A novel site on γ3 subunits important for assembly of GABAA receptors

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1 A novel site on γ 3 subunits important for assembly of GABA A receptors* Sarto I. , Klausberger T. , Ehya N. , Mayer B. § , Fuchs K. , and Sieghart W. Division of Biochemistry and Molecular Biology, Brain Research Institute, University of Vienna and Section of Biochemical Psychiatry, University Clinic for Psychiatry, A-1090 Vienna, Austria and § Institute for Theoretical Chemistry and Molecular Structural Biology, University of Vienna, A-1090 Vienna, Austria To whom correspondence should be addressed: Division of Biochemistry and Molecular Biology, Brain Research Institute, University of Vienna, Spitalgasse 4, A-1090 Vienna, Austria, Tel.:++43-1-4277-62950; Fax: Tel.:++43-1-4277-62959; E- mail:[email protected] Running title: GABA A receptor assembly * This work was supported by grant P12637-Med of the Austrian Science Fund. by guest on April 16, 2018 http://www.jbc.org/ Downloaded from

Transcript of A novel site on γ3 subunits important for assembly of GABAA receptors

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A novel site on γ3 subunits important for assembly of

GABAA receptors*

Sarto I.‡, Klausberger T.‡, Ehya N.‡, Mayer B.§, Fuchs K.‡, and Sieghart

W.‡¶

‡Division of Biochemistry and Molecular Biology, Brain Research Institute, University of

Vienna and Section of Biochemical Psychiatry, University Clinic for Psychiatry, A-1090

Vienna, Austria and §Institute for Theoretical Chemistry and Molecular Structural Biology,

University of Vienna, A-1090 Vienna, Austria

¶To whom correspondence should be addressed: Division of Biochemistry and Molecular

Biology, Brain Research Institute, University of Vienna, Spitalgasse 4, A-1090 Vienna,

Austria, Tel.:++43-1-4277-62950; Fax: Tel.:++43-1-4277-62959; E-

mail:[email protected]

Running title: GABAA receptor assembly

* This work was supported by grant P12637-Med of the Austrian Science Fund.

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GABAA receptors are ligand-gated chloride channels and are the major

inhibitory transmitter receptors in the central nervous system. The majority of these

receptors is composed of two α, two β and one γ subunit. To identify sequences

important for subunit assembly, we generated C-terminally truncated and chimeric γ3

constructs. From their ability to associate with full length α1 and β3 subunits, we

concluded that amino acid sequence γ3(70-84) either directly interacts with α1 or β3

subunits or stabilizes a contact site elsewhere in the protein. The observation that this

sequence contains amino acid residues homologous to γ2 residues contributing to the

benzodiazepine binding site at the α1/γ2 interface suggested that in α1β3γ3 receptors the

sequence γ3(70-84) is located at the α1/γ3 interface. In the absence of α1 subunits this

sequence might allow assembly of β3 with γ3 subunits. Other experiments indicated that

sequences γ3(86-95) and γ3(94-107) that are homologous to previously identified

sequences important for assembly of γ2 subunits, are also important for assembly of γ3

subunits. This indicates that during assembly of the GABAA receptor, more than one N-

terminal sequence is important for binding to the same neighbouring subunit. Whether

the three sequences investigated are involved in direct interaction or stabilize other

regions involved in intersubunit contacts, has to be further studied.

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γ-Aminobutyric acidA (GABA)A1 receptors are the major inhibitory transmitter

receptors in the central nervous system and mediate fast synaptic inhibition by opening an

intrinsic chloride channel (1). These receptors carry binding sites for a number of

pharmacologically and clinically important drugs, such as benzodiazepines, barbiturates,

steroids, anesthetics and convulsants that modulate GABA-induced chloride ion flux by

interacting with separate and distinct allosteric binding sites (2).

The GABAA receptor is a hetero-oligomeric protein consisting of five subunits (3, 4).

So far at least 19 GABAA receptor subunits belonging to several subunit classes (six α, three

β, three γ, one δ, one ε, one π, one θ and three ρ) have been identified in the mammalian

nervous system (5, 6). Although a variety of subunits can be co-expressed within the same

neuron (7, 8), not all receptors that theoretically can be formed are actually formed in the

brain (9-11). Thus, GABAA receptor heterogeneity is limited by the temporal and spatial

expression of subunits (12) and by structural and conformational requirements during

assembly (13). The majority of GABAA receptors is composed of two α, two β and one γ

subunit (4, 11, 14-17) where the γ subunit is located between an α and a β subunit (4, 18).

The assembly of hetero-oligomeric receptors of the ligand gated ion channel

superfamily comprising the nicotinic acetylcholine (nACh) receptor, GABAA receptor,

glycine receptor, and 5-hydroxytryptamine, type 3 receptor, is a complex, multistep process

that also requires conformational changes in the involved subunits (for review see Ref. 19).

The assembly of subunits seems to occur in the endoplasmic reticulum and to involve

interaction with chaperone molecules (20, 21). The processes that lead from single subunits to

completely assembled and pharmacologically functional receptors are still a matter of debate.

For the assembly of nACh receptors a sequential mechanism was proposed (22-24), while for

the GABAA receptors it is not clear whether oligomerization can start randomly from any

possible subunit dimer or follows a strict order of assembly intermediates (25).

In the whole receptor superfamily the major determinants for intersubunit contacts

seem to be located within the N-terminal domains (26-28). For the GABAA receptor, specific

amino acid sequences have been identified in this domain that seem to mediate heteromeric

and homomeric assembly of β subunits (29) or assembly of α with β subunits (30). In

addition, the γ2 amino acid sequences γ2(91-104) and γ2(83-90) were identified as sites

important for assembly with α1 and β3 subunits, respectively (31).

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The amino acid sequence of the γ3 subunit is 64% identical with that of the γ2 subunit

(32, 33) and each of these subunits contributes to the formation of the benzodiazepine binding

site that is located at the interface between the α and γ subunit (34). In the present study it

was investigated whether amino acid sequences homologous to putative assembly sites on γ2

subunits are also important for assembly of γ3 subunits. By using truncated as well as

chimeric constructs a novel amino acid sequence was identified on the γ3 subunit that either

directly interacts with α1 or β3 subunits, or stabilizes a contact site in a different region of the

protein.

In addition, it was demonstrated that sequences homologous to those previously

identified on γ2 subunits are also important in γ3 subunits for interaction with neighbouring

subunits. It was, thus, concluded that during assembly of GABAA receptors more than one N-

terminal γ3-sequence is important for forming contacts with the same adjacent subunit.

