(Received for publication, May 23, 1995; and in revised form, August 11, 1995)
From the
We have investigated the phosphorylation of -aminobutyric
acid type A (GABA
) receptor purified from bovine cerebral
cortex in the absence of added kinases. Incubation of the
affinity-purified receptor with [
-
P]ATP and
500 µM MnCl
yielded incorporation of 0.45 mol
of
P/mol of muscimol binding sites within 2 h at 30
°C. Mn
was much more effective than
Mg
as activator. Phosphorylation of the receptor was
observed on at least three different polypeptides of 51, 53, and 55
kDa. It was predominant on 51- and 53-kDa polypeptides that co-migrate
with the [
H]flunitrazepam photoaffinity-labeled
bands, suggesting that
P incorporation mainly occurs on
-subunits. A monoclonal antibody specific for
-subunits
adsorbed the endogenously phosphorylated GABA
receptor with
a stoichiometry close to 1 mol of phosphate/mol of muscimol. The
phosphorylation of the 51-kDa polypeptide, corresponding to
-subunit, exhibited a micromolar affinity for ATP and
sigmoid kinetics (n
= 2). Major incorporation of
phosphate occurred on serine and threonine residues in roughly
equimolar ratio. By enzyme-linked immunosorbent assay and
immunoblotting studies we also detected a minor incorporation on
tyrosine residues; this was specific for a 55-kDa polypeptide.
Comparison with molecular data suggests that at least
- and
-subunits (Ser and Thr
residues) and possibly
-subunits (Tyr residue) are
endogenously phosphorylated by multiple kinases, with a clear
preference for
-subunit. The
-subunits were not
phosphorylated in our experimental conditions. The corresponding kinase
activities are closely associated to the receptor protein, indicating a
new complexity in the regulation of the GABA
receptor.
-Aminobutyric acid type A (GABA
) (
)receptors are ligand-gated anion channels that mediate
most inhibitory synaptic transmission in the central nervous system.
Molecular studies have identified five distinct subunits with, for most
of them, multiple subtypes; 17 genes have been characterized so
far(1, 2) . Functional studies have used heterologous
expression of several subunits to produce functional GABA
receptors (3, 4) . At the native protein level,
ligand binding heterogeneity of the receptor was demonstrated by
photoaffinity labeling and autoradiography(5, 6) .
Phosphorylation is a common mechanism for the regulation of receptor
function. All subunit subtypes of the GABA receptors
contain some consensus substrate sequences for kinases such as
cAMP-dependent protein kinase, protein-tyrosine kinase, and/or
Ca
-phospholipid-dependent protein kinase
C(7) . Moreover, type 2 calcium/calmodulin-dependent protein
kinase and cGMP-dependent protein kinase phosphorylate the
intracellular domains of
L and
S
fusion proteins(8) .
Using purified GABA receptor, Browning et al.(9) reported that
-subunits could be phosphorylated in the presence of exogenously
added cAMP-dependent protein kinase and
Ca
-phospholipid-dependent protein kinase C.
Ca
-phospholipid-dependent protein kinase C and
cAMP-dependent protein kinase phosphorylation have been reported to
modify the amplitude of GABA-activated currents (4, 10, 11, 12, 13) .
In
acutely dissociated neurons, it has been reported that favorable
conditions for phosphorylation were required to prevent
``run-down'' of GABA currents(14, 15, 16) . The authors
excluded the possibility that
Ca
-phospholipid-dependent protein kinase C,
cAMP-dependent protein kinase, or type 2 calcium/calmodulin-dependent
protein kinase could be operative; they proposed that the maintenance
of normal GABA currents required the activity of a unique kinase
specific for the GABA
receptor.
Although many molecular
and biochemical analyses have shown that 1- (4) ,
3-(17) , and
2-subunits (18) of
GABA
receptors are good substrates for several exogenously
added kinases, there is little information on possible endogenous
phosphorylation by kinase activities associated to the receptor. In
GABA
receptor purified from rat cerebellum, it has been
reported that an
-subunit could be phosphorylated by a
receptor-associated protein kinase(19) . In the present study,
we describe multiple endogenous kinase activities that co-purify with
the GABA
receptor protein purified from cerebral cortex.
Figure 1:
Effect of incubation time on endogenous
phosphorylation of the GABA receptor. Stoichiometry was
calculated as number of moles of phosphate incorporated per mole of
muscimol bound in the presence of 5 mM Mg
(squares) or 500 µM Mn
(circles). The maximal incorporation (120 min) of
phosphate in the presence of Mg
and Mn
was of 0.12 and 0.45 mol of
P/mol of
[
H]muscimol
respectively.
