Vol. 183, No. 2, 1992 March
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Molecular Cloning of a cDNA Encoding a Novel Member of the Mouse Glutamate Receptor Channel Family Masatoshi Yamazaki1*2,Kazuaki Araki’, Akira Shibata2, and Masayoshi Mishina’* ‘Department of Neuropharmacology, Brain Research Institute, and 2First Department of Internal Medicine, School of Medicine, Niigata University, Niigata 951, Japan Received
February
6, 1992
The primary structure of a novel putative subunit of the mouse glutamate receptor channel, designated as 61, has been deduced by cloning and sequencing the cDNA. The 61 subunit shows 21-25% amino acid sequence identity with previously characterized rodent glutamate receptor channel subunits and thus may represent a new subfamily of the glutamate receptor channel. 0 1992Academic pTe55,Inc.
Glutamate receptor (GluR) channels mediate most of the fast excitatory synaptic transmission in the central nervous system [ 1,2] and play a key role in synaptic plasticity, thought to underlie memory and learning as well as development of the nervous system [3,4].
Furthermore, abnormal activation of GluR channels has been suggested to lead
neuronal cell death observed in various acute and chronic brain disorders such as ischemia, stroke, Alzheimer’s dementia and Huntington’s disease [5,6].
Based on the
pharmacological and electrophysiological properties, GluR channels have been classified into three major subtypes, that is, receptors for kainate, a-amino-3-hydroxy-5-methyl-4isoxazole propionic acid (AMPA) and N-methyl-n-aspartate (NMDA)
[ 1,2]. Numbers
of GluR channel subunits have been characterized by cloning and functional expression of cDNAs [7-171 and these studies reveal a great molecular heterogeneity of the GluR *To whom correspondence should be addressed. Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole GluR, glutamate receptor; NMDA, N-methyl-D-aspartate.
propionic acid;
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channel. Here, we report that the cloning and sequencing of a cDNA encoding a novel putative subunit of the mouse GluR channel, designated as 61. Materials and Methods Total RNA was extracted from ICR mouse forebrain and cerebellum by the guanidine thiocianate method [18], and oligo(dT)- and random-primed cDNA libraries were constructed in hgtl0 as described previously [ 10,161. Screening of a random-primed forebrain cDNA library (-1 x lo5 clones) under low stringency conditions yielded one hybridizationpositive cloneYA91 carrying apart of the coding sequence of the 61 subunit. The screening of cDNA libraries was effected by hybridization at 37°C in the presence of 30% formamide using a mixture of 0.6 kilobase-pair (kb) BumHI, 1.2 kb PstI/EcoRI, 1.5 kb EcoRI and 1.Okb EcoRI fragments from pKCR30 [lo], pKCR24 [ lo], pS 130 and pS121 [Morita et al., submitted], respectively, as probes. The hybridization solution contained 30% formamide, 5 x SSC (1 x SSC = 150 mM NaCl and 25 mM sodium citrate), 250 pg/ml denatured salmon sperm DNA, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine semm albumin, 1% SDS and 32P-labelled probes (-1 x lo6 cpm/ml each). The filters were washed at 45 “C in 0.3 x SSC containing 0.1% SDS. The cDNA insert of hYA91 was subcloned into the EcoRI site of the plasmid pBluescript II SK(-) (Stratagene) to yield the plasmidpYA9 1. A full-length cDNA clone hYA9 l-5 was isolated by screening of a oligo(dT)-primed forebrain cDNA library (-1 x lo5 clones) under high stringency conditions using 2.1 kb EcoRI fragment from pYA91 as a probe. The cDNA insert of LYABl-5 was subclonedintotheEcoRIsiteoftheplasmidpBluescriptIISK(-)(Stratagene) to yield the plasmid pYA91-5. The nucleotide sequence (residues -108 to 3073) of the cDNA clone YA91-5 was determined on both strands by the dideoxy chain termination method [ 191using appropriate primers prepared with an automatic DNA synthesizer (Applied Biosystems); nucleotide residues are numbered in the 5’ to 3’ direction, beginning with the codon specifying the amino-terminal residue of the mature 61 subunit, and the preceding residues are indicated by negative numbers. Analysis of nucleotide and amino acid sequences was carried out using GENETYX software (SDC). Amino acid sequence identity between GluR channel subunits was calculated using Gene Works (IntelliGenetics). Total RNA from mouse forebrain and cerebellum was electrophoresed on a 1.5% agarose gel containing 2.2 M formaldehyde [20] and transferred [21] to nitrocellulose membrane (Schleichen & Schnell) . The hybridization was carried out at 42°C for 20 hr in a solution containing 50% formamide, 50 mM sodium phosphate buffer (pH 6.5), 5 x SSC, 250 pg/ml denatured salmon sperm DNA, 10% dextran sulphate, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin and -1 x 106cpm/ml 32P-labelledprobe. The probe was 0.74 kb BamHI fragment from pYA9 l-5. The blots were washed at 55°C in 0.2 x SSC containg 0.1% SDS. Remits and Discussion Screening under low stringency conditions of mouse forebrain cDNA libraries using mouse GluR al -a4 subunit cDNAs asprobes led to the identification of cDNA encoding 887