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EXPERIMENTAL PROCEDURES

AntibodiesThe antibodies anti-peptide α1(1-9), anti-peptide β3(1-13), anti-peptide

α1(328-382), anti-peptide β3(345-408) and anti-peptide γ3(1-35) were generated and affinity

purified as described previously (4, 9). None of these antibodies exhibited any cross-reactivity

with any other GABAA receptor subunits as demonstrated in Western blot and

immunoprecipitation studies using recombinant GABAA receptors (9).

Generation of cDNA constructsFor the generation of recombinant receptors, α1, β3 and

γ3 subunits of GABAA receptors from rat brain were cloned and subcloned into pCDM8

(Invitrogen, San Diego, CA) and subsequently into pCI (Promega, Madison, WI) expression

vectors as described previously (4). Truncated subunits were constructed by PCR

amplification using the full-length subunit as a template. The PCR-Primers contained EcoRI

and XhoI restriction sites, which were used to clone the fragments into pCI vectors. The

truncated subunits were confirmed by sequencing. Chimeras were constructed using the ”gene

SOEing” technique (35) and were cloned into pCI vectors using the EcoRI and XhoI

restriction sites of the primers.

Culture and Transfection of HEK 293 cellsTransformed human embryonic kindney

(HEK 293) cells (CRL 1573; American Type Culture Collection, Rockville, MD) were

cultured as described (4). 3 x 106 cells were transfected with 20 µg of subunit cDNA for

single subunit transfection using the calcium phosphate precipitation method (36). On co-

transfection with two or three different subunits, for each subunit 10 µg or 7 µg cDNA per

subunit was used, respectively. A total of about 20 µg cDNA per transfection and a cDNA

ratio of 1:1:1 seemed to be optimal for the expression of GABAA receptors under the

conditions used as judged by receptor binding studies in cells transfected with α1, β3 and γ3

subunits.

The cells were then harvested 36 h after transfection. At this time point the number of

[3H]Ro 15-1788 binding sites formed per mg protein was at its maximum for cells transfected

with α1, β3 and γ3 subunits. Results obtained, however, did not change when cells were

harvested 34 - 48 h after transfection. In addition, judged by Western blot analysis, expression

levels of full length, truncated or chimeric subunits were comparable at all harvesting times.

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Purification and immunoprecipitation of complete, truncated and chimeric subunitsThe

culture medium was removed from transfected HEK 293 cells and cells from 4 culture dishes

(diameter 9,4 cm) were extracted with 800 µl of a Lubrol extraction buffer (1% Lubrol PX,

0.18% phosphatidylcholine, 150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, pH 7.4,

containing 0.3 mM phenylmethylsulfonylfluorid (PMSF), 1 mM benzamidine and 100 µg/ml

bacitracin) for 8 h at 4oC. The extract was centrifuged for 40 min at 150,000 x g at 4oC.

For Western blot analysis, the clear supernatant was incubated overnight at 4oC under

gentle shaking with 15 µg antibodies directed against the full length subunit. After addition of

Immunoprecipitin (Life Technologies, Gaithersburg, MD; preparation see (4)) and 0.5%

nonfat dry milk powder and shaking for additional 3 h at 4oC, the precipitate was washed

three times with a low salt immunoprecipitation buffer (IP low buffer) (50 mM Tris-HCl,

0.5% Triton X-100, 150 mM NaCl, and 1 mM EDTA, pH 8.0). The precipitated proteins were

dissolved in sample buffer (108 mM Tris-sulfate, pH 8.2, 10 mM EDTA, 25% (w/v) glycerol,

2% SDS and 3% dithiothreitol). SDS-PAGE and Western blot analysis with digoxygenized

antibodies were performed as described (4).

All truncated or chimeric constructs used in this study could be expressed to a comparable

extent upon single transfection into HEK cells. On co-transfection of different constructs,

however, the stability of fragments that could not bind stably to each other was reduced. This

might have been caused by proteolytic degradation due to an unstable or unproductive

interaction of the fragments. In all control experiments the extent of expression of fragments

was therefore determined in singly transfected HEK cells (13).

For [3H]muscimol binding studies, the extracts of transfected HEK cells were incubated

overnight at 4oC under gentle shaking with either 2,5µg, 10µg or 20µg γ3(1-35) antibodies,

or in a separate control experiment with a mixture of 5µg α1(1-9), 10µg β3(1-13) and 10µg

γ3(1-35) antibodies per culture dish. After addition of Immunoprecipitin and 0.5% nonfat dry

milk powder and shaking for additional 3 h at 4oC, the precipitate was washed three times

with IP low buffer. The precipitated proteins were resuspended in 50 mM Tris/citrate buffer,

pH 7.4 containing 0,1% Triton X-100, at a protein concentration in the range of 0.1 - 1 mg/ml

as measured with the BCA protein assay kit (Pierce Chemical) with bovine serum albumin as

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standard. Aliquots of the suspension were then incubated for 60 min at 4oC in a total of 1 ml

of a solution containing 50 mM Tris/citrate buffer, pH 7.4, 0,1% Triton X-100, and 20nM of

[3H]muscimol (28.1 Ci/mmol, DuPont NEN) in the absence or presence of 1 mM GABA.

Precipitates were then filtered through Whatman GF/B filters, and the filters were washed

twice with 3.5 ml ice-cold 50 mM Tris/citrate buffer and were then subjected to scintillation

counting. Unspecific binding in the presence of 1 mM GABA was subtracted from total

[3H]muscimol binding, to result in specific binding (37).

Immunoprecipitation of receptors expressed on the cell surfaceThe culture medium was

removed from HEK 293 cells transfected with cDNA (21 µg per 3 x 106 cells) of GABAA

receptor subunits (cDNA ratio 1:1:1) and the cells were washed once with phosphate buffered

saline (PBS: 2.7 mM KCl, 1.5 mM KH2PO4, 140 mM NaCl, and 4.3 mM Na2HPO4, pH 7.3).

Cells were then detached from the culture dishes by incubating with 2.5 ml of 5 mM EDTA in

PBS for 5 min at room temperature. The resulting cell suspension was diluted in 6.5 ml of

cold Dulbecco´s modified Eagle medium (DMEM; Life Technologies, Gaithersburg, MD)

and centrifuged for 5 min at 1000 x g.

For Western blot analysis, the pellet from two dishes was incubated with 30 µg α1(1-9) or

γ3(1-35) antibodies in 3 ml of the same medium for 30 min at 37oC. Cells were again pelleted

and free antibodies were removed by washing twice with 10 ml of PBS buffer. Then receptors

were extracted with IP low buffer containing 1% Triton X-100 for 1 hr under gentle shaking.

