Brain Research, 569 (1992) 57-62 Elsevier Science Publishers B.V.

57

BRES 17280

A prominent epitope on G A B A A receptors is recognized by two different monoclonal antibodies Markus Ewert 1, Angel L. de Blas 2, Hanns M6hler 3 and Peter H. Seeburg 1 1Laboratory of Molecular Neuroendocrinology, ZMBH, University of Heidelberg, Heidelberg (ER.G.), 2Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri-Kansas City, MO 64110-2499 (U.S.A.) and 3Pharmakologisches Institut der Universitlit Z~rich, Ziirich (Switzerland)

(Accepted 6 August 1991) Key words: mAb; GABA A receptor; In vitro expression; eDNA; Epitope characterization

The monoclonal antibody 62-3G1 raised against the GABAA/benzodiazepine receptor complex was tested for its subunit selectivity using recombinantly expressed GABA A receptor subunits. The antibody bound selectively to/32 and t3 but not fll nor any other GABA A receptor subunit. Using enzyme-linked immtmosorbent assay, the epitope on t2 and t3 subanits was determined to be residues 1-3. MAb bd 17, which displays identical subunit selectivity as mAb 62-3G1, was seen to bind to the same epitope. These results resolve the subtmit selectivity of mAbs 62-3G1 and bd 17 and reveal the identity and localization of a prominent immunogenie epitope on the GABAAfoenzodiazepine receptor. INTRODUCTION y-Aminobutyric acid ( G A B A ) mediates inhibitory neurotransmission in m a m m a l i a n central nervous systems by activating a chloride conducting channel intrinsic to its receptor ( G A B A A R ) . GABA-elicited channel activity can be allosterically modulated by a variety of compounds which are in wide therapeutic use, such as benzodiazepines and barbiturates 17. The G A B A A R complex has been purified from several species using affinity chromatography on the immobilized benzodiazepine Ro 7-1986/124,30,33 and using the monoclonal antibody 623G1 is. Gel electrophoretic separation of this complex revealed two major bands with Mr 50 and 55 k o termed a and fl respectively, as well as several minor bands 7. GABAARS consist of several different subunits forming a hetero-oligomeric complex of unknown subunit composition and stoichiometry. Molecular cloning has revealed a family of different variants which, according to their sequence similarities, are grouped into a, t , 7 and 6 subunit classes (which are further subdivided in cq_6, fll-3, Yl,2, 6). Depending on subunit composition, different pharmacological and physiological characteristics are displayed by the G A B A A R complex 5'14'19'2°'32. To resolve the topography of G A B A A R constituents in the mammalian brain, in situ hybridization as well as radioligand- and immunohistochemical methods have been applied successfully 3'9'10'15'21'25'27'29'34. The value of immunological studies depends signifi-

antly on the characterization of the antibodies used in respect to specificity and selectivity to G A B A A R subunits 6. Two m A b s , 62-3G1 and bd 17 raised in two of our laboratories 24'a3 have been used extensively in characterizing GABAARS 3'7'9'10'11'16'1s'21'23'25'31'33-36. The G A B A A R subunit selectivity of m A b bd 17 has been previously determined 6, whereas information on the selectivity of m A b 62-3G1 was restricted to its recognition of a 57 k o polypeptide of the G A B A A receptor complex in immunoblots. Using an array of cultured cells transiently expressing 12 different G A B A A R subunits, we demonstrate that 62-3G1 reacts with rat t 2 and fl3, but not fll nor any other rat G A B A A R subunit. This investigation not only revealed identical recognition patterns for m A b 62-3G1 and m A b bd 176 but additionally determined epitopes of these widely used mAbs.

