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The EMBO Journal vol.8 no.6 pp. 1665 - 1670, 1989
GABAA receptor subunit heterogeneity: functional expression of cloned cDNAs
Sanie Ymer, Peter R.Schofield', Andreas Draguhn2, Pia Werner, Martin Kohier and Peter H.Seeburg3 Laboratory of Molecular Neuroendocrinology, ZMBH, University of Heidelberg, INF 282, D-6900 Heidelberg, FRG 'Present address: Pacific Biotechnology Ltd, 72-76 McLachlan Avenue, Rushcutters Bay 201 1, Australia 2Max-Planck-Institut for Biophysical Chemistry, Am Fassberg, D-3400 Gottingen, FRG 3Corresponding author
Communicated by P.Seeburg
Cloned cDNAs encoding two new (3 subunits of the rat and bovine GABAA receptor have been isolated using a degenerate oligonucleotide probe based on a highly conserved peptide sequence in the second transmembrane domain of GABAA receptor subunits. The (32 and (3 subunits share 72% sequence identity with the previously characterized (31 polypeptide. Northern analysis showed that both (32 and (33 mRNAs are more abundant in the brain than (31 mRNA. All three ( subunit encoding cDNAs were also identified in a library constructed from adrenal medulla RNA. Each ( subunit, when co-expressed in Xenopus oocytes with an a subunit, forms functional GABAA receptors. These results, together with the known a subunit heterogeneity, suggest that a variety of related but functionally distinct GABAA receptor subtypes are generated by different subunit combinations. Key words: GABAA receptor/l8 subunit/receptor subtypes/ molecular cloning/oocyte expression -
affinity labelling (Mohler et al., 1980; Sieghart et al., 1983; Fuchs et al., 1988) studies. However, the '(3 band' was regarded as being homogeneous (Haring et al., 1985; Mamalaki et al., 1987). This band is so far molecularly characterized by only one cloned cDNA that encodes a subunit ((1) with significant sequence similarity to the a subunits and, when co-expressed with these in Xenopus oocytes, produces functional GABAA receptors (Schofield et al., 1987; Levitan et al., 1988). Sequence homology also extends, in part, to other ligand-gated ion channels, reflecting the existence of a receptor superfamily (Schofield et al., 1987; Grenningloh et al., 1987a). Comparison of the polypeptide sequences of the three GABAA receptor a subunits, the (31 subunit as well as the 48 kd subunit of the glycine receptor revealed that the highest sequence identity resides in the four putative transmembrane segments (Ml -M4). In particular, a contiguous sequence of eight amino acids rich in threonines is found in M2 of all these polypeptides (Grenningloh et al., 1987b) which, by analogy to M2 of nicotinic acetylcholine receptor (Imoto et al., 1988; Leonard et al., 1988), is thought to form part of the channel lumen. We have used a highly degenerate oligonucleotide probe encoding this peptide sequence to screen for additional GABAA receptor subunits. We report the isolation of two new ( subunit encoding cDNAs, (32 and (33, from both rat and bovine brain cDNA libraries and show that these new (3 subunits are more abundant in brain than the (31 subunit. Functional expression in Xenopus oocytes demonstrates that these (3 subunits are capable of combining with an a subunit to form GABAA receptor chloride channels.
Results Introduction GABA (-y-aminobutyric acid), the major inhibitory neurotransmitter in the vertebrate brain, mediates neuronal inhibition by opening a chloride channel integral to the GABAA receptor, which is also the target for a variety of therapeutically important drugs (reviewed in Olsen and Venter, 1986). Affinity-purified receptor is electrophoretically resolved into two major bands (at, 48-53 kd and 55-57 kd). The smaller band can be photoaffinitylabelled by benzodiazepine derivatives and the larger one by GABA agonists (reviewed by Stephenson, 1988). Molecular cloning of cDNAs encoding GABAA receptor subunits was facilitated by peptide sequences derived from purified receptor (Schofield et al., 1987; Levitan et al., 1988). Analysis of these cDNAs established that the 'a band' is heterogeneous, consisting of several variants of the ca subunit (Levitan et al., 1988) and of other subunits (Pritchett et al., 1989). This confirmed the molecular heterogeneity of GABAA receptors, postulated on the basis of pharmacological (Squires et al., 1979; Braestrup and Nielsen, 1981; Unnerstall et al., 1981; Cooper et al., 1987) and photo(3,
©IRL Press
Cloning of ,32 and /33 subunit cDNAs A cDNA library constructed from bovine brain RNA was screened with the degenerate oligonucleotide probe and numerous hybridizing signals were obtained. Among these were cDNAs encoding the known GABAA receptor subunits acl, a2, a3 and (31 (Schofield et al., 1987; Levitan et al., 1988), which were identified by subunit specific oligonucleotides. The remaining hybridizing clones were sequenced. Preliminary sequence data were obtained either directly from X DNA or from recombinant M13 DNA, using the degenerate oligonucleotide as a primer. Clones encoding new subunits were identified by homology of their deduced amino acid sequence with the third transmembrane region of previously characterized GABAA receptor subunits. Two cDNA clones were isolated that encoded ditferent polypeptides showing high sequence identity to the GABAA receptor (31 subunit. These polypeptides were designated as (32 and (3 subunits. By comparison with the (31 polypeptide, the encoded (2 subunit sequence lacked an initiation codon and the (3 subunit sequence lacked the N-terminal 45 amino acids of the complete polypeptide. Full-length
1665
S.Ymer et al.