Cell debris was removed by centrifugation (30 min; 150,000 x g; 4oC). After addition of

Immunoprecipitin and 0.5% nonfat dry milk powder to the clear supernatant and shaking for 3

h at 4oC, the precipitate was centrifuged for 10 min at 10,000 x g and washed three times with

IP low buffer. The precipitated proteins were dissolved in sample buffer and subjected to

SDS-PAGE and Western blot analysis using digoxygenized antibodies. Secondary antibodies

(Anti-Digoxygenin-AP, Fab fragments; Roche Diagnostics GmbH, Mannheim, Germany)

were visualized by the reaction of alkaline phosphatase with CSPD (Tropix, Bedford, MA,

USA). Protein bands were quantified by densitometry of Kodak X-Omat S films with the

Docu Gel 2000i gel documentation system using RFLP scan software (MWG Biotech,

Ebersberg, Germany). The linear range of the detection system was established by

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determining the antibody response to a range of antigen concentrations following

immunoblotting. The experimental conditions were designed such that immunoreactivities

obtained in the assay were within this linear range, thus permitting a direct comparison of the

amount of antigen applied per gel lane between samples. Different exposures of the same

membrane were used to ensure that the measured signal was in the linear range of the x-ray

film.

For radioligand binding studies, the pellet from six dishes was incubated with a mixture of

20µg α1(1-9), 60µg β3(1-13) and 60µg γ3(1-35) antibodies or with 60µg γ3(1-35) antibodies

in 6 ml of DMEM medium for 45 min at 37oC. Cells were again pelleted and free antibodies

were removed by washing twice with 10 ml of PBS buffer. Then receptors were extracted

with IP low buffer containing 1% Triton X-100 for 1 h under gentle shaking. Cell debris was

removed by centrifugation (30 min; 150,000 x g; 4oC). After addition of Immunoprecipitin

and 0.5% nonfat dry milk powder and shaking for 3 hr at 4oC, the precipitate was centrifuged

for 10 min at 10,000 x g and washed three times with IP low buffer. The precipitated proteins

were resuspended in 50 mM Tris/citrate buffer, pH 7.4 containing 0,1% Triton X-100, at a

protein concentration in the range of 0.1 - 1 mg/ml and subjected to [3H]muscimol binding

assay as described above.

To verify that only receptors on the cell surface were labeled by the antibodies, parallel

samples were incubated with antibodies directed against the intracellular loop of GABAA

receptor subunits (experiments not shown). These antibodies could not precipitate any

GABAA receptor subunits under the conditions used. A possible redistribution of the

antibodies during the extraction procedure could be excluded by an experiment performed

analogous to that described (31).

Secondary structure predictionThe EMBL PredictProtein server (38) was used to

align γ3 sequences to homologous sequences available in the SwissProt database and then to

predict the secondary structure based on the set of aligned sequences by the PHDsec method

(39, 40). The significance of this neural network – based secondary structure prediction for

the sequences of interest was evaluated by force field calculations within a Dynamic Monte

Carlo (DMC) optimization scheme (41). In this procedure the structure optimization started

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with the sequence in extended conformation (φ, ψ = 180°). Within one DMC step a single φ,

ψ or side chain dihedral angle was updated in the range [-180,180], the conformational energy

was calculated within the ECEPP/3 force field (42) and the free energy of solvation was

computed based on a continuum solvation model. 2 x 105 DMC steps were performed. The

final acceptance probability was given by a modified Metropolis criterium considering both,

conformational and solvation energies. Details on the optimization algorithm are given in

(43).

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RESULTS

Truncated γ3 constructs are able to assemble with full length α1 and β3

subunitsRecently, it has been demonstrated that γ2(1-113) was the shortest γ2 fragment that

still could assemble with full length α1 and β3 subunits (31). In the present study it was

investigated whether the same holds true for fragment γ3(1-116) that is homologous to γ2(1-

113) (Fig. 1A). For this, fragment γ3(1-116) was either singly transfected into HEK cells or

co-transfected with full length α1 or β3 subunits. Expressed subunits were extracted from

these cells and immunoprecipitated either with γ3(1-35), or with α1(328-382) or β3(345-408)

antibodies, respectively. The precipitate was subjected to SDS-PAGE and Western blot

analysis using digoxygenized γ3(1-35) antibodies. Fragment γ3(1-116) contained a single

glycosylation site and migrated as two protein bands with apparent molecular mass 14 and 16

kDa that presumably represented the unglycosylated and glycosylated fragments, respectively

(Fig. 1B). As shown in Fig. 1C+D, fragment γ3(1-116) could be co-precipitated with α1 or β3

subunits from appropriately transfected HEK cells. These data indicated that binding sites for

α1 and β3 subunits are located on homologous N-terminal fragments of γ2 and γ3 subunits.

To identify amino acid sequences important for binding, the even shorter N-terminal

γ3 fragments γ3(1-84) and γ3(1-72) were generated and their interaction with full length α1

and β3 subunits was investigated after co-transfection into HEK cells. Fragments γ3(1-84)

and γ3(1-72) exhibit a molecular mass of 9.4 and 8.2 kDa, respectively, and migrated as

single protein bands (Fig. 1B) consistent with the observation that these fragments do not

contain a putative glycosylation site (33). Whereas γ3(1-84) still could be co-precipitated with

α1 and β3 subunits (Fig. 1C+D), no co-precipitation with these subunits could be detected for

the shorter fragment γ3(1-72). Although expression of fragment γ3(1-72) was reduced

compared with γ3(1-84) (Fig. 1B), a stable binding of this fragment to α1 or β3 subunits

would have been detected. The inability of α1(328-382) or β3(345-408) antibodies to co-

precipitate the fragment γ3(1-72) or to precipitate the fragment γ3(1-116) after single

transfection into HEK cells (experiments not shown) confirmed the conclusion that these

antibodies did not cross-react with the γ3 fragments used.

The observation that γ3(1-84) still could be co-precipitated with α1 and β3 subunits

was surprising, because this fragment no longer contained the sequences γ3(86-93) and γ3(94-

107) that are homologous to the sequences γ2(83-90) and γ2(91-104) that previously have

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been identified to be important for binding of γ2 to β3 and α1 subunits, respectively (Fig. 1A).

This indicated that γ3 subunits possibly use sequences different from those of γ2 subunits for

assembly with α1 and β3 subunits and that these γ3 sequences are located within the N-

terminal 84 amino acids of the mature γ3 peptide.

Amino acid sequence γ3(70-84) is important for binding to α1 or β3 subunitsIn

order to identify amino acid sequences important for binding to α1 and/or β3 subunits, it was

investigated which γ3 sequences could induce binding to these subunits after incorporation

into a fragment that originally could not bind to α1 or β3 subunits. The fragment α1(1-68)

seemed to be suitable for this purpose because it is homologous to γ3(1-84), but could not be

co-precipitated with α1 or β3 subunits after co-expression in HEK cells (Fig. 2A).

