(Received for publication, March 3, 1997, and in revised form, May 12, 1997)
From the Metabotropic glutamate receptors, which are
members of a G protein-coupled receptor family, mediate the glutamate
responses by coupling to the intracellular signal transduction pathway. We herein report that calmodulin (CaM) interacts with the metabotropic glutamate receptor subtype 5 (mGluR5) in a
Ca2+-dependent manner in
vitro. CaM is capable of binding on two distinct sites in the
COOH-terminal intracellular region of the receptor with different
affinities. The CaM binding domains are separated by an alternatively
spliced exon cassette present in one of the splicing isoforms of
mGluR5. By using fusion proteins and synthetic peptides we showed that
protein kinase C phosphorylates both CaM binding regions. This
phosphorylation is inhibited by the binding of CaM to the receptor, and
conversely the binding is inhibited by the phosphorylation. These
antagonisms of the CaM binding and phosphorylation thus suggest the
possibility that they regulate the receptor responses in
vivo.
Glutamate is the main excitatory neurotransmitter in the mammalian
brain and is important in memory acquisition and learning. Glutamate
receptors are categorized into two groups: ionotropic receptors and
metabotropic receptors. The ionotropic glutamate receptors function as
a glutamate-gated cation channel, whereas the metabotropic glutamate
receptors (mGluRs)1 are
coupled to G proteins (1) and evoke a variety of functions by mediating
intracellular signal transduction (2). Eight members of the mGluRs have
been identified so far, and metabotropic glutamate receptor subtype 1 (mGluR1) and subtype 5 (mGluR5) were shown to activate phospholipase C
(3-5).
Three splice variants called mGluR1a to -1c have been described (6, 7).
The mGluR1a possesses a long COOH-terminal intracellular domain which
is homologous to mGluR5 in the amino acid sequence. The COOH-terminal
domain of mGluR1b is much smaller than that of mGluR1a since an
additional exon containing an in-frame stop codon is inserted into the
domain. In mGluR1c, a distinct insertion results in a similar
truncation of the COOH terminus. In contrast to the large fast
transient responses induced by mGluR1a, the shorter proteins mGluR1b
and mGluR1c elicited a small and more slowly generated oscillatory
current in Xenopus oocytes (7). Moreover, the subcellular
distribution of mGluR1b expressed in cells was different from that of
mGluR1a (8). We previously reported the presence of a variant of mGluR5
and named it mGluR5b (9). An extra 96-base pair exon was inserted into
mGluR5a, which had originally been reported as mGluR5. Because of this insertion, the mGluR5b cDNA is able to encode a protein longer than
mGluR5a by 32 amino acids. The mRNA levels of the two isoforms were
regulated in a development-specific manner (10). No difference of these
two isoforms could be seen regarding their pharmacological properties
(11, 12). The two mGluR5 variants revealed common functional
characteristics with mGluR1a: the generation of the rapid and transient
responses when expressed into Xenopus oocytes and the
transduction mechanism and subcellular localization in transfected
cells (12). Although it was suggested that the long COOH-terminal
domain of these three receptors may play a similar role, their
structural determinants for the properties still remain unknown. The
identification of proteins which interact with the COOH-terminal domain
of mGluR5 may give a clue for the role of the domain and may suggest
the biological function of the spliced cassette of mGluR5.
In the present study, we created several fusion proteins of glutathione
S-transferase (GST) which possess various portions of the
COOH-terminal intracellular region of mGluR5 and used them to detect
proteins which could interact with them. A 17-kDa protein associating
with them was identified as calmodulin (CaM). To our knowledge, none of
the G protein-coupled receptors have been reported to interact with CaM
directly although several ion channels are regulated by direct CaM
binding (13, 14). We characterized the two distinct CaM binding sites
which apparently have different affinities for CaM. The two binding
sites are separated by the alternatively spliced exon cassette of
mGluR5b. We further showed that protein kinase C (PKC) phosphorylates
the fusion proteins and the peptides which have one of the two CaM
binding regions. PKC phosphorylation is inhibited by CaM binding, and
conversely the formation of the complex with CaM is blocked by this
phosphorylation. These results suggest that the direct binding of CaM
on the COOH-terminal region of the mGluR5 may thus affect the
PKC-mediated modulation of the receptor while also indicating that the
phosphorylation state may regulate the effect of CaM binding on the
receptor.
cDNA
fragments were obtained from cloned cDNA encoding human mGluR5a or
mGluR5b (11) by restriction enzyme digestion, and a BamHI
linker was ligated with the 5
P19
cells and NG108-15 cells were cultured as described (10) in 100-mm
plates for each experiment. Differentiated P19 cells were used for the
experiments on day 3 after treatment with retinoic acid. For metabolic
labeling with [35S]methionine and
[35S]cysteine, the cells were preincubated with cystine-,
methionine-, and cysteine-free Dulbecco's modified Eagle's medium
(ICN) supplemented with 1% fetal bovine serum. After a 30-min
incubation in the medium, the cells were treated with 200 µCi/ml
Pro-mix (Amersham) and incubated for additional 4 h at 37 °C.