Figure 2:
Co-electrophoresis in SDS of
phosphorylated and flunitrazepam photoaffinity-labeled polypeptides of
purified GABA receptor samples. Molecular mass of standards
are indicated in A. The main bands of the receptor (51, 53,
55, and 58 kDa) and the co-purified B-36 voltage-dependent anion
channel (36 kDa) are indicated in B. The GABA
receptor was either phosphorylated without added kinases (squares) or photoaffinity labeled with
[
H]flunitrazepam (circles). Abscissa
values represent slice number; ordinate values represent cpm of
P (left) and
[
H]flunitrazepam (right). Arrows indicate the molecular mass of labeled peaks calculated from
standards; they correspond to the 51- and 53-kDa polypeptides and to
their degradation products (45
kDa)(29) .
Figure 3:
Immunoprecipitation of the phosphorylated
GABA receptor by
-subunit-specific monoclonal antibody
(bd 24). Affinity column-purified receptor was incubated with various
dilutions of bd 24. After precipitation with anti-mouse IgG coupled to
agarose, the pellets were assayed for both
[
H]muscimol binding (triangles) and
P incorporation (squares). A nonimmune serum from
rabbit was used as control. Nonspecific precipitation of activity did
not vary significantly with the dilution of control serum. The inset represents a linear relationship (r
= 0.870) between the number of moles of muscimol (abscissa) and phosphate (ordinate) present in the
pellets with different antibody dilutions. The slope gives a
stoichiometry of 0.83 mol of phosphate/mol of
muscimol.
Figure 4:
Effect of ATP concentration, pH, and
temperature on phosphorylation of the 51-kDa polypeptide. A,
phosphate incorporation was plotted as a function of ATP concentration
in the presence of 5 mM Mg. Experimental
conditions were as described in the legend to Fig. 1. The ATP
concentration corresponding to 50% V
was 1.7
µM. B, incorporation of phosphate as a function
of pH. C, incorporation of phosphate as a function of
temperature.
Figure 5:
Phosphoamino acid analysis of
phosphorylated GABA receptor. Affinity-purified receptor
was phoshorylated either in the presence of 5 mM
Mg
alone (A) or in the presence of both
Mg
(5 mM) and Mn
(2
mM) (B). The preparation was hydrolyzed with HCl 6 M. The products were then separated by two-dimensional thin
layer electrophoresis followed by
autoradiography.
There
is a single consensus sequence for tyrosine kinase in the
-subunit (31) that contrasts with the numerous
consensus sequences for serine and threonine phosphorylation
sites(4) . We therefore used more sensitive and specific
immunochemical methods that can detect very low levels of
phosphorylated tyrosine residues. For these experiments we used a
highly specific anti-phosphotyrosine antibody. The results are
presented in Fig. 6. Aliquots of purified receptor were
phosphorylated with 50 µM unlabeled ATP. By enzyme-linked
immunosorbent assay (Fig. 6A), we observed a positive
reaction only when Mn
(2 mM) was present in
the incubation medium. In the presence of 5 mM Mg
alone (i.e. without Mn
), no
phosphotyrosine could be detected by this method. In order to estimate
the molecular mass of the polypeptide that is phosphorylated on
tyrosine, we used the same antibody for Western blot analysis. Under
the same conditions as for enzyme-linked immunosorbent assay, a single
labeled band was recognized at an apparent molecular mass of 55 kDa (Fig. 6B). The intensity of labeling increased with the
concentration of the antibody. No significant cross-labeling was
observed, even at the highest antibody concentration. This apparent
molecular mass is close to the mass of 55.2 kDa deduced from the bovine
-subunit sequence(31) . Interestingly, the
antibody stained neither the 51-53-kDa polypeptides nor any other
part of the blot.
Figure 6:
Detection of a tyrosine protein kinase
activity in purified GABA receptor by both enzyme-linked
immunosorbent assay and Western blotting. A, we used a
nonradioactive tyrosine kinase assay kit from Boehringer Mannheim. This
kit uses specific peptide substrates that are biotinylated at the amino
terminus. The fraction of phosphorylated substrate is determined
immunochemically via a highly specific anti-phosphotyrosine antibody
directly conjugated to peroxidase. The use of this kit also allows
determination of the rate of dephosphorylation (phosphatase activity)
of the peptide substrate. The detection is shown on the purified
receptor either in the presence of Mg
alone (columns 1-3) or in the presence of Mn
(columns 6-8). Columns 1 and 6,
with ATP; columns 2 and 7, without ATP; columns 3 and 8, phosphatase assay; lane e, blank and
autophosphorylation controls; lane d, standard curve; lanes a, b, and c, biotinylated peptide
protein kinase substrate 2; lanes f, g, and h, biotinylated peptide protein kinase substrate 1. This
experiment indicates a tyrosine kinase activity only in the presence of
Mn
for both peptide substrates. B, an
anti-phosphotyrosine monoclonal antibody was used for Western blotting
on purified receptor. Molecular mass standards are indicated in lane a (97.4, 66.0, 45.0, and 31.0 kDa). One band only was
observed at 55 kDa in the presence of 50 µM ATP and 100
µM Mn
. The antibody was used at
different dilutions: 0.01 µg (lane b), 0.1 µg/ml (lane c), and 1.0 µg/ml (lane
d).