Several chimeras were constructed in which the C-terminal part of the α1(1-68)

fragment was replaced by the corresponding γ3 sequence (Fig. 2A). These chimeras were

transfected into HEK cells together with full length α1 or β3 subunits and expressed

fragments were co-precipitated using α1(328-382) or β3(345-408) antibodies, respectively

(Fig 2B+C). These antibodies were directed against amino acid sequences of the intracellular

loop of α1 or β3 subunits, respectively, and only recognized full length subunits but not the

truncated chimeras. The precipitate was subjected to SDS-PAGE and the proteins were

detected using digoxygenized α1(1-9) antibodies in Western blots. The actual expression of

the chimeras was confirmed by precipitation with α1(1-9) antibodies and detection with

digoxygenized α1(1-9) antibodies (Fig. 2D).

Chim1, in which the sequence α1(35-68) of the α1(1-68) fragment was replaced by

the homologous sequence γ3(51-84), could be co-precipitated with α1 as well as with β3

subunits (Fig. 2A-C). Chim2, however, in which the sequence α1(35-53) was replaced by the

homologous sequence γ3(51-69), could not be co-precipitated with full length α1 or β3

subunits. This indicated that the sequence important for binding to α1 and β3 subunits is

located within the amino acid sequence γ3(70-84). This conclusion was confirmed by the

observation that Chim3, in which the sequence α1(54-68) of the α1(1-68) fragment was

replaced by the homologous sequence γ3(70-84), was able to bind to α1 and β3 subunits (Fig.

2A-C).

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The whole sequence γ3(70-84) is necessary for efficient assembly with full length α1

and β3 subunitsTo investigate which part of the γ3(70-84) sequence is important for

assembly with α1 or β3 subunits, four additional chimeras of the α1(1-68) fragment were

constructed (Fig. 3A).

In Chim4, amino acids α1(61-68) were replaced by amino acids γ3(77-84), in Chim5

amino acids α1(57-68) were replaced by γ3(73-84), in Chim6 α1(54-59) was replaced by

γ3(70-75) and in Chim7 α1(54-62) was replaced by γ3(70-78). All these chimeras were

expressed to a similar extent after transfection into HEK cells (experiments not shown). None

of these chimeras could be co-precipitated with full length β3 subunits indicating that the

complete γ3(70-84) sequence is needed for binding to this subunit (Fig. 3B). Interestingly,

Chim6 could be co-precipitated with full length α1 subunits, but the extent of co-precipitation

was only 6±2% (mean±S.E.M., n=3) of that of Chim3 as quantified by densitometry of

protein bands (see Experimental Procedures). Binding of Chim7 to α1 subunits was stronger

than that of Chim6, but the extent of co-precipitation of Chim7 with α1 subunits was still only

19±5% (mean±S.E.M., n=3) of that of Chim3. Thus, the complete γ3(70-84) sequence was

necessary to allow binding to α1 subunits with full efficiency. Interestingly, all these

chimeras contained a single glycosylation site and thus, migrated as two peptides with a

molecular mass of 10 and 12 kDa. On co-precipitation experiments, predominantly the

glycosylated form of Chim3, Chim6 and Chim7 or Chim3 was precipitated with α1 or β3

subunits, respectively (Fig. 3B).

The sequence γ3(70-84) is important for assembly of GABAA receptors composed of

α1β3γ3 subunitsTo investigate the importance of the γ3(70-84) sequence not only for the

assembly of truncated with the full length subunits, but also for assembly of full length

subunits and receptors expressed on the cell surface, a full length γ3 chimera (γ3*) was

constructed in which the sequence γ3(70-84) was replaced by the homologous sequence

ρ1(92-106). The sequence of the ρ1 subunit shares 32,4% sequence identity with the γ3

subunit (Align; Genestream; IGH Montpellier, France) and was chosen because ρ1 was

reported not to assemble with α1 or β3 subunits (44, 45). In control experiments (Fig. 4A) it

was demonstrated that the extent of expression of the γ3* chimera was similar to that of the γ3

subunit in HEK cells. Subunits γ3 as well as γ3* migrated as protein bands with apparent

molecular mass of 43 kDa (46). The weak staining of protein bands with an apparent

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molecular mass of about 51 kDa presumably was caused by a cross-reactivity of the antibody

with a HEK cell protein (Fig. 4A).

HEK cells were co-transfected with α1, β3 and γ3* or with α1, β3 and γ3

subunits. GABAA receptors expressed on the cell surface were labeled by an incubation of

intact cells with γ3(1-35) antibodies. Antibody-labeled receptors were extracted and

precipitated by addition of Immunoprecipitin. The precipitate was subjected to SDS-PAGE

and Western blot analysis using digoxygenized γ3(1-35) antibodies (Fig. 4B). The staining

intensity of the protein bands was quantified and results obtained from two experiments

indicated that the amount of γ3* subunits on the gel was only 19% and 31% of of that

precipitated from α1β3γ3 transfected cells. The Western blot was then stripped and analyzed

using digoxygenized α1(1-9) antibodies, followed by another stripping procedure as well as

detection with digoxygenized β3(1-13) antibodies. The amounts of α1 subunits (51 kDa) co-

precipitated with γ3* subunits were 23% and 28%, and those of β3 subunits (54 kDa) were

23% and 30% of those precipitated from the surface of α1β3γ3 transfected cells. The similar

reduction in α1, β3, and γ3* subunits precipitated by γ3(1-35) antibodies suggested that all

γ3* subunits on the cell surface were associated with α1 and β3 subunits. The reduction in the

amount of γ3* containing receptors formed on the cell surface indicated the importance of the

sequence γ3(70-84) for the assembly of full length subunits.

In similar experiments, GABAA receptors formed on the surface of α1β3γ3* or

α1β3γ3 transfected cells were labeled by an incubation with α1(1-9) antibodies and the

antibody labeled receptors were again extracted, precipitated by Immunoprecipitin and

subjected to SDS-PAGE and Western blot analysis using digoxygenized γ3(1-35) antibodies.

In agreement with results shown in Fig. 4B, the amount of γ3* subunits co-precipitated by the

α1(1-9) antibody was only 22±4% (mean±S.E.M., n=6, from six different transfections) of

that of the wild-type γ3 subunit (Fig. 5).

The Western blots were then again stripped and re-analyzed using digoxygenized

α1(1-9) or β3(1-13) antibodies. Results obtained revealed that comparable amounts of α1 or

β3 subunits could be detected on the surface of cells transfected with α1, β3 and γ3*, or α1,

β3 and γ3 subunits (Fig. 5). Similar results were obtained when the order of detection of

subunits was changed and Western blots were first probed with digoxygenized α1(1-9)

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antibodies and after stripping were re-analyzed with digoxygenized γ3(1-35) or β3(1-13)

antibodies. The finding that in spite of the strong reduction of γ3* containing receptors

comparable amounts of α1 or β3 subunits could be detected on the surface of α1β3γ3* or

α1β3γ3 transfected cells, then seems to indicate that in addition to α1β3γ3* receptors, α1β3

receptors were formed in these cells.