The cells were then scraped and washed with phosphate-buffered saline.
The pelleted cells were resuspended in 1 ml of buffer A (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.5%
Nonidet P-40) containing 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1.5 µM pepstatin, 5 µg/ml aprotinin, and 1 mM sodium orthovanadate and lysed
by incubating for 30 min at 4 °C. The lysates were clarified by
centrifugation at 14,000 × g at 4 °C for 30 min.
Whole brain from an
11-week-old mouse (C57BL) was homogenized in 10 volumes of buffer A
containing protease inhibitor mixture and then lysed and centrifuged as
described above.
The immobilized fusion protein (3 µg) was incubated with the 35S-labeled cell extracts (400 µl), the mouse brain homogenate (400 µl), or bovine CaM (5 µg) in
a total volume of 400 µl for 1-2 h at 4 °C. The incubation buffer
was as follows: buffer A (described above) for the
35S-labeled cell extracts and buffer B (buffer A with 2 mM CaCl2) or buffer C (buffer A with 5 mM EGTA) for the mouse brain homogenate and bovine CaM
(Seikagaku Corp.). The beads were then washed with the incubation
buffer. The retained proteins were removed from the beads with
SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer and then
were resolved by SDS-PAGE. In Fig. 1B, the gels were fixed
with 10% glacial acetic acid and 30% methanol for 20 min and dried.
The radioactivities were then image-analyzed (FUJIX BAS 1000). In Fig.
1, C and D, the samples were subjected to
SDS-PAGE in which the gel and the electrophoresis buffer contained 5 mM EGTA and were then subjected to Western blotting.
Samples resolved by SDS-PAGE were then
transferred to a polyvinylidene difluoride membrane and visualized by
an immunoblot analysis with a Vistra ECF Western blotting system
(Amersham). Immunoblots were performed as described (15) using an
anti-CaM antibody (Seikagaku Corp.).
The fusion protein (25 pmol) or the synthetic peptide (400 pmol) was incubated with or without CaM in a total volume of 5 µl (20 mM Tris-HCl, pH 7.5, and 2 mM
CaCl2) for 40 min at room temperature. The components for
phosphorylation were then added in a total buffer volume of 20 µl.
The phosphorylation buffer contained 20 mM Tris-HCl, pH
7.5, 10 mM MgCl2, 0.5 mM
CaCl2, 0.25% bovine serum albumin, 0.5 mM
dithioerythritol, 100 µg/ml phosphatidylserine, 100 µM
[ For the experiments examining the interaction of phosphorylated fusion
protein with immobilized CaM, fusion protein (10 pmol) was incubated
with PKC (0.005 milliunit) either in the absence or presence of
[ The synthetic peptide (R1, 15 nmol; R2, 3 nmol) was
incubated in the phosphorylation reaction buffer with or without PKC
(0.04 milliunit) for 13 h at 30 °C. The concentration of the
components was the same as the standard condition except that the ATP
concentration was changed to 2 mM and bovine serum albumin
was omitted. The peptide solution was then diluted to varying
concentrations with the reaction buffer. The diluted peptide solution
(10 µl) was added to 300 pmol of CaM (2.5 µl) containing 50 mM Hepes, pH 7.5, and 10 mM CaCl2
and then was further incubated for 40 min at room temperature.
The aliquots were then resolved by 12.5% nondenaturing polyacrylamide
gel containing 50 mM Tris-HCl, pH 8.8, and 2 mM CaCl2 and visualized by Coomassie Blue staining.
We considered the possibility that a COOH-terminal
intracellular domain of mGluR5 specifically interacts with other
cellular proteins and thus regulates the receptor responses. To test
the possibility, we screened for cellular proteins that interact with the mGluR5 COOH terminus fused to GST using undifferentiated P19 cells
and the P19 cells differentiated into neurons and astroglia (18). The
protein-binding assay in Fig.