MATERIALS AND METHODS Transfection of cultured cells Transformed human embryonic kidney cells 293 (ATCC CRL 1573)8 were grown at 37°C on coverglass coated with fibroneetin (25 /~g//~l in phosphate-buffered saline; PBS) in minimum essential medium (MEM, Gibco) supplemented with 10% fetal bovine serum containing 100 units of penicillin (GIBCO) and 100/~g of streptomycin (GIBCO) per ml in a 5% CO2/95% air incubator. Exponentially growing cells were trypsinized and seeded at 2 x 105 per coverglass in 2 ml of growth medium. The transfeetion was performed by the calcium phosphate precipitation t~elmique 4. The cloned cDNAs of rat GABA A receptor subunits (a 1 (13); a2,s,4 (26); as (12); as (14); fll (13); fl2,3 (38); 71 (39); 72 (28); 6 (28) (references given in brackets)), inserted singly into eukaryotic ex-

Correspondence: P.H. Seeburg, ZMBH, Im Neuenheimer Feld 282, D-6900 Heidelberg, ER.G. Fax: (49) (6221) 56-5894.

58 pression vectors, were used for transfection s'19. The cells were incubated in the presence (2 #g/ml) of one or several supercoiled expression plasmids for 12-16 h at 37°C under 3% CO2/97% air. The medium was removed, and the cells were rinsed twice with culture medium, refed, and incubated in the same medium for 24 h at 37°C under 5% CO2/95% air before performing immunological studies.

Immunocytochemistry Medium was removed by aspiration and cells were washed twice with 10 mM phosphate-buffered saline, pH 7.4 (PBS). Fixation was performed in 3% PBS-buffered paraformaldehyde for 5 min. Cells were rinsed twice with PBS and incubated with 50 mM glycine in PBS. In some cases, cells were permeabilized by the addition of 0.3% Triton X-100. After an additional 5 min, 1% blocking serum

Fig. 1. Selectivity of mAb 62-3G1 for rat G A B A A receptor fl subunits expressed in 293 cells, a: fll subunit; b: f12; c: f13; d: surface staining of non-permeabilized f13 expressing cells; e: f13 in the presence of peptide N1; f: f13 in the presence of peptide N9. Cells were permeabilized with 1% Triton X-100 (except in d) prior to reacting with antibody and were photographed under phase-contrast. Bar = 30 ~m.

59 (Veetastain ABC kit, Vector Laboratories, Eugene, OR) was applied for 30 rain. The mAbs were added to cells for 2-3 h in a 1:5 dilution using supernatants of cultured hybridoma cells. Coverglasses with cells were washed gently with PBS and the second biotinylated antiserum (Vectastain 1:200) was added for 60 min. After washing with PBS the avidin-biotinylated horseradish peroxidase complex (1:100) was applied for 1.5 h. Cells were rinsed with PBS and the reaction was started by incubation in PBS containing 0.05% diaminobenzidine/0.03% 1-1202, typically for 5-15 rain. To stop this reaction, cells were again rinsed with PBS. Coverglasses were dipped for a few seconds into distilled water and mounted on glass microscope slides with Moviol (Hoechst). Peptide competition was performed by preincubation of mAbs bd 17 and 62-3G1 with peptides N1, NS, N9 (i/~g/coverslip) for 30 rain at room temperature.

Enzyme-linked immunosorbent assay A 96 well polystyrene microtiter plate (Nunc) was used to immobilize peptides s7 which correspond to the N-terminal amino acid residues of the GABA A receptor/31,/32 and/33 subunitsX3'3s (N1 (/31): CHSSNEPSNMSYVKET; N8 ~2): CQSVNDPSNMSLVKET; N9 ~3): CQSVNDPGNMSFVKET). Cysteine residues in amino acid position 1 exist for technical reasons only in synthetic peptides, not in vivo. Coating of peptides (1/~g/ml) was performed at 40C over 12-16 h in 0.1 M sodium carbonate buffer, pH 7.4, and unspecific sites were blocked with 1% bovine serum albumine in PBS-Tween for 60 rain at 37°C. After subsequent two-fold washes, mAbs were diluted and incubated in duplicate with the immobilized peptides for 12-16 h at 4"C in a humid chamber. Non-bound antibody was removed by 3 washes and antimouse aikaline-phosphase-coupled IgG from goat (1:2000, Promega) was added for one hour at 20°C. This incubation was followed by 3 rinses with PBS-Tween, by two with 0.1 M and one with 1 M diethanolamine, pH 9.6. Buffer was exchanged by substrate solution (1 mg/ml paranitrophenyl-