This conclusion was supported by experiments investigating [3H]muscimol binding on

the cell surface. Previous studies have indicated that [3H]muscimol binding sites on the cell

surface are only formed by receptors composed of αβ or αβγ subunits (37). HEK cells

transfected with α1β3γ3 or α1β3γ3* subunit combinations were incubated either with a

mixture of α1(1-9), β3(1-13) and γ3(1-35) antibodies for labeling of αβ plus αβγ receptors, or

with γ3(1-35) antibodies for labeling of γ3 or γ3* containing receptors. Labeled receptors

were then extracted from the cells, precipitated by the addition of Immunoprecipitin, and

incubated with 20nM [3H]muscimol in the absence or presence of 1mM GABA. The mixture

of antibodies precipitated comparable amounts of specific [3H]muscimol binding sites (and

thus of total receptors) from the surface of α1β3γ3 or α1β3γ3* transfected cells (340±14 or

351±25 fmol/mg protein, respectively; mean±S.E.M., n=3). The number of [3H]muscimol

binding sites precipitated by γ3(1-35) antibodies, however, was only 96±5 or 30±4 fmol/mg

protein, respectively (mean±S.E.M., n=3). The apparently low percentage of [3H]muscimol

binding sites precipitated by γ3(1-35) antibodies from the surface of α1β3γ3 or α1β3γ3*

transfected cells at least partially was due to the relatively low avidity of these antibodies.

These antibodies, however, exhibited the same precipitation efficiency for α1β3γ3 and

α1β3γ3* receptors (experiments not shown), indicating that the difference in receptors

precipitated reflected the difference in receptors expressed on the surface of α1β3γ3 or

α1β3γ3* transfected cells. Since cell surface labeling, extraction and precipitation of

receptors as well as [3H]muscimol binding assays were performed in the same experiments

and under exactly identical conditions, these data indicate that [3H]muscimol binding to γ3*

containing receptors represented only about 31% of that to γ3 containing receptors.

Considering the variability of the methods used, these data were in good agreement with

results from Western blot experiments and suggested the formation of small amounts of

receptors composed of α1β3γ3* subunits on the cell surface of appropriately transfected cells.

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The electrophysiological properties of α1β3γ3* receptors formed could not be

investigated because it was not possible to unequivocally separate these low abundance

receptors from the large surplus of functional α1β3 receptors2.

Secondary structure prediction for amino acid sequence γ3(70-84)To investigate the

secondary structure of the amino acid sequence γ3(70-84), a neural network-based prediction

was performed using PHDsec (39, 40) for the sequence γ3(68-86). This procedure predicted a

low probability for the formation of an α-helix (below 1 in a probability scale [0,10]), a

higher probability (around 6) for the formation of a loop within the sequence γ3(68-72) and a

high probability (around 7) for the formation of an extended structure for the sequence γ3(73-

84) (data not shown). This prediction was essentially confirmed by results from force field

calculations within a Dynamic Monte Carlo optimization scheme. Low energy structures

exhibiting a potential energy of about 10 kcal/mol below random coil structures showed a

loop for residues γ3(70-76), and an extended structure along the residues γ3(77-84) that

potentially could form a β-sheet (data not shown). Overall, the predicted structure is probably

not sufficiently stable to form an autonomous structural element, but could gain induced

structural stability on interaction with intra- or intermolecular partner sequences.

Regions homologous to previously identified putative γ2 assembly sites are also

important for assembly of γ3 subunitsThe identification of γ3(70-84) as a sequence

important for assembly with α1 and β3 subunits does not exclude the presence of additional

sequences with similar importance in this subunit. It was thus investigated whether the

sequences γ3(86-93) and γ3(94-107) that are homologous to previously identified putative

assembly sites on γ2 subunits (Fig. 1A) (31), are also important for assembly of γ3 subunits.

Applying an approach previously used for the identification of the respective

sequences on the γ2 subunits, the sequences γ3(86-95) and γ3(94-107) were incorporated into

the truncated fragment α1(1-100) which per se was not able to bind to α1 or β3 subunits (13,

31) (Fig. 6A). In these experiments, γ3(86-93), one of the sequences homologous to the

previously identified γ2 sequences, was slightly elongated to γ3(86-95) to avoid loss of a

glycosylation site that possibly could be important for assembly (31). The resulting chimeras

Chim8 and Chim9 were then co-transfected into HEK cells with full length α1 or β3 subunits

and co-immunoprecipitated using α1(328-382) or β3(345-408) antibodies, respectively.

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As shown in Fig. 6B, Chim8 could be co-precipitated by the α1(328-382) antibody

after co-transfection with α1 subunits into HEK cells, indicating that the region γ3(86-95) is

sufficient to induce interaction of the chimeric α1(1-100) fragment with α1 subunits. Chim8

contains two glycosylation sites and migrated as two peptides with apparent molecular mass

14 and 16 kDa which represented the mono- and diglycosylated fragments, respectively. The

unglycosylated peptide with apparent molecular mass 12 kDa could only be detected on

extremly long exposures (experiments not shown). Interestingly, Chim8 on co-transfection

with β3 subunits, could also be co-precipitated with β3(345-408) antibodies. Under these

conditions only the diglycosylated form was co-precipitated, possibly indicating that β3

subunits preferentially interacted with the fully glycosylated fragment (Fig. 6C). These results

are similar to those obtained previously with the α1(1-100) chimera containing the

homologous γ2 sequence (31) and indicated that the sequence γ3(86-95) can induce binding to

α1 as well as β3 subunits (Fig. 6A). Unfortunately, migration of the protein bands in the two

blots (Fig. 6B+C) cannot be directly compared because the blots were taken from different

gels.

Similar to Chim8, Chim9 could be co-precipitated by α1(328-382) antibodies on co-

transfection with α1 subunits into HEK cells (Fig. 6B). In Chim9 the glycosylation site γ3N93

is lost (Fig. 6A) and, therefore, this chimera contains only a single glycosylation site. As

shown in Fig. 6B, Chim9 migrated as two peptides with apparent molecular mass 12 and 14

kDa which represented the unglycosylated and glycosylated fragments, respectively. In

contrast to Chim8, however, Chim9 could not be precipitated by β3(345-408) antibodies from

HEK cells co-transfected with Chim9 and β3 subunits (Fig. 6C). This indicated that the

sequence γ3(94-107) cannot induce interaction with β3 subunits and is in agreement with

previous results for the homologous sequence of the γ2 subunit (31). It was, therefore,

concluded that not only the sequence γ3(70-84), but also sequences homologous to those

previously identified on the γ2 subunit, are important for assembly of the γ3 with α1 or β3

subunits.

