(Received for publication, October 13, 1995; and in revised form, February 5, 1996)
From the
The subunit of the
-aminobutyric acid type A
(GABA
) receptor is known to be photoaffinity labeled by the
classical benzodiazepine agonist,
[
H]flunitrazepam. To identify the specific site
for [
H]flunitrazepam photoincorporation in the
receptor subunit, we have subjected photoaffinity labeled GABA
receptors from bovine cerebral cortex to specific cleavage with
cyanogen bromide and purified the resulting photolabeled peptides by
immunoprecipitation with an anti-flunitrazepam polyclonal serum. A
major photolabeled peptide component from reversed-phase high
performance liquid chromatography of the immunopurified peptides was
resolved by polyacrylamide gel electrophoresis in the presence of
sodium dodecyl sulfate. The radioactivity profile indicated that the
[
H]flunitrazepam photoaffinity label is
covalently associated with a 5.4-kDa peptide. This peptide is
glycosylated because treatment with the enzyme,
peptide-N
-(N-acetyl-
-glucosaminyl)asparagine
amidase, reduced the molecular mass of the peptide to 3.2 kDa. Direct
sequencing of the photolabeled peptide by automated Edman degradation
showed that the radioactivity is released in the twelfth cycle. Based
on the molecular mass of the peptides that can be generated by cyanogen
bromide cleavage of the GABA
receptor
subunit and the
potential sites for asparagine-linked glycosylation, the pattern of
release of radioactivity during Edman degradation of the photolabeled
peptide was mapped to the known amino acid sequence of the receptor
subunit. The major site of photoincorporation by
[
H]flunitrazepam on the GABA
receptor
is shown to be
subunit residue His
(numbering based
on bovine
sequence).
The -aminobutyric acid type A (GABA
) (
)receptor mediates the majority of rapid inhibitory
synaptic transmission throughout the mammalian central nervous system.
As a member of the superfamily of ligand-gated ion
channels(1) , the GABA
receptor is believed to be a
hetero-pentameric protein that spans the neuronal membrane to create a
chloride conducting pore. The homologous subunits that assemble to form
the receptor-chloride channel complex are encoded by distinct but
related genes(2) . Six
, four
, four
, one
, and two
subunit isoforms plus splice variants for many of
the genes have been identified and classified by sequence similarity.
However, the precise stoichiometry of subunit isoforms that comprise
native receptors remains unknown.
A multiplicity of neuroactive
drugs have been shown to interact specifically with the GABA receptor complex to modulate inhibitory neurotransmission
throughout the brain(3) . These include the benzodiazepines,
barbiturates, some steroids and general anaesthetics, and possibly
alcohol(4) . Because of the clinical usefulness of the
benzodiazepines as anxiolytics, hypnotics, and anticonvulsants, their
interaction with the GABA
receptor has been extensively
studied. The benzodiazepines are known to be allosteric modulators of
GABA
receptors in that the classical agonists potentiate
whereas the inverse agonists reduce GABA-mediated chloride
conductances.
An area of particular interest has been the
identification of protein domains involved in the interaction of
benzodiazepines with the GABA receptor. Using
multidisciplinary approaches, several groups have identified structural
features of subunit isoforms that are important for ligand recognition
and for the modulatory effects of the benzodiazepines (reviewed in (5) ). To date, site-directed mutagenesis has identified the
amino acids Gly
of the
subunit(6) , His
of the
subunit(7) , and Thr
of the
subunit (8) as residues that play a role in conferring
the differential binding affinities of benzodiazepine ligands for the
GABA
receptor. Biochemical approaches have shown that the
site of photoaffinity labeling by the classical agonist,
[
H]flunitrazepam, is associated with the
subunit of the GABA
receptor (9, 10) within the large extracellular amino-terminal
domain(11, 12) . In addition, partial sequences of
proteolytic fragments from photoaffinity labeled receptors have
indicated that the [
H]flunitrazepam site occurs
within amino acid residues 8-297 of the
subunit(13) , and using subunit specific antibodies, the
site has been predicted to occur within residues 59-158 of the
sequence(14) . We have mapped the
[
H]flunitrazepam photoaffinity labeled peptides
generated by hydroxylamine cleavage to known GABA
receptor
sequences and have demonstrated that the site of photolabeling occurs
within amino acids 1-103 of the
subunit(15) . These
studies, considered together, limit the predicted site of labeling to
within residues 59-103 of the
subunit or within
homologous segments of other
subunit isoforms. In the present
study, we have employed immunoprecipitation and HPLC techniques to
purify a [
H]flunitrazepam photoaffinity labeled
peptide that was generated by cyanogen bromide cleavage of labeled
GABA
receptors from bovine cerebral cortex. It is shown by
peptide mapping and microsequence analysis that the major site of
[
H]flunitrazepam photoincorporation by the
GABA
receptor is likely to be the amino acid His
of the bovine
subunit.
