(Received for publication, December 18, 1996, and in revised form, February 24, 1997)
From the Center for Medical Genetics, Departments of
Pediatrics and ¶ Physiology, The Johns Hopkins
University School of Medicine, Baltimore, Maryland 21287
-Aminobutyric acid type C
(GABAC) receptors identified in retina appear to be
composed of GABA
subunits. The purpose of this study was to
localize signals for homooligomeric assembly of
1 subunits and to
investigate whether the same region contained signals for
heterooligomeric interaction with
2 subunits. In vitro
translated human
1 was shown to be membrane-associated, and
proteinase K susceptibility studies indicated that the N terminus was
oriented in the lumen of ER-derived microsomal vesicles. This orientation suggested the involvement of the N terminus of
1 in the
initial steps of subunit assembly. To test this hypothesis, mutants
were created containing only N-terminal sequences (N-
1) or
C-terminal sequences (C-
1) of
1. Co-immunoprecipitation studies revealed that N-
1, but not C-
1, interacted with
1 in
vitro. When coexpressed in Xenopus oocytes, N-
1
interfered with
1 receptor formation. Together, these data suggested
that signals for
1 homooligomeric assembly reside in the N-terminal
half of the subunit. Sequential immunoprecipitations were then
performed upon cotranslated
1 and
2 subunits which demonstrated
that
1 and
2 interacted in vitro.
Co-immunoprecipitation indicated that N-
1 specifically associated
with
2. Therefore, the N-terminal regions of
subunits contain
the initial signals for both homooligomeric and heterooligomeric assembly into receptors with GABAC properties.
In the brain and retina, inhibitory neurotransmission is
predominantly achieved through activation of receptors for
GABA1 (1). A novel class of receptors for
GABA, termed type C or GABAC, has been identified in the
retinae of a variety of species including white perch, catfish, hybrid
bass, salamander, rat, and cow (2-7). GABAC receptors
share a number of similarities with the well-characterized
GABAA receptors including an integral Cl
channel that can be inhibited by picrotoxin and sensitivity to divalent
metal cations such as Zn2+ (2-4). However, these novel
receptors are insensitive to compounds that have classically been used
to define GABA receptor types (2-3, 8). Furthermore, the
ligand-binding site of GABAC receptors is distinguished
from GABAA and GABAB receptors in its apparent preference for GABA analogues that can adopt a folded conformation, such as cis-4-aminocrotonic acid, over those that are locked
in an extended conformation (9-12). These observations indicate that GABAC receptors may have a molecular structure similar to
their closest relative, GABAA receptors, but with distinct
physical differences that confer unique biophysical and pharmacologic
properties.
Two GABA receptor subunits, 1 and
2, have been isolated from a
human retina library that have amino acid and structural similarity to
members of the ligand-gated ion channel superfamily and to the
GABAA receptor subunits in particular (13-16). However, human
subunits form homooligomeric receptors in Xenopus
oocytes that are insensitive to bicuculline, barbiturates, and
benzodiazepine (13, 15-16). Also,
receptors have a higher affinity
for GABA and little desensitization compared with GABAA
receptors (16). The pharmacologic similarities between
GABAC receptors and
subunits lead to the suggestion
that
subunits are the molecular components of GABAC
receptors (12, 15). The localization of both
subunits by in
situ hybridization and immunochemistry to rat rod bipolar cells
where GABAC responses have been detected further supports the inclusion of
subunits in GABAC receptors (17-18).
Although human
1 and
2 are each capable of forming homooligomeric
receptors, there is functional evidence suggesting rat
1 and
2
may form heterooligomeric receptors (19). Thus, it is possible that
GABAC receptors in vivo are homooligomeric or
heterooligomeric combinations of
1 and
2 subunits.
In this study, in vitro translated protein and
Xenopus oocyte expression were used to identify sequences
involved in subunit assembly. The in vitro translation
system was chosen for two reasons: the ability of protein interaction
from signals in the primary amino acid sequence can be examined
independent of cell-specific factors, and the availability of each
protein for interaction can be verified. A critical question was the
orientation of the in vitro translated subunit protein
relative to the membrane. Using protease-susceptibility studies, we
demonstrated that the orientation of
1 is consistent with the
predicted topology of the ligand-gated ion channel superfamily, with an
extracellular N-terminal domain and a membrane-associated C-terminal
domain. Specific antibodies were developed to test the interaction of
1 with truncated forms of
1 and with
2. Our results
demonstrate that
1 assembles with
2 in vitro, implying
that they are capable of forming a single receptor in vivo,
and that the N terminus of
1 contains signals for homooligomeric and
heterooligomeric assembly.