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DISCUSSION

Amino acid sequence γ3(70-84) is important for assembly with α1 and β3 subunitsIn

the present study, it was investigated, whether γ3 amino acid sequences homologous to

previously identified putative binding sites on the γ2 subunit are important for assembly with

α1 and β3 subunits. It was demonstrated that the C-terminally truncated γ3 fragment γ3(1-84)

could still bind to α1 or β3 subunits in appropriately co-transfected HEK cells, as indicated

by co-immunoprecipitation with subunit-specific antibodies, although this fragment lacked

the putative binding sites found in γ2 subunits. From this it was concluded that residues

important for assembly with α1 and β3 subunits were located within the first 84 amino acids

of the mature γ3 peptide.

Sites important for binding were then identified by incorporating various γ3 sequences

into the α1(1-68) fragment. This fragment is homologous to γ3(1-84) but in contrast to the

latter fragment could not bind to α1 or β3 subunits after co-expression in HEK cells. Results

indicated that replacement of the sequence α1(54-68) of the α1(1-68) fragment by the

sequence γ3(70-84) was sufficient to induce binding of the chimeric construct to full length

α1 or β3 subunits.

To investigate whether it is possible to define distinct amino acid residues in γ3(70-84)

important for binding to α1 or β3 subunits, several additional chimeras were constructed by

incorporating parts of the γ3(70-84) sequence into α1(1-68) fragments. None of these new

chimeras could interact with β3 subunits, indicating that the complete γ3(70-84) sequence

was necessary for inducing interaction with β3 subunits. Although the sequences γ3(70-75)

and γ3(70-78) were sufficient to induce weak binding of the chimeric α1(1-68) fragment to

the α1 subunit, strong binding to the α1 subunit again could only be demonstrated with the

complete γ3(70-84) sequence. This either indicated that only the complete γ3(70-84) sequence

can form the respective contact site to α1 or β3 subunits or that the complete sequence is

necessary for stabilizing other regions elsewhere in the protein that then can interact with the

adjacent subunits.

To investigate the importance of the γ3(70-84) sequence not only for interaction of

subunits, but also for assembly of α1β3γ3 receptors a chimeric γ3 subunit (γ3*) was

constructed in which the γ3(70-84) sequence was replaced by the homologous ρ1(92-106)

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sequence. The γ3* construct was then co-expressed with α1 and β3 subunits in HEK cells.

Immunolabeling and quantification of receptors on the cell surface indicated a 70-80%

reduction of receptors containing α1, β3 and γ3* subunits. The additional observation that the

amount of specific [3H]muscimol binding sites as well as of α1 or β3 subunits on the cell

surface was comparable in α1β3γ3* or α1β3γ3 transfected cells suggested that due to the

reduced efficiency of assembly of γ3* subunits, in addition to α1β3γ3* receptors, receptors

composed of α1β3 subunits had been formed. These results again confirmed the importance

of the γ3(70-84) sequence for assembly of GABAA receptors. The remaining formation of

α1β3γ3* receptors on the cell surface can be explained by the existence of additional

assembly sites on the γ3 subunit that can partially compensate for the loss of one assembly

site caused by the absence of the γ3(70-84) sequence in γ3* subunits.

Amino acid residues contributing to the benzodiazepine binding site are located within

γ3(70-84) The benzodiazepine binding site of GABAA receptors is located at the interface

between α and γ subunits and several amino acid residues have been identified on these

subunits (α1H101, α1Y159, α1T206, α1Y209 and γ2F77, γ2M130, respectively) that

contribute to this site (34, 47). Due to the high sequence homology between γ2 and γ3

subunits (33) it can be assumed that these subunits exhibit a comparable structure and

comparable interactions with neighbouring subunits. In addition, it can be assumed that amino

acid residues homologous to those forming the benzodiazepine binding site in α1β3γ2

receptors will also form this site in α1β3γ3 receptors. Interestingly, the amino acid residue

γ3F80 which is located within the sequence γ3(70-84) important for binding to α1 or β3

subunits, is homologous to γ2F77 that contributes to the benzodiazepine binding site. It is thus

highly likely that this amino acid residue also contributes to the benzodiazepine binding

pocket of α1β3γ3 receptors and is located at the α1/γ3 interface.

A neural network based prediction using PHDsec as well as a Dynamic Monte Carlo

simulation, both performed in the present study, indicated that the sequence γ3(77-84) could

form a β-sheet. This prediction is not only supported by experiments using the substituted

cysteine accessibility method (48) for the homologous sequence γ2(74-81), but also by the

recently published crystal structure of the acetylcholine binding protein (AChBP), that can be

used as a model for the structure of the extracellular part of the GABAA receptor (49).

Altogether this supports the validity of the theoretical calculations performed in this study and

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suggests that every second amino acid residue in this region might be involved in intra- or

intermolecular contacts, whereas the other residues are water accessible (48) and some of

them form part of the benzodiazepine binding pocket. In addition, the crystal structure of the

AChBP indicates that amino acid residue W53, that is homologous to γ3F80 is located at the

minus side of a dimer interface of the AChBP that corresponds with the α1/γ3 interface of the

GABAA receptors. Since γ3F80 might be solvent accessible like the homologous γ2F77

residue (48) and not be buried in the contact zone between the two subunits, nearby residues

might be involved in subunit/subunit interactions.

Interestingly, a second amino acid residue T45 of the AChBP homologous to γ3N72

contributes to the other side of the interface of the subunit, referred as plus side (49). The

observation that incorporation of the sequence γ3(70-73) into α1(1-68) induced a weak

binding to α1, but not to β3 subunits, then possibly indicates that amino acid residues within

this region might allow accommodation of the α1 subunit at the other interface of the γ3

subunit (plus side) when only α1 and γ3 subunits are present. Interestingly, the sequence

γ3(70-73) seems not to be important for binding of β3 subunits. This is surprising, because γ3

subunits should accommodate an α1 subunit at its minus and a β3 subunit at its plus side (13).

It is possible, however, that γ3 subunits use different contacts for interaction with α1 and β3

subunits at the plus side. A similar observation has been made previously for the α1 subunit,

in which distinct plus sides seem to be important for binding to β3 or γ2 subunits (13).

It has to be considered, however, that the AChBP forms homopentamers, while the

receptor studied in this work is composed of α1, β3 and γ3 subunits. It is, thus, possible that

the plus and minus sides of the GABAA receptor γ3 subunit are located slightly different from

those of the AChBP (49) and that the whole sequence γ3(70-84) is located at the α1/γ3

interface and exclusively contributes to the minus side. The observation that incorporation of

γ3(70-84) into the α1(1-68) fragment not only induced binding to α1 but also to β3 subunits

might then indicate that β3 subunits can interact with the minus side of γ3 subunits when no

α1 subunits are available. In the presence of both α1 and β3 subunits, however, the minus

side seems to exclusively assemble with α1 subunits because of energetic and/or steric

reasons. Alternatively, the sequence γ3(70-84) might stabilize an assembly site for β3

subunits at the plus side of γ3 subunits in the absence or presence of α1 subunits.

