Kennedy, 1989). Distribution of Glutamate Receptors in the PNS Little is known about glutamate receptors in the PNS. In immature animals, KA receptors have been described in the C-fibers of the dorsal root ganglia (Agrawal and Evans, 1986), and a substantial propor-

NWVXl

592

GluR5

. ,; I

a

:

Figure 7. Localization of the CIuR4 and GIuR5 Gene Transcripts in the Adult Rat Brain Using In Situ Hybridization Coronal sections through the hippocampal region (left column) and the cerebellum (right column) are shown. Abbreviations: Amg, amygdala; CA1 and CA3, regions in the hippocampus;CbN, cerebellar nuclei; Ci, cingulate cortex; CP, caudate-putamen; DC, dentate gyrus; C, granular cell layer; Hyp, hypothalamus; M, molecular layer; mH, medial habenula, P, Purkinje cell layer; Pir, piriform cortex; rN, reticular thalamic nucleus; S, septal area; Th, thalamus.

tion of dorsal root ganglion neurons from adult rats express NMDA receptors (Lovinger and Weight, 1988). Two reports demonstrate the existence of NMDA receptors in the myenteric plexus of the guinea pig ileum (Moroni et al., 1986; Shannon and Sawyer, l989), and glutamate receptors with a novel pharmacological profile are found in bronchial smooth muscle (Aas et al., 1989). [aH]glutamate binding sites with characteristics of an NMDA receptor are expressed in adrenal glands (Yoneda and Ogita, 1986). No reports are available demonstrating glutamate receptors in the trigeminal ganglion and acoustic ganglia. However, gene transcripts of GIuR5 and the KA/AMPA subunit CIuR4are detected in these regions. Except in the dorsal root ganglia, it appears therefore that the CIuR5 gene expression follows none of the distribution patterns so far described for peripheral glutamate receptors. Molecular genetic studies have demonstrated that the diversity of nAChR, GABAAR, and GlyR is greater than had been anticipated from biochemical, pharmacological, and physiological studies. A similar picture is emerging for glutamate receptors. The KA/AMPA subunit genes GIuRl, R2, R3, and R4 demonstrate that there is subtype heterogeneity and functional diversity within glutamate receptors. This diversity, combined with the possibility that different subtypes of glutamate receptors might share the same subunit,

provides one explanation for the observation that the GluR5 expression pattern does not clearly conform to the distribution scheme determined for a particular glutamate receptor subtype. GluR5 could also represent a previously uncharacterized subtype of glutamate receptors. The GIuR4 and GIuR5 Genes Are Expressed in Areas Where Neuronal Differentiation and Synapse Formation Are Taking Place The expression of the GIuR4 and GIuR5 genes is detected during periods of intense cellular differentiation. Diffuse expression of GIuR4 and GIuR5 transcripts in the entire CNS is first observed at EIO. At this stage of development, the five major brain areas, tel-, di-, mes-, met-, and myelencephalon, have already formed (Rugh, 1968). Phylogenetically old brain structures start to differentiate earlier in ontogeny than the more recently evolved structures (Purves and Lichtman, 1985). Reflecting this scheme, primitive brain regions are first to accumulate CIuR4 and GIuR5 transcripts, thus establishing a rostra1 to caudal gradient of increasing labeling intensity (Figure 5; Figure 6). The highest transcript levels of the GIuR4 and GluR5 genes are found in areas where synaptogenesis is in progress. In the thalamus, for example, GIuR4 and GluR5 transcripts are first detected at E16, when neuronal differentiation in this structure begins (Figure 5;