of all subunits of ligand-gated ion channels (Criado et al., 1986; Schofield et~al., 1987). Notably, all three (3 subunits contain a cAMP-dependent phosphorylation site in a homologous position within their putative intracellular domain, suggesting involvement in the cellular control of
cDNA clones were not identified upon rescreening, but a complete j33 subunit encoding clone was isolated from a bovine adrenal gland cDNA library (see below). We then screened a rat forebrain cDNA library with oligonucleotides based on the bovine (3 subunit coding sequences and isolated full-length cDNA clones for the rat (31, (32 and (33 subunits. The nucleotide and deduced amino acid sequences of the rat subunits, including part of the 59 and 39 non-coding regions, are shown in Figure 1. All three cDNAs encode polypeptides of -450 amino acid residues (Mr 52 kd) with a 25-residue signal peptide (Von Heijne, 1986). The predicted mature polypeptide sequences contain four putative membranespanning regions, three potential N-linked glycosylation sites and a 15-residue-long, cysteine-flanked region, characteristic
receptor activity.
Comparison of the three f3 polypeptides predicted from either rat or bovine cDNAs (Figure 2) shows that 72% of the residues are invariant, reflecting an identity similar to that seen between different ax subunits (Levitan et al., 1988). As for the ca variants, regions of highest homology include the membrane-spanning domains and large extracellular region. Low sequence simnilarity is seen in the signal peptides and in the intracellular domain located between M3 and M4.
AMTIWACAT 900
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CATTGATATCGCCAGCATCGATATGGTTTCTGAAGTCAATATGGACTACACCTTGACCATGTATTTCCAGCAAGCCTGGAGAGATAAGAG R A W F D Y T L
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271 41
TGCGGATCGATGTCGCCAGCATAGACATGGTCTCGGAAGTGAATATGGATTATACACTCACCATGTATTTCCAGCAIkGTCTTGGAAGGACA 0 F
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540 131
AACTACAGCTGCCTGCATGATGGACCTAAGGCGGTATCCACTGGATGAACAAAACTGCACGTTGGAGATCGAAAGCTATGGCTATACAAC A A 0 L A R E 5 Y G Y T T P L 0 F A A C T L E A
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541 132
631 161
CCACCGATGACATCGAATTTTACTGGAATGGAGGAGAGGGAGCAGTAACTGGGGTCAATAAAAATGSAGCTTCCCCAATTTTCAATTGTCG SOAVNK IFE LEA0F S 190 1000D E F YWAN GSGF G A V
720 190
831 TGATGACATTGAGTTTTACTGGCGTGGCGATGACAATGCAGTCACGGGAGTGACAAAGATTGAGCTICCTCAGTTCTCCATTGTAGATTA 5 V 0 Y E L P A E F 0 W A G 0 0 A A V T S V T K 102 0 0
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990 281
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1080 311
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OVA FAMD ElN E KA AE KEA A NAANAE FAMR TGC CATCTTACTCAGCACTCTTGAGATAAAAAATGAGATGGCCACATCAGAAGCAGTAATGGGACTTGGAGACCCCAGGAGCACAATGCT ENE MA T SE A VAIGL GODP R S TIN LA I L LS FL F CTATGATGCCTCCAGCATCCAGTATCGGAAAGCTGGGTTGCCTAGGCATAGTTTTGGCCGCAACGCCCTGGAACGACATGTGGCACAAAAA C Y REKA GS ERANS FGR NARI E R VVAAF Y DA SS GAAAAGTCGCCTGAGGAGACGTGCCTCCCAACTGAAAATCACCATCCCCGACTTGACTGATGTGAACGCCATTGATCGGTGGTCCCGCAT F F SR L R R R A T S DOR WSR I VODL T DVANA KI
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GABAA receptor /31 (a), /32 (b) and /33 (c) subunits. Nucleotide and amino acid signal peptide residues. Potential N-linked glycosylation sites carry asterisks, the /3-structural ioop line and the four transmembrane regions arc underlined. Putative regulatory sites in the intracellular circles. UJnderlined residues in /32 (positions 336-352) correspond to a chemically determnined peptide
numbers refer to
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brain libraries. In rat brain, the hippocampus and cerebellum contain the highest levels of ( subunit mRNAs while the cortex shows only small amounts of (-specific RNA. (2 subunit mRNA is altogether more abundant than (33 mRNA. The three (3 subunit encoding mRNAs differ in size, with (31 mRNA being the largest (-. 12 kb) followed by (32 mRNA ( - 8 kb) and (3 mRNA, which is represented by two size forms
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A subunit localization The extent of ( subunit expression in the brain was investigated by Northern blot analysis. RNA samples were prepared from rat and calf total brain as well as from the cortex, hippocampus and cerebellum. Northern blots of these RNAs were hybridized with 32P-labelled oligonucleotides complementary to DNA sequences encoding the divergent intracellular domain of the three ( subunits. The autoradiographs (Figure 3) document that in the rat and bovine brain both the (2 and (3 subunits are considerably more abundant than the (1 subunit. The relative abundance of the (3 subunit mRNAs parallels that of the respective cDNA clones in the
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substituted between the rat and bovine homologues. A similarly high degree of sequence conservation has been
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