Glutamate receptors of Drosophila melanogaster: Cloning of a kainate-selective subunit expressed in the central nervous system. (neurotrasmntter/gand-gted ...
Proc. Nati. Acad. Sci. USA Vol. 89, pp. 10484-10488, November 1992 Neurobiology
Glutamate receptors of Drosophila melanogaster: Cloning of a kainate-selective subunit expressed in the central nervous system (neurotrasmntter/gand-gted Ion dc neb/Xenopuu oocyts/Drosophila deveopment/invertebrate)
ANDREAS ULTSCH, CHRISTOPH M. SCHUSTER, BODO LAUBE, PATRICK SCHLOSS, BERTRAM SCHMITT, AND HEINRICH BETZ* Abteilung Neurochemie, Max-Planck-Institut fMr Hirnforschung, Deutschordenstrasse 46, D-6000 Frankfurt/Main 71, Federal Republic of Germany
Communicated by Erwin Neher, July 29, 1992 (received for review May 13, 1992)
i charWe report the solation and f ABSTRACT acterization of cDNAs ening a Drosophila kainate-selective glutamate receptor. The deduced mature 964-residue protein (DGluR-I) is 108,482 Da and exhibits sgn t homology to mnammalian glutamate receptor subunits. Injection of DGluR-I cRNA Into Xenopus oocytes generated kainate-operated ion channels which were blocked by the selective non-N-methyl-Daspartate receptor antagonist 6-cyano-7-nitro-quinoxallnets are differ2,3-dione and philanthotoxn. DGluR-I t entially expressed during Drosophila development and, in late embryogenesis, accumulate in the central nervous system.
Glutamate receptors (GluRs) are the major mediators of excitatory neurotransmission in the vertebrate central nervous system. These receptors play a key role in synaptic plasticity and complex brain functions such as learning and memory and are thought to be causally involved in the pathogenesis of various neurological and neurodegenerative diseases (1-3). Based upon pharmacological and electrophysiological studies, GluRs are currently classified into metabotropic and ionotropic subtypes (2, 4). The ionotropic receptors harbor an intrinsic cation channel and are named after and selectively activated by their agonists N-methyl-Daspartate (NMDA), a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate. cDNAs encoding different ionotropic GluR proteins have been isolated from rodent brain (reviewed in ref. 5); the deduced polypeptides display common structural features characteristic of the ligand-gated ion channel superfamily (6). In invertebrates, glutamate serves as a transmitter at the neuromuscular junction (7, 8). When applied locally, glutamate activates both excitatory (D-type) and inhibitory (Htype) ion channels on locust muscle cells (9) and on larval muscle of the fruit fly Drosophila melanogaster (10). GluRs from invertebrate muscle have also been expressed and characterized in Xenopus oocytes (11). A cDNA encoding a GluR polypeptide expressed in Drosophila muscle has been isolated; this protein (DGluR-II) represents a distantly related homolog of the vertebrate GluR family (12). Little is known about the existence and function of GluRs in the invertebrate central nervous system. Cultured cockroach and locust neurons respond to glutamate, aspartate, and the glutamate agonists kainate and quisqualate (13-15). Three distinct types of GluRs were identified on molluscan neurons, and the effect of glutamate on these neurons was to open either chloride or potassium channels (16). In this report, we describe the structure and characterization of a cDNA encoding a Drosophila GluR subunit (DGIuR-I).t Heterologous expression of this protein results in the formation of homooligomeric ion cannels which are activated by
the glutamatergic agonist kainate. Expression ofthe DGluR-I gene is developmentally regulated and confined to the central nervous system of Drosophila. These data provide the basis for a genetic approach to GluR function in the fruit fly model system.