Expression of Glutamate Receptors in the PNS and CNS 593

Niimi et al., 1962). Some parts of the CNS, such as the olfactory bulb, the dentate gyrus of the hippocampus, and the cerebellum, undergo neurogenesis postnatally (Purves and Lichtman, 1985). In the olfactory bulb, new synapses are established throughout life, since receptor cells in the mucosa are continuously replaced (Purves and Lichtman, 1985). In all these areas, high levels of CIuR4 and GIuR5 transcripts are found both during development and in mature animals. An exception is the dentate gyrus, where GIuR5 transcripts are detected only weakly in adults (Figure 7). In the PNS, where GIuR4 and GIuR5 are expressed during embryonic and postnatal development, synaptogenesis and rearrangement of synapses occur during postnatal development as well as in adult animals (Purves and Lichtman, 1985). In summary, the temporal and spatial expression patterns of the GluR4and GIuR5 genes are compatible with their gene products playing a role during neuronal development. The Expression Pattern for GIuR5 in Adult Rat Brain Is Different from That of CIuRl, R2, R3, and R4 Although the GIuR5 gene expression pattern is largely overlapping with the KAIAMPA subunit patterns (Boulter et al., 1990; KeinHnen et al., 1990), significant differences in regional hybridization intensities are apparent. These differences are most notable in the hippocampal region (Figure 7). The GIuRl, R2, and R3 probes produce strong labeling throughout Ammon’s horn. GIuR4 transcript levels are high in CA1 but diminish in CA3. In contrast, GIuR5 transcripts, albeit observable throughout Ammon’s horn, are not abundant in the hippocampus or in the medial habenula of the epithalamus and in the caudate-putamen. In general, lower hybridization signal intensities, reflecting differences in the abundance or stability of the mRNAs, and more restricted transcript distributions are observed with GIuR5 probe than with the KA/ AMPA subunit probes. Conclusions The identification of GluR5 brings the number of members in the glutamate receptor subunit gene family to five and leads to the description of a second class of glutamate receptor subunits. As with the KA/AMPA subunits, the class represented by GIuR5 has no sequence identity and little conserved structural characteristics in common with other ligandgated ion channels. This casts doubt on the proposal that all ligand-gated channels are evolutionary related. The GIuR5 protein is able to form a glutamate-gated ion channel in Xenopus oocytes. The fact that oocytes expressing GIuR5 are responsive to L-glutamate but not to quisqualate or AMPA might indicate that this protein can participate in the formation of receptors with a different pharmacological profile than those formed by the KAIAMPA subunits. This hypothesis is supported by the in situ hybridization study, During embryonic and postnatal development, the CIuR5 gene is expressed in subsets of neuronal cells in the

CNS and PNS and the spatial and temporal expression pattern of the GIuR5 gene is largely overlapping with that of the KAIAMPA subunit GIuR4. However, in adult brains, the GIuR5 gene is expressed in a pattern distinct from those of the KAIAMPA subunit genes, consistent with the hypothesis that GIuR5 represents a subtype of glutamate receptors different from the KAIAMPA receptors. Experimental Procedures Isolation of the cDNAs for CIuR4 and GluR5 Using a fragment of the GluRL cDNA (Boulter et al., 1990) as probe and a low stringency hybridization protocol, several GIuR4 and GIuR5 clones were isolated from a rat forebrain library. Sequence analysis demonstrated that none of the cDNA clones contained the entire open reading frame. Northern blots with mRNA from different adult rat brain tissues indicated that the GluR4 and CIuR5 transcripts were most abundant in the cerebellum. Consequently, using the partial CIuR4 and GIuR5 sequences as probes and high stringency screening conditions, cDNAs encoding large open reading frames were isolated from an adult rat cerebellum cDNA library. The rat forebrain and cerebellum cDNA libraries were constructed with the lambda ZAPII system (Stratagene). The 32P-labeled probe used for the low stringency screening was derived from the GIuR2 cDNA clone LRB14 (Boulter et al., 1990). Plaques (106) of each library were plated. The hybridization solution used was 1 M NaCI, 50 mM Tris (pH 8.0), 0.5% SDS, 0.1% sodium pyrophosphate, 100 pglml denatured herring sperm DNA, 0.1% (w/v) each of Ficoll, polyvinylpyrrolidone, and bovine serum albumin. For low stringency screening, the hybridization temperature was 50°C and the filters were washed in 2x SSPE (lx SSPE is 180 mM NaCI, 9 mM Na*HPO,, 0.9 mM NaH2P04, 1 mM EDTA [pH Z4]) containing 0.5% SDS at room temperature. For high stringency screening, the temperature was adjusted to 65°C and the filters were washed at this temperature in 0.2x SSPE containing 0.5% SDS. Filters were exposed for 18-48 hr. cDNA clones encoding the GIuR4 gene were isolated as described above. Two such cDNA clones were obtained (ILERIIZ and XER121B); these encoded only portions of the GluR4 gene but possessed sufficient overlap to engineer a full-length, expressible construct in the PBS SK (+) vector (Stratagene Cloning Sytems). The nucleotide sequence of this construct, designated pK45, was determined and the deduced amino acid sequence is presented in Figure 2. The amino acid sequence of the protein encoded by the CIuR4 subunit gene is virtually identical to that reported by Keina’nen et al. (1990) for CIuR-D. Among the 29 GIuR5 cDNAs isolated from the cerebellum library we have identified three clones, specified IRB12, hRB15, and LRB20, encoding an identical large open reading frame. The sequence of cDNA clone hRB20 is shown in Figure 1. LRB15 and LRB12 are shorter than hRB20 at the 5’ end. The ;IRB20 cDNA consists of a 187 bp 5’ untranslated region, a continuous open reading frame of 2760 bp, and a 3’untranslated region of 303 bp. The 5’ untranslated region ends with the sequence AAGATGC, which is characteristic of a translational start site (Kozak, 1983). We have examined three additional cDNA clones that were originally isolated from the forebrain library. The sequences of these cDNAs are identical to IRB20 in the predicted translation initiation site region. Sequence analysis revealed that two variants of the GIuR5 cDNA are represented in the forebrain and the cerebellum libraries. This heterogeneity derives from an insertion of 45 nucleotides found in hRB20 and 17 of the 29 cDNA clones isolated (Figure 1). This insertion does not interrupt the open reading frame. Furthermore, consensus splice donor and acceptor sites (Breathnach and Chambon, 1981) are absent, which suggests that the insertion does not arise from an unspliced intron and is, most likely, the result of an alternative splice event.

