The binding of y-aminobutyric acid (GABA) to the. GABAA receptor leads to the rapid opening of a chloride ion channel which is an integral part of the receptor.
9s Alternative splicing heterogeneity.
increases
GABAA
receptor
A.
ANNElTE LASHAM. ALAN N. BATESON and MARK G. DARLISON
p4 GAAAAGAG E K R
TTGACCCT k D P
MRC Molecular Neurobiology Unit, MRC Centre, Hills Road, Cambridge CB2 2QH, U.K. The binding of y-aminobutyric acid (GABA) to the G A B A A receptor leads to the rapid opening of a chloride ion channel which is an integral part of the receptor complex. This receptor is of particular interest because it contains a variety of binding sites for pharmacologicallyimportant compounds such as the benzodiazepines, barbiturates. 8-carbolines and picrotoxin. Electrophysiological and pharmacological studies indicate that more than one type of GABAA receptor exists [1,2]. Cloning of GABAA receptor subunit cDNAs has revealed the existence of many subunits and subunit isoforms [reviewed in 31, some of which have been shown to be expressed in a cell-specific manner in different regions of the vertebrate brain [4]. It is likely, therefore, that the differential expression of particular subunit genes is the molecular basis for this receptor heterogeneity. A cDNA library was constructed in XgtlO using mRNA that was isolated from a combination of 12- and 16-day-old embryonic chick whole brain. This was screened with a full-length bovine p 1-subunit cDNA [5] at low stringency. Filters were hybridised in a solution containing 6 x SSC (1 x SSC = O.15M sodium chloride, 0.015M sodium citrate) at 60OC for 16 hours and then washed in 2 x SSC at 37OC for 20 minutes prior to autoradiography. Five clones were isolated [6]. 3 of which, when sequenced, were found to encode a polypeptide that exhibited no greater than 77% identity to any of the published p-subunit sequences [7]. These 3 cDNA clones are not full length; they lack that part of the sequence that encodes the fourth putative transmembrane domain. This is due to the presence of a naturally-occuring E c o R I site at this position. The deduced polypeptide shows very little homology to other p subunits in the signal peptide and in the presumptive intracellular loop region. We believe, therefore, that this represents a novel GABAA receptor 8 subunit which we designate 84. DNA sequencing revealed that one of the clones contains an insertion of 12bp in the region encoding the intracellular loop. The extra 12bp encodes the 4 amino acids: V-R-E-Q (one-letter code) (Figure IA). By sequencing the corresponding genomic DNA, it was found that there is an intron at the point of divergence of the two cDNA sequences (Figure 1B). The 12bp insertion is contiguous with the previous exon and includes a 5' donor splice point, such that the different cDNA sequences arise by the selection of one of two different 5' splice sites. Strikingly, a similar situation has been found for cDNAs that encode human tyrosine hydroxylase [8], where there is a 12 base insertion in one alternativelyspliced mRNA that gives rise to the extra amino acids: VR-G-Q. This extra 12bp has also been shown to be contiguous with the previous exon. These additional amino acids in tyrosine hydroxylase are postulated to result in the formation of a consensus phosphorylation site, which may be involved in modulation of the activity of the enzyme [8]. Although the extra 4 amino acids in one form of the chicken GABAA receptor 84 subunit (termed 84') does not result in either the addition or deletion of a consensus phosphorylation site, this sequence may still have some functional significance. Interestingly, we have also found an insertion, in a similar location, in a cDNA clone that encodes the chicken GABAA receptor y2 subunit [9], although we have not yet sequenced the corresponding genomic DNA. G A B A A receptor heterogeneity is currently believed to arise from the association of different subunit types and subunit isoforms to form receptor subtypes. Our findings
B. E
K
R
V
R
E
Q
GAAAAGAGGGTGAGAGAGCAGgtttgtccccttcttt
Figure 1. Alternative s d i c i n e of the chicken GABAA K c e m o r D4-subunit transcrim. A. Part of the cDNA and the corresponding deduced amino-acid sequences (one-letter code) of the p4 and p 4 ' subunits. The extra 12bp (4 amino acids) found in p4' are indicated. B. The DNA sequence across the intron/exon boundaries involved in the alternative splicing of the 8 4 subunit transcript. The arrows indicate the two different splice points. Lower case DNA sequence indicates intronic sequence. Note that the sequence between the arrows is intronic in p4 and exonic in p4'. The deduced amino-acid sequence of the 3' end of the 5' exon is also indicated (one-letter code). suggest, however, that alternative splicing mechanisms may generate even greater receptor heterogeneity. A.L. holds an MRC Research Studentship. We thank Drs Andrew Hicks and Detlef Giissow for the chicken cDNA and genomic libraries, respectively, and Professor Eric Barnard for his interest in and support of this work. 1.
Akaike, N., Jnoue. M. & Krishtal, O.A. (1986) J. Physiol.
379, 171-185 2. 3. 4. 5.
6.
7. 8. 9.
Olsen, R.W. & Venter, J.C. (1986) BenzodiazepinelGABA Receptors and Chloride Channels: Structural and Functional Properties, Alan R. Liss. New York Olsen. R.W. & Tobin, A.J. (1990) FASEB J. 4. 1469-1480 Wisden, W.. Moms, B.J.. Darlison, M.G., Hunt, S.P. & Barnard. E.A. (1988) Neuron 1, 937-947 Schofield, P.R., Darlison. M.G., Fujita, N.. Burt, D.R., Stephenson, F.A.. Rodriguez, H., Rhee. L.M.. Ramachandran. J.. Reale, V., Glencorse. T.A., Seeburg, P.H. & Barnard, E.A. (1987) Nature (London) 328, 221-227 Bateson. A.N., Ultsch. A., Harvey, R.J.. Sutherland. M.L., Lasham, A., Glencorse, T.A., Barnard, E.A. & Darlison. M.G. (1990) in Regulation of Gene Expression in the Nervous System (Giuffrida Stella, A.M., Perez-Polo, J.R. and De Vellis, J.. eds.). in press, Alan R. Liss, New York Ymer, S . , Schofield, P.R., Draguhn, A., Werner, P., Kohler, M. & Seeburg, P.H. (1989) EMBO J. 8, 1665-1670 Le Bourdelles, B., Boularand, S.. Boni, C.. Horellou, P., Dumas, S . , Grima, B. & Mallet, J. (1988) J. Neurochem. 50, 988-991 Glencorse. T.A., Bateson, A.N. & Darlison, M.G. (1990) Biochem. SOC. Trans. (this Volume)