ratbetal ratbeta2 ratbeta3 Consensus

N11 H S S N E P S N M S Y V K E ~ N81QSVNDPSNMSLVKE~ N9 Q S V N D P G N M S F V K E ~ -S-N-P-NMS-VKE~

diphosphate (Merck, Darmstadt), 1 mM MgC12 in 1 M diethanolamine, pH 9.6), and the reaction was performed at 370C. Color change indicating binding affinity of mAbs to peptides was determined in a spectrophotometer at 405 vs 690 nm (Titertek Multiskan MCC/340, EFLAB, Finland). RESULTS

Recombinant G A B A A receptor subunits can be transiently expressed in transformed human embryonic kidney cells (293 cells). These receptors have repeatedly been investigated for their pharmacological and electrophysiological characteristics. We have used this expression system as described previously 6 to investigate the selectivity of mAb 62-3G133 in its binding to 12 different G A B A A receptor subunits. Cells transiently expressing single subunits were fixed and incubated with the antibody, and antibody binding was visualized by the avidinbiotinylated HRP method. Mock-transfected cells did not react with mAb 62-3G1. The antibody displayed high specificity and selectivity only with rat t2 and, even more, with rat t3 GABAAR subunits (Fig. lb,c). Despite the high amino acid sequence homology of higher than 75% between all 3 known rat fl subunits 38, the antibody failed to immunoreact with rat fll (Fig. la). The same lack of responsiveness was observed with other rat G A B A A receptor subunits (al-6, •1,2, 6). MAb 62-3G1 showed high

60 VDRLLKGYDIRLRPDFGGPPVDVGMRIDVASIDMVSEVNMDYTLT VDRLLKGYDIRLRPDFGGPPVAVGMNIDIASIDMVSEVNMDYTLT VDKLLKGYDIRLRPDFGGPPVCVGMNIDIASIDMVSEVNMDYTLT VD-LLKGYDIRLRPDFGGPPV-VGM-ID-ASIDMVSEVNMDYTLT

ratbetal ratbeta2 ratbeta3 Consensus

120 M Y F Q Q S W K D K R L SYS G IPLNLT L D N R V A D Q L W V P D T Y F L N D K K S F V H G V T V K N R M I R L H P M Y F Q Q A W R D K R L SYNVI P LNLT LDNRVAD QLWVPD T Y F L N D K K S F V H G V T V K N R M I R L H P M Y F Q Q Y W R D K R L A Y S G IP L N L T L D N R V A D Q L W V P D T Y F L N D K K S F V H G V T V K N R M I R L H P M Y F Q Q -W -DKRL -Y -- I P LNLT L D N R V A D Q L W V P D TYFLNDKKSFVHGVTVKNPd~IRLHP

ratbetal ratbeta2 ratbeta3 Consensus

DGTVLYGLRI DGTVLYGLRI DGTVLYGLRI DGTVLYGLRI

ratbetal r atbet a 2 ratbeta3 Consensus

IELPQFS I V D Y K M V S K K V E F T T G A Y P R L S IE LPQFS IVDYKL ITKKVVF STGSYPRLS IELPQFS I V E H R L V S R N V V F A T G A Y P R L S I E L P Q F S I V ........ V - F - T G - Y P R L S

............... T T T A A C M M D L R R Y P L D E Q N C T L E IE SYGYTTDD T T T A A C M M D L R R Y P L D E Q N C T L E IE S Y G Y T T D D T T T A A C M M D L R R Y P LDEQNCT LE IE S Y G Y T T D D T T T A A C M M D L R R Y P LDEQNCT LE IE S Y G Y T T D D

~ 180 IE F Y W N G G E G A V T G V N K IE F Y W R G D D N A V T G V T K IE F Y W R G G D K A V T G V E R IEFYW -G .... V T G V - -