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al
51
a2
51
6,
59
a1
116
02
116
61
139
a1
SI
a2
SLIEVVQYDKF
61
RLVTELR#&YF~SEVDlRGF~SF~~
IDH~K~T~VI~DRGLSVL~V~~EKNUQ~AVN----ILTTTEEGVRMSF/-----QD~KKKERL--VVVD VL
DISDRGLSTLIQAVLD1S
185
EKKYQNKAINvGNINNDKKDETvRs~F~-----QD RLGLDbdLQK----VDK~ISHVFTS~TTMKTEE~RVRDTLRRAILL
a1
ELKKERR--vlLD
189 215
263
61
261 295
a1
342
a2
346
61
313
a1
417
a2
421
61
448
(II a2
491
61
521
al
656
a2
660
61
678
a2
501
al
729
a2
133
61
756
al
808
a2
812
61
836
aI
888
a2
86'2 916
61
889
al
L
61
PEQSSHCTSRTLSSGPSSNLPLPLSSSATMPSlQCKHASPNGGLFRPSPVKTPlPMSFQPVPCGVLPEALDTSHCTSI
994
Figure 1. The deduced amino acid sequences of the mouse GluR 61 subunit and alignment with those of the al and a2 subunits. Amino acid residues are numbered beginning with the ammo-terminal residue of the mature subunit and the preceding residues are indicated by negative numbers. Numbers of the amino acid residues at the right-hand end of the individual lines are given. Sets of identical amino acid residues in the homologous region are enclosed. The putative transmembrane segments (Ml-M4) are indicated. The asparagine residues as potentialN-glycosylation sites in the predicted extracellular domain are marked below with asterisks. Consensus phosphorylation sites for Ca2+-calmodulin dependent protein kinase type II in the predicted intracellular domain are marked with open circles. The positions of the point mutations al-K445E [29] and a2-R586Q [2830] affecting the agonist binding and channel properties, respectively, are indicated by arrowheads. The nucleotide sequence data will appear in the DDBJ, EMBL and GeneBank Nucleotide Sequence Database under the accession number D10171. 888
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a novel putative GluR subunit, designated as 61. Figure 1 shows the deduced amino acid sequence of the 61 subunit, in comparison with those of the al and a2 subunits of the AMPA-selective GluR channel [lo].
The initiative methionine is assigned to the first The nucleotide sequence
methionine residue found in a large open reading frame.
surrounding the proposed initiation codon agrees reasonably well with the consensus sequence [22]. Analysis and comparison of the local hydropathicity profile of the 61 subunit suggest the presence of an amino-terminal signal peptide and four putative transmembrane segments (Ml-M4)
in the carboxyl-terminal half of the molecule, as proposed for the
GluR al and a2 subunits [lo].
The proposed mature 61 subunit is composed of 994
amino acids with a calculated molecular weight of 110,440. The mouse 61 subunit shares 21-25% amino acid sequence identity with previously characterized mammalian GluR channel subunits [7-171 (Table 1). The putative transmembrane regions as well as the proposed agonist binding region preceding segment Ml [ 10, 111 are well conserved among GluR subunits. Four of five potential N-glycosylation sites [23] are found in the proposed extracellular domain. The putative cytoplasmic domain between segments M3 and M4 contains potential phosphorylation sites for Caz+-calmodulin dependent protein
Table 1.
Percent amino acid sequence identity between GluR channel subunits
CLSubfamily Mouse (&Rl) Mouse al Mouse a2 Rat GluRC Rat GluRD Rat GluR5-1 Mouse p2 Rat KA-1 Mouse fl Mouse 61
Mouse &lR2) 66
p Subfamiiy
Rat GluRC (GluR3)
Rat GluRD (GluR4)
Rat GluR5-1
MOW
p2 (GluR6)
ysubfamily Rat KA-1
Mouse fl
6 Subfamily
< Subfamily
Mouse 61
Mouse (NZDARl)
64
66
36
31
33
32
25
18
12
70
36
37
31
30
23
20
71
36
38
32
31
22
19
36
36
31
30
23
20
74
38
38
23
21
38
37
21
20
67
24
20
24
20 19
Sequence data are taken from [lOI (mouse al and (x2), [81 (rat GluRC and GluRD), [121 (rat GluRS-l), Morita er al., submitted (mouse PZ), cl31 (rat KA-1). [161 (mouse 112) and [17] (mouse cl). Essentially the same values (differences are within 2%) are obtained for the rat counter&wts of the mouse al, ~2, P2 and cl subunits, that is, the GluRl, GluRB (GluR2). GluR6 and NMDARl subunits, respectively.