To characterize the site of photoincorporation by
[H]flunitrazepam on the GABA
receptor, a photoaffinity labeled peptide component from bovine
cerebral cortex was purified by immunoprecipitation with a polyclonal
antiserum raised against free flunitrazepam and with the precipitating
reagent, protein G-linked Sepharose. In addition to quantitatively
precipitating free [
H]flunitrazepam, the
anti-flunitrazepam serum was shown to immunoprecipitate photoaffinity
labeled peptides by specifically recognizing the
[
H]flunitrazepam ligand covalently associated
with the GABA
receptor. After confirming (by SDS-PAGE) that
the photoincorporation of [
H]flunitrazepam with
the GABA
receptor from bovine cerebral cortex was
associated with a major 53-kDa protein, previously defined as the
subunit(s)(9, 10) , and that there was no significant
labeling of other species, the photoaffinity labeled protein was
solubilized and subjected to specific chemical cleavage at Met residues
by treatment with CNBr. The concentration dependence of
immunoprecipitation of [
H]flunitrazepam
photoaffinity labeled CNBr peptides with anti-flunitrazepam serum was
investigated to assess the optimal antibody concentration for use in
large scale purification. A maximum immunoprecipitation of 70 ±
9% was achieved with less than 1 µl of neat antiserum/pmol of
[
H]flunitrazepam photolabeled CNBr peptide.
Nonspecific adsorption of labeled peptides to the protein G matrix was
measured in the absence of antiserum and represented less than 1% of
the total yield.
Batch immunoprecipitation was used to purify
[H]flunitrazepam photoaffinity labeled CNBr
peptides in quantities sufficient for further characterization. About
50% of the total yield of immunoprecipitated product could be
specifically eluted from the antibody-protein G complex by incubation
with free flunitrazepam. The radioactivity profile of the
immunopurified peptides resolved by Tricine SDS-PAGE (Fig. 1A) demonstrated that CNBr cleavage of the
GABA
receptor preparation generated a
[
H]flunitrazepam photoaffinity labeled peptide of
5.5-kDa molecular mass, with a minor component that resolved as a
2.5-kDa species. Unfortunately, the inadequate amount of purified
protein precluded the use of silver stain for protein detection. The
elution profile from reversed-phase HPLC of the immunoprecipitated
peptides (Fig. 1B) displays three apparent peaks of
radioactivity, arbitrarily marked (i), (ii), and (iii). Although the
H peak marked (iii) invariably represented 55-60% of the total radioactivity
loaded onto the column, the relative amount of radioactivity in the
other two peaks varied between peptide preparations. The percentage of
H that eluted with the peaks ranged from 17 to 35% for (i) and from 8 to 26% for (ii). However, the combined
cpm in peaks (i) and (ii) routinely represented
40-45% of the total radioactivity.
Figure 1:
Immunoprecipitated
[H]flunitrazepam photoaffinity labeled CNBr
peptides resolved by SDS-PAGE and HPLC. A, representative
radioactivity profile of immunopurified
[
H]flunitrazepam photolabeled peptides resolved
by Tricine SDS-PAGE is shown. Determination of the cpm per gel slice
was performed as described under ``Experimental Procedures.''
The numerals above each arrow indicate the relative
position of the molecular mass standards (expressed in kDa). The major
peak of radioactivity corresponds to a peptide band with approximate
molecular mass of 5.5 kDa, whereas the smaller component resolves as a
2.5-kDa peptide. The data shown are typical of several preparations of
immunopurified peptides. B, immunoprecipitated
[
H]flunitrazepam photolabeled peptides were
resolved by reversed-phase HPLC as described under ``Experimental
Procedures.'' The data are shown as the representative
radioactivity profile of the elution of [
H] in
1-ml fractions as determined by scintillation counting of 100-µl
aliquots. Approximately 14,000 cpm of the immunoprecipitated peptides
were loaded directly onto the column, and the recovery of
[
H] was greater than 93%. The peak marked as (i) eluting at fraction 21 contained 4,700 cpm, the small peak
marked (ii) eluting at fraction 26 contained 1,100 cpm, and
the largest peak (iii) eluting at fraction 33 contained
approximately 7,250 cpm.