METHODS Cloning of the Genomk Fragments gDl and gD2. Cloning of the genomic polymerase chain reaction (PCR) product gD1 has been described (12). To generate gD2, PCR was performed on Drosophila genomic DNA with the oligonucleotide primers 5'-ATG-CAG-CAG-GGA-TGC-GAT-ATT-3' (sense, nucleotides 2122-2142) and 5'-GTA-GTA-GAT-CCCGGC-CAC-ATT-G-3' (antisense, nucleotides 2730-2751) (12). This resulted in the amplification of a 796-base-pair (bp) fragment, gD2, which was subcloned into pBluescript (Stratagene) for sequence analysis. Cloning of the Full-Length DGluR-I cDNA. Drosophila cDNA libraries (prepared from late embryonic and early adult tissues) were screened with the gDl probe at moderate stringency (12). A second probe used was a 500-bp EcoRIBamHI fragment (positions 2342-2843), which originated from a single cDNA clone encoding the putative transmembrane region M4. Two cDNA clones, cD1 and cD2, were isolated and sequenced on both strands; cDl covered the sequence from the 5' untranslated region of DGluR-I to a unique internal EcoRI site at positions 2338-2342, cD2 extended from this EcoRI site to the poly(A) tail (see Fig. 1A). The exon containing the EcoRI site was sequenced from genomic DNA, and this confirmed that the two clones were indeed contiguous. Subsequently, this restriction site was used to generate a full-length DGluR-I cDNA construct. Genomc Southern Blot Analysis. Drosophila genomic DNA was digested with restriction endonucleases, resolved in a 0.7% (wt/vol) agarose gel, blotted onto Hybond-N (Amersham), and immobilized by UV crosslinking. The blot was hybridized to a 32P-labeled cDGluR-I fragment (12). Expression in Xenopus Oocytes. Capped cRNA was synthesized in vitro from an Xho I-linearized pSP64T-DGluR-I construct by using SP6 RNA polymerase. Preparation and injection of oocytes were performed as detailed previously (17). Control recordings were performed on uninjected oocytes. RNA Isolation and Northern Blot Analysis. Poly(A)+ RNA was isolated at various developmental stages (FastTrack mRNA isolation kit, Invitrogen), electrophoresed in a 1% agarose/formaldehyde gel (5 pug per lane), and transferred Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4isoxazolepropionate; CNQX, 6-cyano-7-nitro-quinoxaline-2,3-dione; GluR, glutamate receptor; NMDA, N-methyl-D-aspartate. *To whom reprint requests should be addressed. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. M97192).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Neurobiology: Ultsch et al. onto Hybond-N membrane. After staining with 0.04% methylene blue in 0.5 M sodium acetate (pH 5.2), the filter was hybridized to a 32P-labeled DGluR-I cDNA fragment (nucleotides 909-1883) at 650C (12). In Situ Hybridization. DGluR-I transcripts were localized in late embryos by whole-mount in situ hybridization (18) using a 909-bp digoxygenin-labeled cDNA fragment corresponding to nucleotides 1-909.