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Origin-
2.4-
1.4-
0.24-
Figure 2. Autoradiograph of blot hybridization analysis of total RNA (10 pg each) from mouse forebrain (lane 1) and cerebellum (lane 2) with GluRiSl subunit cDNA as a probe. RNA ladders from Bethesda Research Laboratories were used as size markers (size in kilobases).
kinase type II [24, 251 and protein kinase C [26, 271. The proposed transmembrane topology model is supported by the recent finding that the Ca*+ permeability of AMPAselective GluR channels is determined by the arginine residue 586 in M2 segment of the a2 subunit [28,29]. RNA blot hybridization analysis of total RNA shows two major hybridizable RNA species of -3400 bases and -6200 bases (Fig. 2). The content of the 61 subunit mRNA is comparable between forebrain and cerebellum. Numbers of GluR channel subunits have been characterized by cloning and expression of the cDNAs [7-171. These GluR channel subunits can be classified into the a, p, yand < subfamilies according to amino acid sequence homologies [lo, 16,171 (Table 1). The members within the a, p or y subfamily share about 70% amino acid sequence identity, whereas identity between subunits belonging to different subfamilies are about 20-40%. The newly found 61 subunit shares 21 to 25% amino acid sequence identity with the GluR subunits of the a, p, yand c subfamilies, and thus may represent a novel subfamily of the 890
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GluR channel. The members of the a subfamily form homomeric and heteromeric channels selective for AMPA [7-l 11,whereas the p andysubfamilies represents the kainate-selective GluR channels [ 12-14,161. The recently identified member of the 6 subfamily (the mouse 61 and the rat NMDARl)
forms homomeric GluR channels endowed with most of the
characteristics of the NMDAreceptor channel [ 15,171. The identification of the 61 subunit may imply further molecular diversity of the GluR channel family. Acknowledgments We thank Miss Mutsuko Motokoshi for help in the preparation of the manuscript. This investigation was supported in part by research grants from the Ministry of Education, Science and Culture of Japan, the Institute of Physical and Chemical Research, the Ministry of Health and Welfare of Japan, the Naito Foundation, and the Yujin Memorial Grant. References 1. Mayer, M. L., and Westbrook, G. L. (1987). Prog. Neurobiol. 28, 197-276. 2. Monaghan, D.T., Bridges, R. J., and Cotman, C. W. (1989). AMU. Rev. Pharmacol. Toxicol. 29,365-402. 3. Collingridge, G. L., and Bliss, T. V. P. (1987). Trends Neurosci. 10,288-293. 4. McDonald, J. W., and Johnston, M. V. (1990). Brain Res. Rev. 15,41-70. 5. Choi, D. W. (1988). Neuron 1,623-634. 6. Olney, J. W. (1990). AMU. Rev. Pharmacol. Toxicol. 30,47-71. 7. Hollmann, M., O’Shea-Greenfield, A., Rogers, S. W., and Heinemann, S. (1989). Nature 342, 643-648. 8. Keinanen, K., Wisden, W., Sommer, B., Werner, P., Herb, A., Verdoom, T. A., Sakmann, B., and Seeburg, P. H. (1990). Science 249,556-560. 9. Boulter, J., Hollmann, M., O’Shea-Greenfield, A., Hartley, M., Deneris, E., Maron, C., and Heinemann, S. (1990). Science 249, 1033-1037. 10. Sakimura, K., Bujo, H., Kushiya, E., Araki, K., Yamazaki, M., Yamazaki, M., Meguro, H., Warashina, A., Numa, S., and Mishina, M. (1990). FEBS Lett. 272, 73-80. 11. Nakanishi, N., Shneider, N.A., and Axel, R. (1990). Neuron 5, 569-581. 12. Bettler, B., Boulter, J., Hermans-Borgmeyer, I., O’Shea-Greenfield, A., Deneris, E. S., Moll, C., Borgmeyer, U., Hollmann, M., and Heinemann, S. (1990). Neuron 5,583-595. 13. Werner, P., Voigt, M., Keinanen, K., Wisden, W., and Seeburg, P. H. (1991). Nature 351,742-744. 14. Egebjerg, J., Bettler, B., Hermans-Borgmeyer, I., and Heinemann, S. (1991). Nature 351,745-748. 15. Moriyoshi, K., Masu, M., Ishii, T., Shigemoto, R., Mizuno, N., and Nakanishi, S. (1991). Nature 354,31-37. 891
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