cDNA Constructs
An 8-residue synthetic epitope (FLAG: DYKDDDDK)
recognized by the mouse monoclonal M2 antibody (Eastman Kodak) was
inserted in-frame at the C terminus of 1 by a three-way ligation of
two annealed oligonucleotides with MaeI and NcoI
sites (sense: 5
-TACGACTACAAGGACGACGATGACAACAAGTAGC-3
; antisense:
5
-CATGGCTACTTGTCATCGTCGTCGTCCTTGTAGTCG-3
), a
KpnI/MaeI fragment containing the
1 cDNA,
and a KpnI/NcoI-digested pBluescript vector
(Stratagene). The amber termination codon of
1 was replaced by an
amber codon in the oligonucleotides. Oocyte injection and current
recordings demonstrated that
1FLAG formed homooligomeric GABA-gated
receptors with properties indistinguishable from native
1 (22).
A triplicated 9-residue epitope of the influenza
virus hemagglutinin (HA tag: TPYDVPDYA) recognized by the monoclonal
antibody 12CA5 (20) was ligated into either the N terminus or C
terminus of the human 2 subunit. Kpn2I recognition sites
were included in PCR primers complementary to the 5
and 3
ends of the
HA tag (5
HA-Kpn2I 5
-ATCCGGATCTTTTACCCATAC-3
,
restriction site is underlined; 3
HA-Kpn 2I
5
-TTCCGGAGAGCAGCGTAATCTGG-3
). Following PCR and subcloning into pCRII (Invitrogen), a 120-bp Kpn2I fragment
with the correct HA tag sequence was ligated into the Kpn2I
site of
2 at nucleotide 229 in the N terminus of
2, 30 residues
downstream from the predicted signal peptide cleavage site. Inserting
the tag at this position was predicted to not alter
2 channel
function on the basis of a chimera domain-swapping strategy which
excluded this region from being necessary for robust homooligomeric
expression (21). To introduce the HA epitope to the C terminus of
2,
StyI sites were included in PCR primers HA-forward
(5
-TTCCAAGGGCCGCATCTTTTAC-3
) and HA-reverse
(5
-ATCCTTGGCTAGCACTGAGCAGCGTAATC-3
). The 118-bp PCR
product was subcloned into the StyI site at the
2
termination codon (nucleotide 1474). The resulting construct was
sequenced in entirety. Both
2-5
HA and
2-3
HA were tested for
interaction with
1 and will be referred to as
2HA for
simplicity.
N-1 was created by replacing the tyrosine
residue of
1 at codon 256 with an amber termination codon (TAG)
using the mutagenic oligonucleotide
5-CTGTGCTGCTCTAGAAAGCCA-3
. The resulting mutant
1 is
truncated 18 residues prior to the predicted first transmembrane domain
(TM) and represents the N-terminal half of
1. C-
1 was created by
deleting the 700-bp NlaIV-RsaI fragment
containing the entire N terminus of
1, leaving the four putative
hydrophobic segments and the sequences between each. This strategy
retained the native canonical methionine and the putative signal
peptide of
1. The mutations were confirmed by sequencing.
In Vitro Transcription and Translation
cRNA was synthesized from linearized 1 and
2 cDNA
using the appropriate RNA polymerase and the Megascript In
Vitro Transcription Kit (Ambion). The cRNA was purified by
phenol/chloroform extraction, precipitated by ethanol, and resuspended
in diethyl pyrocarbonate-treated water. The integrity of the cRNA was
verified on a 1.5% agarose-formaldehyde gel. GABA
subunits were
synthesized using the Flexi Rabbit Reticulocyte Translation System
(Promega), in the presence of canine pancreatic microsomal membranes
(Promega, Boehringer Mannheim). Synthesized protein was detected by
incorporation of 4 mCi/ml (specific activity, 1180 mCi/mmol)
[35S]methionine and [35S]cysteine (DuPont
NEN), and resolved on SDS-PAGE (10%) gels using one-fifth of each
translation reaction mixed with an equal volume of sample buffer (15%
glycerol, 5%
-mercaptoethanol, 4.5% SDS, 100 mM
Tris-Cl, pH 6.8, 0.03% bromphenol blue). Gels were fixed, dried, and
exposed to Hyper-film (Amersham Corp.) for 16 h to 4 days. The
molecular masses (in kDa) of the translated proteins were derived using
standard curves generated from protein size standards (Amersham
Corp.).