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At least two distinct γ3 sequences are important for assembly with α1 subunitsThe

identification of γ3(70-84) as a novel site important for assembly of GABAA receptors does

not exclude the existence of additional assembly sites on γ3 subunits. In other experiments it

was, therefore, investigated whether the sequences γ3(86-95) and γ3(94-107), that are

homologous to previously identified putative intersubunit contact sites on γ2 subunits (31),

are important for binding to α1 or β3 subunits. These sequences were incorporated into the

α1(1-100) fragment which per se could not bind to α1 or β3 subunits. Incorporation of the

sequence γ3(94-107) induced binding to α1 but not to β3 subunits, indicating that this

sequence facilitates a selective assembly with α1 subunits. These data are in agreement with

the finding that the homologous site on the γ2 subunit also is important for specific assembly

with α1 subunits (31). The observation that amino acid residues S75 and P77 of the AChBP

contribute to the minus side (that is homologous to the α/γ interface of GABAA receptors),

supports the conclusion that the corresponding GABAA receptor residues γ3S103 and

γ3M105 are located at the α1/γ3 interface. The sequence γ3(94-107), or part of it, might thus

be directly involved in forming the α1/γ3 interface.

Incorporation of the sequence γ3(86-95) into the α1(1-100) fragment induced binding

to α1 as well as β3 subunits. These results again are consistent with results obtained for the

homologous sequence on the γ2 subunit (31). Based on the AChBP structure, the γ3(86-95)

region does not appear to be in a position to directly interact with the α1 or β3 subunits, but

this sequence contains several amino acid residues highly conserved in the whole receptor

superfamily (50). Such residues might be important for stabilizing the fold of the subunit and

thus, might stabilize the conformation of assembly sites elsewhere in the protein.

Thus, interestingly, at least two amino acid sequences (γ3(70-84 and γ3(94-107)) have

been identified on the γ3 subunit in the present study that seem to be located at the same

interface (49) and either can directly bind to α1 subunits or stabilize such binding sites. This

is the first time that a GABAA receptor subunit has been shown to contain more than one N-

terminal sequence important for contacts with the same neighbouring subunit during assembly

of the receptor. The formation of multiple intersubunit contacts in this receptor superfamily is

supported by the crystal structure of the AChBP that indicates that there are seven

topologically distinct regions on a subunit forming contacts with the same adjacent subunit

(49). Further experiments will have to be performed to clarify whether the sequences

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investigated in the present study are only important for primary contacts of subunits or also

for contacts in the completely assembled receptor.

In different subunits homologous regions might be important for assemblyAlthough

several different putative assembly sites have been identified so far on GABAA receptor

subunits (13, 29-31, 50), it is not known whether in different subunits homologous sequences

are important for interaction with neighbouring subunits. Here, it was demonstrated that

sequences γ3(86-95) and γ3(94-107) that are homologous to previously identified sequences

important for assembly of γ2 subunits, are also important for assembly of γ3 subunits. In

addition, we have demonstrated that the γ2 sequence homologous to the newly identified

γ3(70-84) sequence seems also to be important for assembly of γ2 subunits3. These data for

the first time demonstrate that in different subunits of the same subunit class homologous

sequences are important for assembly.

Interestingly, it has been demonstrated previously that α1(58-67), a sequence

homologous to part of the newly identified γ3(70-84) sequence (see Fig. 6A for sequence

comparison), mediated binding to β3 subunits (30). This finding confirms the importance of

the newly identified sequence γ3(70-84) for assembly and supports the conclusion that even

in subunits belonging to different subunit classes homologous sequences are important for

intersubunit contacts. It is highly likely, however, that different combinations of subunits

exhibit slightly different interactions (13), ensuring binding selectivity and a fixed receptor

stoichiometry.

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AcknowledgmentsWe want to thank Dr. Roman Furtmüller for preliminary

electrophysiological experiments, Susanne Karall and Elisabeth Dögl for excellent technical

support and Nahid Fathi for cloning the full length γ3 subunit.

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1 The abbreviations used are: GABA, γ-aminobutyric acid; nACh, nicotinic acetylcholine;

HEK, human embryonic kidney; PAGE, polyacrylamide gel electrophoresis; IP low buffer,

low salt immunoprecipitation buffer; PBS, phosphate buffered saline; DMEM, Dulbecco´s

modified Eagle medium; DMC, Dynamic Monte Carlo; AChBP, acetylcholine-binding

protein. 2 (R. Furtmüller, unpublished experiments) 3 (Sarto et al., in preparation)

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FIGURE LEGENDS

FIG. 1. Co-immunoprecipitation of truncated γ3 fragments with full length α1 and β3

subunits. A, schematic drawing of C-terminal sequences of γ2 and γ3 fragments. The

sequence of the fragments is indicated by the amino acid numbers given in parentheses. 1

represents the first amino acid of the mature subunit. The sequences shown are indicated by

the numbers above the amino acid single letter code representing the position of the

respective amino acid residue. The previously identified putative γ2 assembly sites for α1 or

β3 subunits as well as the homologous sites on the γ3 subunit are boxed. + indicates co-

precipitation, and - indicates absence of co-precipitation of these constructs with full length

α1 or β3 subunits. B, in control experiments the truncated γ3 fragments were transfected into

HEK cells, extracted with Lubrol extraction buffer and immunoprecipitated using γ3(1-35)

antibodies. The precipitate was subjected to SDS-PAGE and the constructs were detected

using digoxygenized γ3(1-35) antibodies. The protein fragment γ3(1-116) exhibited an

apparent molecular mass of 14 and 16 kDa (the un- and monoglycosylated peptide,

respectively), whereas fragments γ3(1-84) and γ3(1-72) migrated as bands of 9.4 kDa and 8.2

kDa, respectively. C, HEK cells expressing the respective C-terminally truncated fragments

together with full length α1 subunits were extracted, proteins were precipitated with α1(328-

382) antibodies and subjected to Western blot analysis using digoxygenized γ3(1-35)

antibodies. D, HEK cells expressing the C-terminally truncated fragments together with full

length β3 subunits were extracted, proteins were precipitated with β3(345-408) antibodies

and subjected to Western blot analysis using digoxygenized γ3(1-35) antibodies. All

experiments were performed three times with comparable results.