GABA receptor function is regulated by
phosphorylation(4, 13, 14, 15, 16, 32) .
The receptor protein contains consensus sequences for phosphorylation
by protein kinases(7) . In the present work, we describe kinase
activities associated to the affinity-purified GABA
receptor from cerebral cortex. In order to characterize
endogenous activities, the purification procedure had to be improved.
Indeed, we observed that the use of high detergent and urea
concentrations during the purification procedure drastically decreased
the rate of phosphorylation. Moreover, freezing the purified protein
nearly completely abolished this activity. In contrast, phosphate
buffer seems to have a protective effect during the solubilization of
the protein. These technical differences might explain why a
significant level of endogenous phosphorylation was not previously
detected(9) .
We observed a major phosphorylation in the
51-53-kDa bands. Maximum incorporation of P was in
the 51-kDa polypeptide. We also detected a minor incorporation of
P in a band of higher molecular mass (55 kDa). When we
studied the effect of ATP concentration on 51-kDa polypeptide
phosphorylation, we obtained a sigmoid curve. This suggests that the
endogenous protein kinase has allosteric properties with at least two
cooperative binding sites for ATP. Similar effects were reported for
the insulin-stimulated autophosphorylation of the insulin receptor (33) .
Immunoprecipitation of the phosphorylated receptor
was obtained with a monoclonal antibody specific for -subunits,
confirming that phosphorylation occurs on these subunits. The 51- and
53-kDa bands co-migrated with two bands photoaffinity-labeled by
[
H]flunitrazepam. These two bands appear
identical to those previously identified by partial sequencing and
Western blotting as corresponding to the
and
gene products(29) . However, data currently
available are not yet sufficient to provide definitive proof of their
identity. In an earlier study, Sweetnam et al.(19) ,
using a partially purified GABA
receptor preparation from
rat cerebellum, suggested that an
-subunit was the preferred
substrate for endogenous phosphorylation (which was observed only on
serine). These results did not rule out the possibility that endogenous
phosphorylation might also occur on other types of subunits. Indeed, it
was shown later (34, 35) that
-,
-,
-,
-,
-, and
-subunits are expressed in cerebellum but
the other subunit subtypes are not. The absence of expression of
is in agreement with the lack of 53-kDa polypeptide
in cerebellum(5, 6) . Our preparation, purified from
bovine cortex, contains
- and
-subunits, and endogenous phosphorylation was observed
in
- but not in
-type subunits. In contrast,
phosphorylation by cAMP-dependent protein kinase and
Ca
-phospholipid-dependent protein kinase C never
occurs on
-subunits but only on
-subunits(9, 36) . The nature of
-subunit
subtypes affects both the sensitivity of the receptor to GABA and the
affinity of different ligands for the benzodiazepine binding
site(5, 6) . It is thus reasonable to propose that the
-subunits can regulate the channel conductance in two different
ways: the binding of agonist ligands to the NH
-terminal
extracellular domain (37) and the endogenous phosphorylation in
the long intracellular loop. The physiological consequences of
phosphorylation on
-subunits by exogenous kinases are different
from those proposed for endogenous phosphorylation(4) .
We
measured a roughly equimolar P-incorporation in Thr and
Ser residues in
-subunits, whereas a previous study (19) suggested an endogenous phosphorylation of
-subunit
only on serine. A possible explanation for this apparent discrepancy is
that the latter preparation (19) was from cerebellum. The
additional phosphorylation on Thr residue might reflect phosphorylation
on other
-subunits (
) expressed in the
neocortex(38) .
When our preparation was incubated with ATP
+ Mn, we also detected the presence of
phosphotyrosine on a single 55-kDa polypeptide. The identification of
the band(s) labeled at 55 kDa is made difficult by the presence of
several
-,
-, and
-subunit subtypes sharing similar
molecular mass(1) . This incorporation may occur at least
partially on the
-subunit as in the bovine brain, this
subunit is the only one containing a unique consensus sequence for
tyrosine phosphorylation (located on Tyr
(31) ).
The apparent molecular mass has been estimated at 55 kDa with a highly
specific phosphotyrosine antibody. This corresponds to the deduced mass
from the
gene. However, the apparent molecular mass
of the
gene product is still uncertain due to
post-transcriptional events; specific antibodies also recognized a
broad band at 41-47 kDa(39, 40, 41) .
Thus, the identification of the 55-kDa polypeptide with the
-subunit of the GABA
receptor is possible
but not clearly demonstrated.
The parallels between the conditions
reported previously and the present study are worth noticing. It has
been suggested that GABA receptor current is maintained by
phosphorylation involving an unknown kinase and an unknown substrate (14) . Our results bring new insights on the complexity of the
- and possibly
-subunit pharmacology and indicate that the
nature of the subunit subtypes composing the GABA
receptor
may also directly affect the properties of the GABA-activated chloride
channel. The present work suggests an additional heterogeneity of
receptor function due to modulation by multiple endogenous
phosphorylations.