Topological Analysis of in Vitro Translated 1
In vitro translated 1
was transferred to polycarbonate tubes and centrifuged at 80,000 rpm in
a Beckman TLA-100 rotor for 2 h at 4 °C to pellet the
microsomal membranes. The pellet and an aliquot of the supernatant were
resuspended in sample buffer and subjected to electrophoresis. As a
control for membrane association the hydrophobic water channel
aquaporin-1 (cRNA provided by Dr. J. Reeves, Johns Hopkins University)
was translated and centrifuged in parallel.
One-half of the translate was incubated with 0.1 mg/ml proteinase K (Life Technologies, Inc.) alone, and the other half was incubated with proteinase K and 1% Triton X-100. Both digestions were carried out for 60 min at 0 °C and then terminated by the addition of the protease inhibitor phenylmethylsulfonyl fluoride (1.0 mM) for 10 min. The reactions were mixed with sample buffer and loaded onto a 10% polyacrylamide gel.
Development of 1-Specific Antibodies
The 1-specific peptide (QRQRREVHEDAHK)
representing a 13-residue epitope at the N terminus of
1 was
conjugated at its C terminus to the carrier protein keyhole limpet
hemocyanin prior to emulsification and injection. Peptide synthesis,
keyhole limpet hemocyanin conjugation, and rabbit handling were
performed by Research Genetics, Huntsville, AL. All experiments
described here were performed with 10-week bleed antisera.
PCR primers 5 TM3-TM4-BamHI
(5
-ACGGATCCGTCAACTACCTGACC-3
) and 3
TM3-TM4-EcoRI (5
-CAGAATTCAATGGCGTGGGTGTC-3
)
were used to amplify the TM3-TM4 loop of
1 from residues 358 to 445. The 280-bp product was subcloned into pCRII (Invitrogen) and sequenced and then shuttled into pGEX-2T (Pharmacia Biotech) using
BamHI and EcoRI sites to create a glutathione
S-transferase fusion protein. A 200-ml HB 101 bacterial
culture (Life Technologies, Inc.) expressing the fusion protein was
pelleted and lysed in 10 ml of lysis buffer (1% Nonidet P-40 in PBS)
with 0.5 mg/ml lysozyme (Sigma) for 30 min at room temperature. The
lysate was sonicated with a 5-mm diameter probe for 4 × 10 s
and then clarified by centrifugation. The clarified sonicate was mixed
overnight with 1 ml of a 50% slurry of glutathione-Sepharose (Sigma)
with 1 mM phenylmethylsulfonyl fluoride at 4 °C. The
fusion protein bound to Sepharose beads was pelleted at 500 × g for 2 min at 4 °C, washed four times with cold 1%
Triton X-100 in PBS, and then washed three times with cold PBS. The
fusion protein was eluted from the beads using an equal volume of 15 mM reduced glutathione, 50 mM Tris-Cl, pH 8.0, for 1 h at room temperature or with sample buffer for 10 min at 100 °C. The eluted product was cut from a preparative 15% SDS-PAGE gel and sent to Research Genetics for purification and injection of
rabbits. Antisera obtained 10 weeks post-immunization were used in all
experiments.
The specificity of the anti-1 antibodies was tested by cell
staining. A mammalian cell line (HEK 293) that stably expresses human
1 (22) was used as the protein source. Wild-type HEK 293 cells and
1-expressing cells were seeded onto glass coverslips (VWR) coated
with purified collagen (Collagen Corp.) and grown to approximately 75%
confluency. The cells were washed 2 or 3 times with PBS and then fixed
in 3% paraformaldehyde solution for 20 min at room temperature. The
fix was removed, and the cells were rinsed in PBS and then
permeabilized in 1% Triton X-100/PBS for 5 min. The cells were
immunostained with the polyclonal antisera, and
1 protein was
detected using a horseradish peroxidase staining method. In the first
method, endogenous horseradish peroxidase was inhibited by incubating
the cells in 1 × blocking solution for 10 min at room temperature
and removed by washing in two changes of PBS. The primary antibody
(hASH-1 or hASH-2) diluted 1:50-1:500 was added to the cells for 20 min and then the cells were washed three times in PBS. The secondary
antibody, biotin-conjugated anti-rabbit IgG (Boehringer Mannheim), was
added at 1:100-1:1000 dilution for 20 min and then washed as above. A
1:1000 dilution of streptavidin-horseradish peroxidase (Boehringer
Mannheim) was added for 20 min, followed by the True-Blue substrate
(Kirkegaard & Perry Laboratories) for 10 min. The cells were washed in
water and then air-dried before being analyzed.