The pooled fractions from
each of the radioactive HPLC elution peaks were resolved by Tricine
SDS-PAGE for determination of the molecular mass of the
[H]flunitrazepam labeled CNBr peptide components.
In addition, the samples were treated with the deglycosylation enzyme, N-Glycanase, to assess whether the radiolabeled peptides
contained asparagine-linked oligosaccharides. On SDS-PAGE, HPLC peak (iii) resolved as a radiolabeled 5.4-kDa peptide (Fig. 2A), i.e. a profile similar to the
unfractionated peptides (Fig. 1A). This peptide was
glycosylated because after N-Glycanase treatment of peak (iii), the apparent molecular mass of the labeled peptide was
reduced to 3.2 kDa (Fig. 2B). Reversed-phase HPLC of
deglycosylated peak (iii) also showed a small but consistent
shift to a higher percentage of acetonitrile required for the elution
of this peptide from the column. In both the SDS-PAGE and HPLC
profiles, some undigested peak (iii) apparently remained after N-Glycanase treatment, and this accounted for approximately
20% of the total radioactivity.
Figure 2:
Peak (iii) from reversed-phase
HPLC of immunopurified [H]flunitrazepam CNBr
peptides, resolved by SDS-PAGE before and after treatment with N-Glycanase. The
H profiles of native and
deglycosylated peptides of HPLC peak (iii) resolved by
SDS-PAGE are shown as described in the legend to Fig. 1. A, peak (iii) from reversed-phase HPLC of
immunoprecipitated peptides resolves by SDS-PAGE to an apparently
single radiolabeled peptide with molecular mass of about 5.4 kDa. B, following treatment of the peptides eluted in peak (iii) with N-Glycanase as described under
``Experimental Procedures,'' the major radiolabeled species
resolves to a 3.2-kDa peptide with some residual 5.4-kDa peptide
evident.
When HPLC peak (ii) was
resolved by SDS-PAGE, it ran as a broad band of radioactivity with
apparent molecular mass ranging from 8.7 to 12.1 kDa. After digestion
with N-Glycanase, this peak resolved to a more distinct but
still broad band of H that corresponds to a radiolabeled
peptide of about 9 kDa. It was not possible to resolve the
radioactivity present in HPLC peak (i) to a distinct protein
band by electrophoresis. In repeated attempts, the radioactivity showed
a diffuse migration pattern spanning the middle portion of the gel that
did not vary when the pore size of the gel was altered. The incubation
of peak (i) with N-Glycanase had no effect on the
apparent inability of this fraction to be resolved by gel
electrophoresis.
The [H]flunitrazepam
photoaffinity labeled peptides purified by HPLC (see Fig. 1B) were subjected to direct automated sequencing
to measure the release of radioactivity during each cycle. The pattern
of release of radioactive PTH amino acids generated by Edman
degradation of the [
H]flunitrazepam photolabeled
peptides present in HPLC peak (iii) shows that a maximum
release of
H occurred in sequencer cycle 12 (Fig. 3). The release of radioactivity remained elevated for
about four sequencer cycles before returning to baseline levels. The
H elution seen in cycles 13-16 is most likely due to
NH
-terminal cleavage from residual peptide containing the
[
H]flunitrazepam-associated amino acid,
originally residue 12, and/or incomplete extraction of the modified
amino acid. It is probable that the covalent incorporation of the
ligand with a particular amino acid adversely affects the efficiency of
the Edman degradation reaction. The moderate level of radioactivity
seen in the first cycle likely results from desorption of peptide from
the filter cartridge system of the sequencer. The pattern of
radioactive release from automated sequencing of HPLC peaks (i) and (ii) were indicative of nonspecific elution during
the Edman degradation cycles (Fig. 4). Therefore, duplicate
aliquots of these samples were processed in the same manner as for
standard sequencing, except that PITC was omitted from the reaction
chemistry. The elution of
H during the cycles from sham
sequencing of peaks (i) and (ii) was parallel to that
obtained for the standard Edman reaction cycles.
Figure 3:
Release of radioactive amino acids during
automated Edman degradation of
[H]flunitrazepam-labeled CNBr peptides from HPLC
peak (iii). The amount of
H associated with the
PTH amino acids generated by each cycle of Edman degradation of the
photolabeled peptides present in HPLC peak (iii) was
determined as described under ``Experimental Procedures.''
The radioactivity shown is that observed and has not been corrected for
repetitive yield of sequencer cycles. Approximately 30% of the
radioactivity loaded onto the sequencer was recovered in the fractions
collected from each cycle; about 25% remained on the filter and
cartridge seal, and the remainder was presumably lost in the
washes.