TMI 2,0 LSFRLKRNI GYF I LQTYMP STL IT I LSWVSW L S F K L K R N I GYF I LQTYMP S I L IT I LSWVSW LSFRLKRNI GYF I LQTYMPS IMI T I LSWVSW LSF-LKRNI GYF I LQTYMP S-- I T I LSWVSW

Fig. 2. Amino acid sequence comparison of the N-terminal domain of rat GABAA receptor/31, /32 and/33 subunits. Numbers denote amino acid residues (single letter code) and start at the predicted N-terminus of the mature polypeptides. The common disulfide-bonded/3-structurai loop is indicated by a dotted line, and the first transmembrane region is overlined. Amino acid residues identical in all three polypeptides are listed below the respective sequences. Arrows indicate positions with identical residues in/32 and/33, but different in/31. The boxed area depicts amino acid residues (1-15) contained in the peptides N1, N8 and N9 from rat/31,/32 and/33, respectively, which were used for ELISA.

60 a

mAb 62-3G1

100

b

mAb bd 17

to f12 and/33 in the presence of N1 (Fig. le), but recognition was inhibited by peptides N8 and N9 (Fig. lf). MAb bd 17 displayed identical characteristics (not shown).

~,~,,,,~ !1il ~

DISCUSSION

4OOant] 1/[mAb-supernatant] 1/[mAb-supernat 250

5O0

1000 2OO0

5O

100

20O

Fig. 3. Enzyme-like immunosorbent assay with mAbs 62-3G1 (a) and bd 17 (b). Depicted is the reactivity of mAbs to peptides N1 (squares), N8 (circles), and N9 (triangles), comprising the first 15 amino acid residues of rat ill,/32, and f13 GABAA receptor subunits. The ordinates show normalized reactivity of alkaline phosphatase coupled to mAbs, determined by spectrophotometric quantitation of the end-product para-nitrophenylamine. Abscissae indicate dilutions of hybridoma supernatants. Data show one of 3 independent experiments with identical results.

specificity, i.e. very low reactivity to cells other than those expressing f12 or/33. This antibody therefore displayed the same high selectivity and specificity to rat f12 and f13 GABAA R polypeptides as mAb bd 176. In non-permeabilized cells the antibodies have access only to epitopes at the outer surface of the plasma membrane. Under these conditions, mAb 62°3G1 and bd 17 immunoreacted with rat/32 and f13 polypeptides (Fig. ld), indicating that both antibodies located an epitope(s) on the extracellular part of these fl subunits. Assuming the proposed topology, this epitope could be either -- and most likely -- on the N-terminal part of the subunit, or between membrane spanning regions TM2 and 3, or at the very C-terminus. Amino acid sequence inspection for amino acids identical in BE and f13 but different in ill, left 6 potential antibody binding sites in the N-terminal part and 1 in the C-terminus of these proteins (Fig. 2). Because the monoclonal anti-GABAAR antibody bd 24 binds to the very N-terminus of the human and bovine al subunit 6, we used 15 amino acid residue long peptides of the rat fit (N1), f12 (N8), and f13 (N9) GABAAR subunits for enzyme-linked immunosorbent assay (ELISA). We detected strong reactivity with mAbs bd 17 and 62-3G1 to peptide N9 (f13), a 3°fold weaker reactivity to N8 (f12), and no recognition of N1 (fl~) (Fig. 3). These results show conclusively that mAbs 62-3G1 and bd 17 bind to the same epitope located on peptides N8 and N9 but not on N1. This epitope should include amino acid positions 1 and 3 where peptides N8 and N9 differ from peptide N1. We also used peptide inhibition of antibody recognition on cells expressing/32 or f13 subunits. Both mAbs were incubated with these cells in the presence or absence of peptides N1 (ill), N8 (f12), and N9 (/33). Under these conditions, mAb 62-3G1 showed immunoreactivity