Immunoprecipitation
In vitro translation reactions were diluted to 200 µl with buffer A (10 mM Tris-Cl, 140 mM NaCl, 0.5% Triton X-100, and 0.1% bovine serum albumin) and 10 µl of a 50% slurry of protein-A Sepharose CL-4B (Pharmacia) was added. The resulting solution was mixed for 3 h at 4 °C and then centrifuged; antibody was added to the clarified supernatant at the following concentrations: 2 µg of anti-FLAG mouse monoclonal antibody M2 (Eastman Kodak), 1:40 dilution of polyclonal antisera hASH-1 or hASH-2, or 1:1000 dilution of anti-HA mouse monoclonal antibody 12CA5 (Boehringer Mannheim). The solution was mixed for 16 h at 4 °C and then 30 µl of 50% protein A-Sepharose was added. The antibody-antigen complex was pelleted by centrifugation at 200 × g, and the supernatant containing unbound proteins was saved. The pellet was washed twice with buffer A, twice with buffer B (10 mM Tris-Cl, 140 mM NaCl), and once with 50 mM Tris-Cl, pH 6.8. The proteins were eluted in sample buffer by heating at 56 °C for 10 min prior to SDS-PAGE electrophoresis. To evaluate the synthesis reaction, aliquots of the supernatant, or the translate prior to precipitation, were included for each immunoprecipitation. For sequential immunoprecipitations, the immunoprecipitation pellet was washed as above and then eluted in 0.1 M glycine, pH 3.0, neutralized by 0.25 volume of 1 M Tris-Cl, pH 7.4, and brought up to a volume of 200 µl with buffer A. The second antibody was added to precipitate complexed proteins by the standard protocol described above.
Oocyte Expression of Wild-type and Mutant 1
Cytoplasmic injection of cRNA and the protocol for
electrophysiologic studies were as previously published (16). For
assembly studies, oocytes were co-injected with 5 ng of wild-type 1
cRNA and an equimolar amount or a 2- and 5-fold molar excess of N-
1 or C-
1 cRNA. Seventy-two hours after injection, whole-cell current in response to 5 µM GABA was measured by two
microelectrode voltage clamp in ND96 solution and compared with maximum
currents obtained in oocytes injected with
1 alone. ND96 solution
contains 96 mM NaCl, 2 mM KCl, 2 mM
CaCl2, 1 mM MgCl2, and 5 mM Hepes-NaOH, pH 7.4. Student's t test was
used to determine the statistical significant difference between two
sample groups. Independent sampling and comparison were used, and
p < 0.01 was set as a significant level.
-A cell-free synthesis
system was utilized to produce sufficient levels of subunit protein for
study. The predominant in vitro translated 1 protein
resolved at approximately 62 kDa (range 52-65 kDa), which is 10 kDa
larger than the size predicted from amino acid composition (Fig.
1). In addition to the major
1 62-kDa polypeptide,
less intense bands of 58, 56, and 48 kDa were occasionally present (see
Fig. 5). In vitro translated
2 protein resolved at
approximately 58 kDa (range 50-60 kDa), which is 7 kDa larger than the
predicted size for this protein. A second, less intense, band at 54 kDa
was occasionally observed. To aid in the analysis of the subunits,
1
and
2 were tagged with epitopes:
1 was tagged with FLAG at the C
terminus (20), and two
2 constructs were made with the influenza
hemagglutinin antigen tag (HA) (23) at either the N or C termini. The
electrophoretic migration patterns of in vitro translated
1FLAG and
2HA were indistinguishable from
1 and
2,
respectively. Two
1 mutants were used in this study. N-
1 included
the
1 N terminus sequence up to residue 256, and C-
1 contained
the signal peptide, the native initiating methionine, and the four
hydrophobic domains (Fig. 2). N-
1 and C-
1
synthesized in vitro migrated as single bands close to their predicted sizes of 30 and 26 kDa, respectively (Fig. 1).
By analogy to other members of the ligand-gated neurotransmitter
receptor family, GABA subunits are predicted to have four hydrophobic domains that are associated with membranes (Fig. 2). We
evaluated membrane association of the
1 subunit in vitro
by translating proteins in the presence of microsomal vesicles derived from the endoplasmic reticulum of canine pancreatic cells. Microsomal membranes and their associated proteins were then pelleted by high-speed centrifugation. The water channel aquaporin-1, which is
heavily hydrophobic and has been shown to be membrane-associated (24),
was used as a control in this study. As expected, in vitro translated aquaporin-1 was found predominantly in the membranous pellet
fraction (Fig. 3A). Similarly, in
vitro translated
1FLAG partitioned predominantly in the
membrane fraction, with a minor portion in the soluble fraction (Fig.