Figure 4:
Release of radioactivity from HPLC peaks (i) and (ii) subjected to automated Edman
degradation. The amount of H that eluted with each cycle
during standard conditions of Edman degradation and when PITC was
omitted from the automated reaction was investigated (see
``Experimental Procedures''). Equivalent amounts of
radioactivity were loaded onto the sequencer for all instances. A, automated sequencing of HPLC peak (i) in the
presence (
) and absence (
) of PITC. Of the
H
recovered, 28% was released during the cycles of Edman degradation, 4%
was left on the filter and cartridge seal, and 68% was presumed lost in
washes. B, automated sequencing of HPLC peak (ii) in
the presence (
) and absence (
) of PITC; the recovery of the
radioactivity loaded onto the sequencer was 17% in the normal reaction
cycles, 3% remained on the filter, and 80% was lost in washes. The
radioactivity released from peaks (i) and (ii) during
the sham sequencing cycles (minus PITC) was slightly reduced, whereas
the amount of
H presumed to be lost in washes was increased
compared with that for the standard sequencer
runs.
Several structural determinants required for the allosteric
modulation of GABA receptors by the benzodiazepines have
been characterized by site-directed mutagenesis of recombinant
receptors (see Introduction). Using this information, the
benzodiazepine binding domain has been modelled as a composite of these
structural features(5) . However, the identity of specific
amino acid residue(s) in native GABA
receptors that are directly involved in benzodiazepine binding has remained an
area of intense interest. One approach used extensively has been to
specifically and irreversibly label the benzodiazepine binding site
with the photoactivatable agonist,
[
H]flunitrazepam, as first described by
Möhler et al.(21) . Although
strong evidence has been reported to show that the major site of
[
H]flunitrazepam photoaffinity labeling occurs on
the GABA
receptor
subunit(5) , the precise
position of the photolabel on the polypeptide has not previously been
established.
To identify the [H]flunitrazepam
photoaffinity labeling site on the GABA
receptor, a
purified preparation of labeled peptide was required in sufficient
quantities to allow for characterization by conventional biochemical
techniques. The development of an anti-flunitrazepam
immunoprecipitation assay has provided a highly specific method to
purify the photoaffinity labeled peptides from a crude brain
preparation. The [
H]flunitrazepam photoaffinity
labeled peptides generated by CNBr cleavage were precipitated with an
antiserum directed against free flunitrazepam that also specifically
recognized the covalently attached ligand. Whereas the major component
evident in the immunopurified preparation was a 5.5-kDa photolabeled
peptide when resolved by SDS-PAGE, three distinct peaks of
radioactivity were resolved by reversed-phase HPLC.
The
[H]flunitrazepam present in HPLC peak (iii) represented the majority (approximately 60%) of the total
immunoprecipitated radioactivity and was shown by SDS-PAGE to be
associated with a peptide of apparent molecular mass 5.4 kDa. It was
also shown that this photolabeled peptide contains asparagine-linked
carbohydrate, because after N-Glycanase digestion, it migrated
as a 3.2-kDa peptide. Recognizing that: 1) CNBr specifically cleaves
peptide bonds on the carboxyl side of Met residues with high
efficiency, except for Met-Thr and Met-Ser bonds, which are
essentially resistant to CNBr and 2) asparaginyl-linked glycosylation
exclusively occurs at the consensus sequence of Asn-Xaa-Thr or
Asn-Xaa-Ser, the origin of the photolabeled peptide can be mapped to
the known amino acid sequence of the NH
-terminal domain of
the GABA
receptor
subunit (see Fig. 5). Considering the potential sites for cleavage by CNBr
and for asparaginyl glycosylation, the only peptide that could be
generated from the
subunit to contain Asn-linked carbohydrate and
to resolve by electrophoresis as described above is
Ala
-Met
. This peptide has a predicted
molecular mass of 3.1 kDa without consideration given for
glycosylation, which is close to the estimates obtained by SDS-PAGE
analysis. The other CNBr peptide that could be generated from the
subunit to contain Asn-linked oligosaccharide is
Gln
-Met
, which would have a molecular
mass after deglycosylation of greater than 6.5 kDa. Because previous
attempts to sequence the
subunit indicated that the polypeptide
has a blocked NH
terminus(1) , the peptide
beginning with Gln
would also be refractory to the Edman
chemistry. Furthermore, the peptide bonds carboxyl to Met
and Met
have previously been shown to be
susceptible to CNBr cleavage, and sequencing of the peptides
Ala
-Ala
and
Pro
-Thr
provided the information
necessary for the first cloning of the GABA
receptor
subunits(1) . The [
H]flunitrazepam
photolabeled peptide has been mapped to the
subunit,
because this isoform has been shown to occur in the vast majority of
native GABA
receptors from bovine cerebral
cortex(22) . Although other, less abundant, isoforms of the
subunit have been identified in cortical GABA
receptors, the interpretation for the origin of the photolabeled
peptide is not compromised, because the isoforms possess a high degree
of sequence identity throughout this domain.