We have analyzed the specificity and selectivity of the widely used mAb 62-3G1 towards 12 different rat GABA A receptor subunits expressed mammalian cells using immunocytochemical methods. The antibody is selective for the f12 and f13 subunits and neither recognizes rat fll nor any other GABAAR subunit (Fig. la,b,c). The expression of all subunits was controlled through immunocytochemical reacion of mAb bd 28 with cells transfected in parallel. This antibody recognizes all GABA A receptor subunits, except ill, which is only poorly detected by bd 286. The expression of this subunit was shown by electrophysiological means 2. The selectivity of 62-3G1 is identical to that of mAb bd 176. Interestingly, both antibodies stain rat/32 weaker than fla. We have observed this phenomenon repeatedly but the difference in staining intensity is difficult to reproduce (Fig. lb,c) due to loss of signal during photographic processing. Furthermore, it is difficult to quantify the staining of immunocytochemical reactions. Non-permeabilized cells expressing rat GABAAR f12 and f13 subunits were also stained by both mAbs, indicating the localization of their common epitope on the extracellular side (Fig. ld). Reaction products could only be visualized on the cell surface and thus staining intensity was clearly weaker, due to the smaller number of subunit molecules located in the outer membrane than in the whole cell membrane (including endoplasmic reticulum, golgi apparatus and endosomes). The latter situation was visualized in permeabilized cells where antibodies had access to all epitopes. Strong immunoreactivity could be observed intracellularly, indicating high amounts of receptors either transferred to the cell surface or endocytosed for degradation. The failure of mAbs 62-3G1 and bd 17 to recognize rat ill, as well as their ability to immunoreact with the extracellular region of GABAA receptors, together with the high amino acid sequence homology of over 95% in this region between all 3 fl GABAAR subunits, were indicators for a small number of potential binding sites. These had to fulfill the requirement of amino acid residue identity between f12 and f13 but different from fll (Fig. 2). Conservative amino acid substitutions such as an aspartate for a glutamate were not considered. To identify the one site out of potentially 7, we focused on the very N-terminus of the fl subunits, because mAb bd 24 binds to the homologous region in human and bovine

61 G A B A A R al subunits 6. We successfully tested this region of the fl subunits with synthetic 16-mer peptides representing amino acids 1-15. Enzyme-linked immunosorbent assay ( E L I S A ) on these immobilized peptides clearly showed binding to N8 and N9 (/32 and f13) but not to N1 (fl0 (Fig. 3a,b). This supported not only the resuits obtained with transfected cells but also revealed the epitope for m A b s 62-3G1 and bd 17. It remains unknown whether giutamine at position 1 or valine at position 3 or even both determine the antibodies' ability to bind to this region. E L I S A data show that both m A b s strongly bind to the rat f13 subunit specific peptide, 3-fold weaker to f12 peptide and not at all to the fl~ peptide (Fig. 3). Binding affinity differences of 62-3G1 and bd 17 to f12 and f13 could be explained by the influence of amino acid residues at positions 7 (S, G) and 11 (L, F). Because m A b s 62-3G1 and bd 17 display the binding ratio of 3:1 with f13:f12, the conclusion that both antibodies share a comm o n epitope is additionally strenghtened. These findings indicate the presence of a prominent epitope on f12 and f13 which seems to be similarly immunogenic as the epitope of bd 24 with its homologous location on a~. Possibly, all other G A B A A receptor polypeptides are likely to display similar 3-dimensional structures and

therefore the same peptide region will be accessible for raising other subunit selective antibodies. Recently, a c D N A encoding a fourth fl subunit of the G A B A A receptor has been cloned from chicken 1, which so far has not been detected in the mammalian central nervous system. A comparison of the epitope sequence recognized by bd 17 and 62-3G1 with the respective sequence of f14 reveals only low homology. Hence both m A b s are unlikely to recognize the f14 G A B A A receptor subunit even if the hypothetical mammalian f14 subunit were to differ slightly from the one of chicken. Nothing is known about functional differences of the 3 G A B A A receptor fl subunits. It has been shown that they are necessary participants in the G A B A A receptor complex for displaying pharmacological and electrophysiological profiles similar to those of the in vivo receptors.