3A).
To assess the topology of 1FLAG relative to the microsomal vesicle
membranes, proteinase K susceptibility studies were performed. The
lipid bilayer of the vesicle membrane acts as a physical barrier, protecting portions of polypeptides translocated into the vesicle lumen
from digestion by proteinase K. Regions of a protein that have an
extracellular orientation in vivo will be enclosed within the lumen of the microsomal vesicle in this in vitro assay.
1FLAG was reduced in size to about 32 kDa following treatment with
proteinase K (Fig. 3B, 2nd lane). Upon disruption of the
microsomal membrane,
1FLAG was completely degraded (3rd
lane). Thus, about half of the
1 protein was protected by the
membrane of the microsomal vesicle. To determine which part of
1FLAG
was protected from degradation, protease susceptibility studies were
performed upon the N-
1 and C-
1 mutants. In the presence of
microsomal vesicles, N-
1 was completely protected from proteinase K
degradation (Fig. 3B, 5th lane). Disruption of membranes
with detergent resulted in degradation of N-
1 (6th lane)
indicating that the protease was able to cleave N-
1. Conversely,
microsomal membranes did not protect C-
1 from digestion (Fig.
3B, 8th lane). Together, these results indicate that the
N-terminal half of full-length
1 is located in the lumen of the
microsome and predicts that this region will have an extracellular
orientation in vivo.
The nucleic analysis software
program PCGene (IntelliGenetics, Inc.) was used to identify the most
antigenic region specific to 1 in the N terminus of the protein (see
Fig. 2). A 13-residue peptide corresponding to amino acids 37 to 49 of
1 was synthesized, conjugated to keyhole limpet hemocyanin, and
injected into rabbits to produce the polyclonal antisera hASH-1. A
second polyclonal antisera, hASH-2, was obtained from rabbits injected
with a glutathione S-transferase fusion protein containing
the putative intracellular loop between TM3 and TM4 of
1 (Fig. 2).
The specificity of both antisera was tested by immunodetection of
1
protein expressed in a mammalian cell line. An HEK 293 cell line
transformed with an expression vector containing the human
1
cDNA (293-
1) (22) was incubated with an antibody specific for
subunits of several vertebrate and invertebrate species (18). The
1-expressing cells, and not the parental HEK 293 cells, were
immunostained (Fig. 4A, left panel). Next,
the polyclonal anti-
1 antibodies were tested for their ability to
detect
1 protein in the 293-
1 cells. The hASH-1 and hASH-2
antisera specifically immunostained 293-
1 cells but not the parental
HEK 293 cells (Fig. 4A, middle and right panels).
The signal was distributed mainly at the periphery of the cells;
perinuclear staining observed in some cells may represent protein
within the ER. Incubation with primary antibody alone or secondary
antibody alone did not immunostain 293-
1 cells. Furthermore,
immunostaining was markedly reduced by preabsorbing hASH-1 with 10 µg/ml peptide used to create the antisera or by preabsorbing hASH-2
with in vitro translated
1 protein (data not shown).
When tested against in vitro translated proteins, hASH-1
immunoprecipitated 1 (Fig. 4B, 2nd lane). The N terminus
mutant N-
1, which contains the hASH-1 epitope, was also precipitated (Fig. 4B, 6th lane). Proteins that did not contain the
hASH-1 epitope,
2 and C-
1, were not immunoprecipitated by hASH-1
(4th and 8th lanes). Furthermore, precipitation
could be blocked by preabsorbing the hASH-1 antisera with 2 µg/ml of
the 13-residue peptide. As expected, hASH-2 antisera immunoprecipitated
1 and C-
1 but not N-
1 or
2 (Fig. 4B). Preimmune
sera did not immunoprecipitate any of the
proteins. We also
confirmed that the epitope-tagged
subunits,
1FLAG and
2HA,
were immunoprecipitable by the appropriate monoclonal antibodies,
M2-antiFLAG and 12CA5-antiHA, respectively. As noted above, in
vitro translated
1 occasionally had minor electrophoretic forms
(58, 56, and 48 kDa) in addition to the major form at 62 kDa. The two
1-specific polyclonal antibodies and the M2-antiFLAG monoclonal
antibody precipitated all electrophoretic variants.