Figure 5:
Partial amino acid sequence of the bovine
GABA receptor
subunit. The GABA
receptor sequence of the large extracellular amino-terminal
domain of the
subunit from Gln
to the
first putative transmembrane domain is shown (1) . Met residues
are in bold type with the potential cleavage sites for CNBr
indicated by down arrows, the potential sites for
asparagine-linked glycosylation are underlined (Asn-Xaa-Thr),
the large bracket between Asn
and Gly
marks the only potential site in the subunit for specific
cleavage by hydroxylamine, and the putative membrane spanning region is
marked by the shaded box above the initial residue
Phe
.
The pattern of release
of radioactive PTH amino acids obtained from automated Edman
degradation of the major photolabeled peptide
(Ala-Met
) indicated the
[
H]flunitrazepam is covalently associated with
the twelfth residue, which corresponds to His in position 102 of the
subunit. The photoincorporation of
[
H]flunitrazepam with His
is
consistent with the findings of hydroxylamine cleavage experiments that
demonstrated photolabeling occurred prior to Asn
, as well
as previous reports that predicted the site occurred within limited
subunit domains (see Introduction). The involvement of a histidine
residue in the interaction of benzodiazepines with the GABA
receptor was implicated in earlier studies that investigated the
effects of chemical modification and the pH dependence of radioligand
binding(23, 24) . In addition, His
of
the
subunit is the residue that was shown by point
mutation to be required for the high affinity binding of benzodiazepine
agonists in recombinantly expressed GABA
receptors (7) .
The chemical nature of the radioactivity present in
HPLC peaks (i) and (ii) has not been established.
HPLC peak (ii), which accounted for 10-25% of the total
radioactivity, resolved by SDS-PAGE to a molecular mass ranging from 9
to 12 kDa, with deglycosylation causing a marginal shift in the gel
profile that indicated a 9-kDa photolabeled peptide. The radioactivity
profile obtained from direct sequencing of peak (ii) did not
suggest the [H]flunitrazepam label was associated
with a particular amino acid residue. Automated sequencing in the
absence of PITC demonstrated that the release of radioactivity seen
during the first few cycles of Edman degradation was not due to the
generation of radiolabeled PTH amino acids resulting from
NH
-terminal cleavage of a labeled peptide. Although a
reliable prediction for the origin of the photolabeled peptide is
difficult, we can speculate that a 9-kDa peptide containing
[
H]flunitrazepam-labeled His
could
result from incomplete cleavage at one or more of the potential CNBr
sites in the NH
-terminal domain of the
subunits.
Alternatively, the peptide may have been generated from a different
domain of the
subunit or from another subunit subtype, thereby
representing a minor fraction of photolabel incorporated with a residue
other than His
. Recent studies have suggested that the
benzodiazepine binding domain is made up of determinants from both the
and
subunits. Site-directed mutagenesis has found that
Thr
of the
subunit is involved in conferring the
modulatory effects of benzodiazepine ligands (8) , and previous
photoaffinity labeling experiments have shown data that suggest some
[
H]flunitrazepam label may incorporate with the
subunit(14) . The apparent inability to resolve the
radioactivity present in HPLC peak (i) by electrophoresis
suggested the [
H]flunitrazepam was not associated
with a distinct peptide. In addition, the radioactivity profiles
obtained from automated sequencing of peak (i) were not
consistent with the release of labeled PTH amino acids from the Edman
degradation reaction. It is possible that the peak (i) fraction of the immunopurified product may represent free
[
H]flunitrazepam or ligand incorporated with
carbohydrate or some other nonprotein molecule.
In conclusion, we
have presented evidence that photoaffinity labeling of the GABA receptor with [
H]flunitrazepam leads to the
covalent association of the ligand with His
of the
subunit. Despite our intention to characterize the photolabeled peptide
components by microsequence analysis, current technical limitations and
insufficient yields of the purified peptides have precluded the
identification of the PTH amino acids generated during each cycle.
Therefore, although His
of the
subunit is likely to
be the major site of photoincorporation, it is not possible to
substantiate the possible existence of other amino acids that may be
photolabeled by [
H]flunitrazepam.