FIG. 2. γ3(70-84) is important for binding to α1 and β3 subunits. A, C-terminal sequences

of the γ3(1-84) and α1(1-68) fragments as well as of different chimeras are shown

schematically. Amino acid sequences of the γ3 subunit are boxed. HEK cells were co-

transfected with these constructs together with full length α1 or β3 subunits, extracted

proteins were precipitated with α1(328-382) (designed as α1L-Ab) or β3(345-408) antibodies

(designed as β3L-Ab), respectively, and subjected to SDS-PAGE and Western blot analysis

using digoxygenized α1(1-9) antibodies (designed as α1N-Ab). A possible co-

immunoprecipitation was investigated as described under „Results“. + indicates co-

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precipitation, and - indicates absence of co-precipitation of these constructs with the

respective full length subunits. B+C, Western blots demonstrating co-precipitation of

different chimeras with full length α1 or β3 subunits. All chimeric constructs contained a

single glycosylation site. The precipitated constructs, however, migrated predominantly as the

glycosylated form with an apparent molecular mass of 12 kDa. The protein with apparent

molecular mass 51 kDa in B represents the full length α1 subunit. All experiments were

performed three times with comparable results. D, Western blots demonstrating comparable

expression of chimeric fragments. HEK cells expressing the chimeric fragments alone were

extracted, proteins were precipitated with α1(1-9) antibodies (designed as α1N-Ab), and

subjected to SDS-PAGE and Western blot analysis using digoxygenized α1N-Ab antibodies.

FIG. 3. Co-immunoprecipitation of chimeric constructs with full length α1 or β3

subunits. A, the C-terminal sequences of Chim3, of the α1(1-68) fragment, as well as of

different chimeras are shown schematically. Amino acid sequences of the γ3 subunit are

boxed. HEK cells were co-transfected with these constructs together with full length α1 or β3

subunits, extracted proteins were precipitated with α1(328-382) or β3(345-408) antibodies,

respectively, and subjected to SDS-PAGE and Western blot analysis using digoxygenized

α1(1-9) antibodies. A possible co-immunoprecipitation was investigated as described under

„Results“. +++ indicates strong co-precipitation, + indicates weak co-precipitation, +/-

indicates very weak co-precipitation and - indicates absence of co-precipitation of these

constructs with the respective full length subunits. B, Western blots demonstrating co-

precipitation of chimeric constructs with full length α1 or β3 subunits. All chimeric

constructs contained a single glycosylation site, but all co-precipitated constructs migrated

predominantly as the glycosylated form with an apparent molecular mass of 12 kDa. All

experiments were performed three times with comparable results.

FIG. 4. Western blot analysis of GABAA receptors labeled on the surface of HEK cells

using γ3(1-35) antibodies. A, In control experiments, HEK cells were co-transfected with

α1, β3, and γ3* or α1, β3, and γ3 subunits. The γ3* chimeric construct was a full length γ3

subunit in which the sequence γ3(70-84) was replaced by the homologous sequence ρ1(92-

106) of the ρ1 subunit. Proteins were extracted from cells, immunoprecipitated by γ3(1-35)

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antibodies and subjected to Western blot analysis using digoxygenized γ3(1-35) antibodies. γ3

subunits as well as γ3* subunits were expressed to a comparable extent and exhibited an

apparent molecular mass of 43 kDa. The proteins with apparent molecular mass of about 51

kDa were labeled due to a cross-reactivity of the antibody with HEK cell proteins. B, HEK

cells were co-transfected with α1, β3, and γ3*, or with α1, β3, and γ3 subunits. GABAA

receptors expressed on the surface were immunolabeled by an incubation of intact cells with

γ3(1-35) antibodies, and were then extracted, precipitated by Immunoprecipitin and subjected

to SDS-PAGE and Western blot analysis using digoxygenized γ3(1-35) antibodies. Blots were

then stripped and re-analyzed with digoxygenized α1(1-9) antibodies. Then blots again were

stripped and re-analyzed with digoxygenized β3(1-13) antibodies. The experiment was

performed twice with comparable results. α1, β3, and γ3 subunits migrated as protein bands

of 51 kDa, 54 kDa and 43 kDa, respectively.

FIG. 5. Western blot analysis of GABAA receptors labeled on the surface of HEK cells

using α1(1-9) antibodies. HEK cells were co-transfected with α1, β3, and γ3*, or with α1,

β3, and γ3 subunits. GABAA receptors expressed on the surface were immunolabeled by an

incubation of intact cells with α1(1-9) antibodies, and were then extracted, precipitated by

Immunoprecipitin and subjected to SDS-PAGE and Western blot analysis using

digoxygenized γ3(1-35) antibodies. Blots were then stripped and re-analyzed with

digoxygenized α1(1-9) antibodies. Then blots again were stripped and re-analyzed with

digoxygenized β3(1-13) antibodies. Comparable results were obtained when the order of

detection of subunits was changed. These experiments were performed six times with

comparable results. α1, β3, and γ3 subunits migrated as protein bands of 51 kDa, 54 kDa and

43 kDa, respectively. The average amount of γ3* subunits detected with γ3(1-35) antibodies

in the α1-precipitate of receptors present on the cell surface was 22±4% (n=6) of γ3 subunits.

The intensity of γ3* subunit staining shown on this blot represents 15% of γ3 subunit staining.

FIG. 6. Amino acid sequences γ3(86-95) and γ3(94-107) are also important for assembly

of γ3 subunits. A, The C-terminal sequences of the α1(1-100) and γ3(1-116) fragments as

well as of different chimeras are represented schematically. The position of amino acid

residues is given by numbers on top of the single letter code and amino acid sequences of the

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γ3 subunit are boxed. HEK cells were co-transfected with these constructs together with full

length α1 or β3 subunits, extracted proteins were precipitated with α1(328-382) or β3(345-

408) antibodies, respectively, and subjected to SDS-PAGE and Western blot analysis using

digoxygenized α1(1-9) antibodies. A possible co-immunoprecipitation was investigated as

described under „Results“. + indicates co-precipitation, and - indicates absence of co-

precipitation of these constructs with the respective full length subunits. B, Western blots

demonstrating co-precipitation of chimeric constructs with full length α1 subunits. C,

Western blots demonstrating co-precipitation of chimeric constructs with full length β3

subunits. Migration of the protein bands in Fig. 6B+C cannot be directly compared because

the blots were taken from different gels. All experiments were performed three times with

similar results.

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FIG 1.

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FIG. 2

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FIG. 3

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FIG. 4

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FIG. 5

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FIG. 6

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Page 36: A novel site on γ3 subunits important for assembly of GABAA receptors

Werner SieghartIsabella Sarto, Thomas Klausberger, Noosha Ehya, Bernd Mayer, Karoline Fuchs and

3 subunits important for assembly of GABAA receptorsγA novel site on

published online June 13, 2002J. Biol. Chem. 

  10.1074/jbc.M203597200Access the most updated version of this article at doi:

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