To localize the sequences that specify 1
subunit homooligomeric assembly,
1 mutants were tested for
interaction with full-length
1 subunits by immunoprecipitation of
cotranslated proteins and by recordings of GABA-gated currents of
co-injected Xenopus oocytes. The initial interaction between
most proteins occurs in the lumen of the ER (25). Thus, the
localization of the N terminus of in vitro translated
1
to the lumen of microsomal vesicles, a region analogous to the lumen of
the endoplasmic reticulum (ER), suggested that the N terminus of
1
was involved in subunit assembly. We therefore investigated whether the
N-terminal half of
1 (N-
1) interacted with either
1 or
2
subunit. The C-
1 construct, containing the four transmembrane
domains, was used as a control.
The epitope-tagged version of 1 (
1FLAG) was used to facilitate
its identification since the M2-antiFLAG monoclonal antibody immunoprecipitated only
1FLAG and did not cross-react with the
1
truncation mutants.
1FLAG was cotranslated with N-
1 or C-
1 and
then immunoprecipitated with M2-antiFLAG.
1FLAG and mutant
1
proteins were identified on the basis of size; co-immunoprecipitation of the proteins indicated an interaction. Immunoprecipitation of
cotranslated N-
1 and
1FLAG using the M2-antiFLAG monoclonal antibody precipitated both proteins (Fig. 5, 2nd
lane). However, when N-
1 and
1FLAG were individually
translated, solubilized, and then mixed, only
1FLAG was
immunoprecipitated with the M2-antiFLAG antibody (Fig. 5, 4th
lane). The dependence of this interaction upon cotranslation
suggests that both proteins had to be present in the same microsomal
vesicle for oligomerization to occur and excludes the possibility that
the coprecipitation of N-
1 and
1FLAG was the result of
nonspecific protein aggregation. Furthermore, C-
1 was not
precipitated by M2-antiFLAG antibody when cotranslated or mixed with
1FLAG (Fig. 5, 6th and 8th lanes). Together,
these results indicate that the precipitation of N-
1 with
1 was
due to a specific interaction.
The effect of N-1 and C-
1 upon the ability of full-length
1 to
form functional GABA receptors was investigated in Xenopus oocytes. Neither N-
1 nor C-
1 formed GABA-gated chloride channels when the respective cRNAs were injected singly into oocytes. However, co-injection of N-
1 with full-length
1 resulted in a
dose-dependent inhibition of
1 current. A 2:1 ratio of
N-
1 to
1 cRNA decreased the current by about 90%
(n = 5) maximal
1 current, and a 5:1 ratio
eliminated the
1 current entirely (n = 5) (Fig.
6). In contrast, co-injection of C-
1 with full-length
1 did not inhibit
1 current at any ratio (n = 5 for each) (Fig. 6). Based upon evidence that N-
1 physically
interacts with
in vitro, we propose that the same
phenomenon occurred in Xenopus oocytes, and the resulting
N-
1/
1 oligomers were either non-functional or re-routed to a
degradation pathway. In this scenario,
1 subunits that did not
interact with N-
1 were able to form functional homooligomeric GABA
receptors. Increasing the ratio of N-
1 to
1 RNA favored the
formation of non-functional N-
1/
1 heterooligomers, thereby reducing the number of functional
1 homooligomeric receptors and
decreasing the whole-cell currents elicited by GABA. Therefore, the N
terminus of
1 is critical for
1 receptor assembly, based on its
luminal location, its co-immunoprecipitation with
1, and its ability
to interfere with
1 receptor formation.
To determine whether 1 physically interacts
with
2,
1 was cotranslated with
2 and then sequentially
immunoprecipitated. Two immunoprecipitation steps were necessary
because the in vitro translated
1 and
2 proteins could
not easily be distinguished on the basis of size. Since
2-specific
antibodies are not available,
2 with the HA epitope (
2HA) was
used. As noted above,
2HA is specifically immunoprecipitated by the
anti-HA antibody 12CA5. In the first study,
1-specific antisera
(hASH-2) was used in the first immunoprecipitation, and the anti-HA
monoclonal antibody (12CA5) was used in the second immunoprecipitation
(Fig. 7A). Since the hASH-2 antisera was
highly specific for
1, we expected protein to be precipitated by
12CA5-antiHA only if
2 was associated with
1 in the first
immunoprecipitation. Indeed, protein was not present following double
immunoprecipitation of
1 translated alone (Fig. 7A, right
panel, 1st lane), but proteins of the size appropriate for both
subunits were present after double immunoprecipitation of
1
cotranslated with
2 (Fig. 7A, right panel, 2nd lane). In the second experiment, the HA-specific antibody (12CA5) was used in the
first immunoprecipitation, and the
1-specific antibody (hASH-2) was
used in the second immunoprecipitation (Fig. 7B). Again,
proteins of the size expected for
1 and
2 subunits were present
following the second immunoprecipitation step only in the cotranslation
(Fig. 7B).