complete sequence determination allowed the construction of a full-length clone, cDGluR-I. Both gD1 and a second genomic DGluR-I PCR product, gD2, as well as the DGluR-I cDNA, are shown in Fig. 1A. The cDGluR-I cDNA was 3314 bp long (not including the polyadenylate tail), 2973 bp of which encoded the complete sequence of a Drosophila GluR. The corresponding open reading frame defined a polypeptide of 991 amino acids (Fig. 1B), and analysis of the N-terminal stretch of 27 amino acids disclosed features characteristic of a signal peptide (21). The predicted posttranslational cleavage of this signal peptide yields a mature DGluR-I protein of 964 amino acids with a calculated mass of 108,482 Da. Structure of DGluR-I. Hydropathy analysis of the deduced mature DGluR-I protein revealed four putative membranespanning segments (residues 586-605, 651-669, 680-698, and 869-889; Fig. 1B). This arrangement is identical to that of DGluR-II and the mammalian GluR subunits and suggests a conserved transmembrane topology with a large N-terminal gD2 * gD1 *
RESULTS Molecular Cloning of DGluR-I cDNA. As detailed elsewhere (12), a partial genomic DGluR-I sequence, gD1, was originally obtained by performing PCR on Drosophila genomic DNA. This genomic fragment, which encodes transmembrane regions M1-M3 and a short stretch of the putative extracellular domain, was used here to screen embryonic and adult Drosophila cDNA libraries at moderate stringency. The isolation of overlapping cDNA clones and the subsequent
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DGluRt-I1 DGluR-I GluR3 DGluR-II
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FIG. 1. (A) Schematic representation of the full-length DGluR-I cDNA (cDGluR-I) and a part of the corresponding gene (gDGluR-I). The cDNA clones cD1 and cD2 were used to generate cDGluR-I; E represents the unique EcoRI site. A bar denotes the coding region, and four putative transmembrane regions (numbered 1-4), as well as the putative signal peptide, are indicated in black. The partial exon-intron organization of the DGluR-I gene as deduced from the genomic clones gD1 and gD2 is shown above the cDNA; arrows depict PCR primers, and introns (A-D) are symbolized by stippled boxes. (B) Alignment of the DGluR-I, rat AMPA receptor GluR3 (19), and Drosophila muscle-type GluR DGluR-II (12) amino acid sequences. Gaps were introduced to maximize homology (20); amino acid numbering starts with the predicted mature N termini. The signal peptide (SP) and the putative transmembrane regions (M1-M4) are denoted by thick lines above the amino acid sequences. Identical positions are boxed, and the proposed GluR "core" domain (12) is delineated by arrows.
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extracellular domain and four transmembrane regions for all these proteins. Indeed, comparison of the DGluR-I protein with other vertebrate and invertebrate GluR polypeptide sequences disclosed significant homologies (Fig. 1B and Table 1). The highest overall amino acid identity (41-44%) is seen to the GluRi to GluR4 class of rat GluR subunits (19, 26), whereas the invertebrate DGluR-II (12) and Lymnea stagnalis (25) GluR polypeptides are overall 26% and 40% identical, respectively. Low but significant homology (20%) exists to a rat NMDA receptor protein (24). Most amino acid differences are found in the proposed large extracellular region and C-terminal to the putative fourth transmembrane segment. Without these variable regions, the remaining "core" domain (residues 455-897 of DGluR-I in Fig. 1B) displays approximately 57%, 35%, and 53% amino acid identity to the equivalent regions ofthe rat GluR3 (20), DGluR-II (12), and Lymnea (25) GluR proteins, respectively (Table 1). A Single Gene Codes for DGluR-I. Drosophila genomic DNA was singly digested with four restriction endonucleases. Southern analysis with a mapped DGluR-I cDNA fragment (nucleotides 909-1883) revealed only a single hybridizing fragment for DNA cut with EcoRI ['44.5 kilobases (kb)], HindIII (8.9 kb), or BamHI (3.9 kb) (Fig. 2). Consistent with the presence of a Pst I site in the cDNA probe, two hybridizing fragments were observed for Pst I-cut DNA (0.9 kb and 3.5 kb). These data indicate that a single-copy gene codes for DGluR-I in the haploid Drosophila genome. The cytogenetic localization of the DGluR-I gene was determined by in situ hybridization on salivary gland polytene chromosomes, using a digoxygenin-labeled cDNA probe (27). A single hybridization signal was detected on chromosome 3L at position 65C (data not shown), thus further supporting the notion that only one gene encodes DGluR-I. DGIuR-I Forms a Kalnate-Gated Ion Channel. To investigate whether the DGluR-I subunit could assemble into functional ion channels, RNA was synthesized from the fulllength cDNA and injected into Xenopus oocytes. Under voltage clamp at -100 mV, transient inward currents of up to 10 nA were observed with RNA-injected oocytes in response to 1-100 iLM kainate (n = 15 oocytes; Fig. 3A, I). No response to glutamate (