NelIrfJfl 594

Nucleotide sequence analysis indicates that the two CIuR5 variants are otherwise identical. We did not find a cDNA clone of the shorter splice variant encoding the entire open reading frame. Expression Studies in Xenopus Oocytes The preparation of oocytes, the in vitro synthesis of RNA transcripts from the cloned cDNA, and the methods used for electrophysiology have been reported (Boulter et al., 1987; Hollmann et al., 1989). For the expression studies, we have constructed the cDNA clone lRBD20 (GluRS-2), which does not contain the 45 nucleotide insertion (positions 1203-1248) in hRB20 (GIuR5-1; Figure 1). lRBD20 is a hybrid clone that has the Pstl-Clal fragment of lRB20 (positions 1136-2234) replaced with the corresponding fragment of hRB418, a cDNA clone lacking the insertion. The lRB20 and ARBDZO cDNAs were linearized with Xhol prior to sense-strand RNA transcription with T7 polymerase. Two nanograms of each cRNAwas injected per oocyte. When combinations of cRNAs were assayed, 2 ng of each cRNA was injected per oocyte. The injected oocytes were incubated for 2-4 days before they were analyzed for the presence of functional receptors. In Situ Hybridization Histochemistry In situ histochemistry on adult rat brains was performed as described (Deneris et al., 1989). For the developmental in situ histochemistry studies, we first assessed the specificity of the rat cRNA probes on mouse sections. In situ hybridized brain sections from P21 mice and adult rats were analyzed, and comparable results were obtained in both species (data not shown). Therefore, we have used anti-sense rat cRNA probes to hybridize sections of mice. Fetal and neonatal animals from natural matings between inbred ICR mice (HSD) were collected at the stages indicated in Figure 6. The occurrence of a vaginal plug was defined as EO, and the day of birth as PO.The animals were frozen on dry ice and IO pm sections were prepared on a cryostat. Prehybridization treatment and hybridization were performed as described (Deneris et al., 1989) except that the proteinase K digestion was omitted and the hybridization buffer contained 4x SSC (Ix SSC is 150 mM NaCl, 15 mM sodium citrate [pH ZO]), 1 mM EDTA, 0.05% tRNA, 0.5 mg/ml salmon sperm DNA, 10% dextran sulfate, lx Denhardt’s solution, and 10 mM dithiothreitol. The hybridized sections were exposed to Hyperfilm Betamax (Amersham) for 3 or 10 days. For higher resolution studies, the same slides were dipped in Kodak NTB-2 nuclear emulsion. Dipped sections were developed in D19 (Kodak) after 4 weeks exposure, and the tissue was subsequently stained with Giemsa. The [3sS]UTP-labeled full-length cRNA probes were derived from the cDNA clone 1RB20 (CIuRS-1). As a control of specificity, a second set of hybridizations was performed using 600-900 nucleotide long anti-sense probes derived from regions N-terminal of MSR I (Figure 2). The hybridization patterns were identical to those obtained with the full-length cRNA probes (data not shown). Morphological structures in embryonic mice sections up to El6 were identified by reference to Rugh (1968). Sequence Alignment and Homology Calculations Amino acid sequences were compared using sequence analysis software from the University of Wisconsin Genetics Computer Group (Devereux et al., 1984). The percent sequence identity between paired sequences was calculated by dividing the number of aligned positions with identical amino acids by the total number of aligned positions in the shortest of the sequences examined and multiplying the quotient by 100. Acknowledgments We would like to thank our colleagues, especially R. Duvoisin, E. Freed, and T. Hughes for help and comments on the manuscript and P. Sawchenko and the members of his laboratory for support and advice. This work was supported in part by postdoctoral fellowships from the Schweizerischer Nationalfond (8. B.), the HSFPO (I. H.-B. and U. B.), the Kommission zur F6rderung des Akademischen Nachwuchses des Kantons Ztirich

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Cloning of a novel glutamate receptor subunit, GluR5: expression in the nervous system during development.

We have isolated cDNAs encoding a glutamate receptor subunit, designated GluR5, displaying 40%-41% amino acid identity with the kainate/AMPA receptor ...
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