The next step was to assess whether the N termini of subunits were
involved in the interaction between
1 and
2 subunits. The
1
mutants were tested for interaction with
2 containing the HA epitope
(
2HA). When
2 was cotranslated with N-
1 and immunoprecipitated
with the anti-HA antibody, both
2 and N-
1 were precipitated (Fig.
8, 2nd lane). Furthermore,
2 and N-
1 were both immunoprecipitated when antisera specific for N-
1 (hASH-1) was used (Fig. 8, 4th lane). The same result was obtained
when untagged
2 was cotranslated with N-
1 and precipitated with
antisera specific for
1 (hASH-1) (data not shown). This result
indicated that the HA tag in
2 was not responsible for the
interaction between N-
1 and
2. Conversely, only
2 was
precipitated with the anti-HA antibody when
2 was cotranslated with
C-
1 (Fig. 8, 6th lane). Together, these data indicated
that N-
1 interacts specifically with
2.
GABA and GABAA subunits have similar amino acid
sequences and predicted structures (12-13). However, there are
distinct differences between receptors formed from
subunits and
those formed from GABAA subunits. The pharmacologic and
biophysical properties of GABA
receptors are more similar to
retinal GABAC receptors than GABAA receptors
(12, 15-16). Furthermore, human
1 and
2 subunits both
efficiently form homooligomeric GABA-gated receptors (13, 15), whereas
GABAA subunits must heterooligomerize for efficient receptor formation (26). Interestingly, the overlapping expression patterns of
1 and
2 in the rat retina (17) indicate that
subunits may also heterooligomerize into a single receptor. This concept is supported by functional studies of rat
subunits (19). Using in vitro translated protein and immunoprecipitation
with subunit-specific antibodies, we provide evidence that the primary amino acid sequences of
1 and
2 contain signals for subunit interaction, independent of cell-specific factors. The following observations indicate that this interaction is not the result of
nonspecific aggregation facilitated by the hydrophobic regions of each
protein. First, the signals for interaction were mapped to a region of
the protein shown to be in the appropriate location for oligomerization
to occur (microsomal vesicle lumen). Second, the interaction between
1 and N-
1 was dependent upon cotranslation, consistent with
assembly studies of other multimeric ion channels (27-28). Third, the
hydrophobic portion of
1 (C-
1) did not co-immunoprecipitate with
either
1 or
2 under any condition. We therefore conclude that the
N-terminal region of
1 contains signals for homooligomeric and
heterooligomeric interactions.
The in vitro translation system has been used to
evaluate assembly of a variety of proteins that form multimeric
structures (29-30). In vitro studies of K+
channels have demonstrated the in vivo relevance of this
approach for integral membrane proteins that function as ion channels
(27, 31). The in vitro system has not been used extensively
to study assembly of ligand-gated ion channels. One early report failed to detect association of nACh receptor subunits translated in vitro that were known to interact in vivo (32).
However, as the authors acknowledged, interaction may not have been
observed due to low levels of each subunit expressed from total RNA
(estimated to be 0.5% total translate) (32). In the present study, the use of in vitro synthesized RNA transcripts ensured high
levels of protein expression. In addition, examination of the lysate prior to immunoprecipitation studies enabled us to confirm that each
protein was synthesized and therefore available for interaction. The
apparent molecular mass of the predominant in vitro
translated subunit protein was larger than predicted from the amino
acid sequences (10 kDa greater for
1 and 7 kDa for
2). Similar
discrepancies between observed and predicted sizes have been noted for
GABAA subunit proteins (33). The in vitro
translated subunits also displayed heterogeneous migration. Each of the
electrophoretic variants were immunoprecipitated by antibodies against
the N terminus (hASH-1), an internal domain (hASH-2), and the C
terminus (M2-antiFLAG) of
1, indicating that each variant was an
intact
subunit protein. Therefore, the multiple bands may represent
different conformers of the full-length
proteins or proteolytic
fragments generated after immunoprecipitation. The same phenomenon was
observed for in vitro translated nACh receptor
subunits
(34).