Acknowledgements. We thank our colleagues Anne Herb, Martin K6hler, Dolan B. Pritchett, Bernd Sommer, Rolf Sprengel, Pia Werner and Sanie Ymer for cDNA expression vectors, Sabine Grfinewald for excellent help with cell culture, and Jutta Rami for superb secretarial help. We particularly acknowledge Hartmut Liiddens for this technical advice and interest and Rolf Sprengel for computer analysis. M.E. is the recipient of a doctoral fellowship by the Boehringer Ingelheim Fonds. This research was supported by Deutsche Forschungsgemeinschaft, SFB Grant 317/139, to P.H.S.

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62 of the GABAA/benzodiazepine receptor complex by immunoaffinity chromatography, J. Neurochem., in press. 19 Pritchett, D.B., Sontheimer, H., Gorman, C.M., Kettenmann, H., Seeburg, P.H. and Schofield, P.R., Transient expression shows ligand gating and allosteric potentiation of GABA A receptor subunits, Science, 242 (1988) 1306-1308. 20 Pritchett, D.B., Ltlddens, H. and Seeburg, P.H., 1~pe I and type II GABAA-banzodiazepine receptors produced in transletted cells, Science, 245 (1989) 1389-1392. 21 Richards, J.G., Schoch, P., Hitrin, P., Takacs, B. and M6hler, H., Resolving GABAA/benzodiazepine receptors: cellular and subceUular localization in the CNS with monoclonal antibodies, J. Neurosci., 7 (1987) 1866-1886. 22 Richards, J.G., Schoch, P., Httring, P., Takacs, B. and MOhler, H., Strategies for raising antibodies against benzodiazepine receptors. In F.W. van Leeuwen, R.M. Buijs, C.W. Pool and O. Pack (Eds.), Molecular Neuroanatomy, Elsevier Publishers, 1988, Amsterdam, pp. 159-190. 23 Schmitz, E., Reichelt, R., Walkowiak, W., Richards, J.G. and Hebebrand, J., A comparative phylogenetic study of the distribution of cerebellar GABAA/benzodiazepine receptors using radioligands and monoclonal antibodies, Brain Research, 473 (1988) 314-320. 24 Schoch, P., Hitting, P., Takacs, B., Stithli, C. and M6hler, H., A GABA/benzodiazepine receptor complex from bovine brain: purification, reconstitution and immunological characterization, J. Receptor Res., 4 (1984) 189-200. 25 Schoch, P., Richards, J.G., Hitting, P., Takacs, B., Stithli, C., Staehlin, T., Haefely, W. and MOhler, H., Colocalization of GABAA receptors and benzodiazepine receptors in the brain shown by monoclonal antibodies, Nature, 314 (1985) 168-171. 26 Seeburg, P.H., Wisden, W., Verdoorn, T.A., Pritchett, D.B., Weruer, P., Herb, A., Ltlddens, H., Sprengel, R. and Sakmann, B., The GABAA receptor family: molecular and functional diversity, Cold Spring Harbor Symposia on Quantitative Biology, Vol. LV, 1990, pp. 29-40. 27 S6quier, J.M., Richards, J.G., Malherbe, P., Price, G.W., Matthews, S. and M6hler, H., Mapping of brain areas containing RNA homologous to cDNAs encoding the a and fl subunits of the rat GABAA ),-aminobutyrate receptor, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 7815-7819. 28 Shivers, B.D., KiUisch, I., Sprengel, R., Sontheimer, H., KOhler, M., Schofield, P.R. and Seeburg, P.H., Two novel GABA^ receptor subunits exist in distinct neuronal subpopu-

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A prominent epitope on GABAA receptors is recognized by two different monoclonal antibodies.

The monoclonal antibody 62-3G1 raised against the GABAA/benzodiazepine receptor complex was tested for its subunit selectivity using recombinantly exp...
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