Centrifugation studies indicated that in vitro translated
1 was associated with membranes. Interactions among multimeric integral membrane proteins generally take place in the lumen of the ER
(25). In the in vitro system, the interior of microsomal vesicles provides an environment analogous to the lumen of the ER. It
was therefore important to determine the topology of the in
vitro translated
subunits relative to the microsomal vesicle membrane. Protease digestion of
1 and truncated
1 proteins
synthesized in the presence of microsomal vesicles indicated that the
N-terminal half of
1 was protected from degradation. Our results
suggest that this region of
1 is inserted into the lumen of the
vesicle and predict an extracellular location in vivo. In
contrast, the C-terminal half of
1 was not protected from
degradation. If the C-
1 protein was inserted in the membrane in the
same manner proposed for full-length
1 (Fig. 2), the majority of the
peptide would have been exposed to proteinase K resulting in digestion
of C-
1. Although C-
1 was designed with the native signal peptide
sequence, it is possible that this mutant had an aberrant structure
that prevented insertion into the membrane. Determination of the
membrane topology of the C-terminal half of
1 will require
additional studies. Placement of the N-terminal half of in
vitro translated
1 in an extracellular orientation is
consistent with the identification of an N-terminal histidine residue
(His-156) critical for the inhibitory effect of extracellular
Zn2+ upon functional
1 receptors (35). Furthermore, two
agonist binding domains have been localized to the N terminus of
1
between the cysteine loop and TM1 (36). Therefore, the orientation of GABA
subunits in vitro appears to be similar to the
topology of
subunits within functional receptors and corresponds to
the predicted topology of other subunits in the ligand-gated ion
channel superfamily (37-38).
Although the association of 1 and
2 in vitro predicts
that the subunits will also interact in vivo, we cannot
determine in this study whether the
1/
2 heterooligomeric
receptors are functional. Indeed, oligomerization has been demonstrated
among certain GABAA subunits that do not form functional
receptors (39). However, a functional interaction of
1 and
2 was
suggested using rat
subunits, in which picrotoxin-insensitive
receptors were formed when picrotoxin-sensitive
1 subunits were
coexpressed with
2 subunits (19). The localization of signals for
subunit interaction to the N-terminal region is consistent with
studies of other members of the ligand-gated receptor superfamily, the nACh receptor, and glycine receptors (40-42). It is possible that the
C-terminal half of
1 contains signals for subunit interaction, but
such signals could have been masked by aberrant conformation of the
C-
1 protein. Future studies will clarify whether there is only one
assembly motif in
1 that enables both homooligomerization as well as
heterooligomerization, as seen in the Shaker-like potassium channels
(43). If the signals for homooligomeric receptor formation are the same
as those for heterooligomeric receptors, and operate with the same
efficiency, then the proportion of homooligomeric and heterooligomeric
receptors should be related to the amount of
1 and
2
polypeptides present. Distinct signals may indicate that homooligomeric
and heterooligomeric
receptors are formed in mixed ratios,
regardless of the relative abundance of each subunit. Identifying the
precise amino acid residues for oligomerization of
subunits would
be intriguing as these signals may also be involved in determining the
stoichiometry of
1 and
2 subunits in heterooligomeric
GABAC receptors. This mechanism could be similar to the
"assembly boxes" in the N-terminal luminal domain that determine
the 3
:2
subunit stoichiometry of glycine receptors (42).
Unfortunately, the similar size of each
subunit did not allow
determination of the relative proportion of
1 and
2 in the
immunoprecipitations of heterooligomeric complexes. It is also possible
that other factors influence
receptor assembly. These may include
neuronal proteins, such as the transmembrane chaperone calnexin
involved in nACh receptor
subunit folding (44), or signals for
later stages of assembly, as reported for the C-terminal regions of
nACh receptors (45).
Isolation and subunit analysis of GABAC receptors from
retina tissue will confirm whether the receptors exist as
heterooligomers in vivo. The identification of a third
subunit in the rat (46) and evidence that there are five
subunits
in the perch (47) suggest that a wide variety of GABAC
receptor compositions probably occur. This study indicates that the
assembly properties of cloned subunits that have sequence homology to
(such as rat
3 (46)) or functional similarity to
GABAC (such as the Drosophila Rdl subunit (48))
could be tested in vitro which will guide in vivo studies of GABAC receptor composition. Finally,
manipulation of the cDNAs encoding the
subunits, starting with
the N terminus, can be used to identify specific amino acids involved
in GABAC receptor assembly.
We thank Drs. R. Huganir, A. Hubbard, and S. Penno for constructive criticism of this work and Drs. R. Enz and H. Wässle for the anti-rat 1 antibody.