1B revealed that a 17-kDa protein interacts with the immobilized cytoplasmic domain of both mGluR5a (GST-MGR5a846-1073, Fig. 1A)
and mGluR5b (GST-MGR5b846-1073, Fig.
1A). The undifferentiated and differentiated P19 cells had the 17-kDa protein (Fig. 1B), and the neuroblastoma × glioma hybrid cells NG108-15 also had one (data not shown). To identify
the domains which bind the 17-kDa protein, we examined the ability of
the GST-fusion proteins containing various lengths of the COOH terminus
of mGluR5a (Fig. 1A) to bind the 17-kDa protein using NG108-15 cells. Two distinct regions, the 31-amino acid sequence (at
the residue numbers 846-876 corresponding to human mGluR5b; denoted by
site I We next
tested whether the protein specifically associated with the two regions
of mGluR5 is calmodulin (CaM), a Ca2+-binding protein and
an intracellular Ca2+ transducer that regulates the
activity of a variety of structurally distinct proteins (19). The
fusion protein-immobilized agarose beads were incubated with mouse
brain homogenate. The bound proteins were then analyzed by an
immunoblot analysis using an anti-CaM antibody, which revealed the
17-kDa protein to be CaM (Fig. 1C). In Fig. 1D,
the agarose resin was incubated with CaM from the bovine brain, and the
CaM binding on the affinity resin was detected. This strongly suggests
that the association between CaM and the immobilized fusion protein is
direct. The CaM binding was abolished by the removal of
Ca2+ and the addition of 5 mM EGTA (Fig. 1,
C and D). Thus, the CaM binding to the fusion
protein is Ca2+-dependent. We confirmed that
the signal intensity of CaM control did not decrease by incubation with
EGTA prior to SDS-PAGE (data not shown), which excludes the possibility
that the signals may thus be missing in the +EGTA/ The mobility of CaM shown in Fig. 1, C and D, was
slightly different from that in Fig. 1B. The polyacrylamide
gel and the electrophoresis buffer for the experiments shown in Fig. 1,
C and D, contained EGTA whereas the gel and the
buffer of the experiment of Fig. 1B did not. It is known
that CaM shows Ca2+-dependent shifts in
electrophoretic mobility and also exhibits apparent heterogeneity at
the subsaturation levels of Ca2+ (21) as shown in Fig.
1B.
CaM bound less efficiently to site I To identify the CaM binding sites
as shorter domains and to verify the direct association of the domains
with CaM without any mediating proteins, two synthetic peptides (R1 and
R2) were thus synthesized (Fig.
2A). Although the primary
structures of CaM binding domains on a variety of proteins show little
homology, they do share the same propensity to form a basic amphiphilic
A minor band was observed in the peptide-free lane which may be the
aggregated CaM. This fraction moved upward as the peptide:CaM molar
ratio increased. These bands may represent the complexes of the
aggregated CaM and increasing amounts of peptides.
Many receptors including ion channels and G protein-coupled
receptors are known to be modulated by phosphorylation. No strict consensus sequences are detected among the phosphorylation sites by PKC
except for the fact that basic amino acid residues near the
phosphorylation site may be required (23, 24). The two CaM binding
regions described above possess several threonine and serine residues
which have basic amino acids around them. We thus examined whether the
recombinant fusion protein serves as a substrate of PKC in
vitro. As shown in Fig.
3A, the GST-MGR5site
I and the GST-MGR5site II were phosphorylated by
PKC. In contrast, PKC could not phosphorylate
GST-MGR5bb-specific, which has the mGluR5b-specific region
but does not have either site I or site II. Recently,
PKC-dependent phosphorylation of threonine residue at
position 840 of rat mGluR5 was reported (22). Since GST-MGR5site
I We next determined whether PKC phosphorylation was affected by the
binding of CaM on its target sequence. Fig. 3A shows that the phosphorylation of site I, site I The peptides R1 and R2 could also be phosphorylated by PKC, and the
32P incorporation into the R1 peptide was much slower than
the incorporation into the R2 peptide (Fig.
4A). We quantified the maximal
levels of 32P incorporation into the peptides. The
calculation of stoichiometry indicated that PKC catalyzed the
incorporation of approximately 0.61 mol of phosphate/mol of peptide R1
and 0.79 mol of phosphate/mol of peptide R2 (mean of duplicate
determinations, from two separate experiments). These results thus
indicated that the enzyme phosphorylates at least one residue in
peptides R1 and R2. The phosphorylation of the two peptides was
inhibited by preincubation with CaM (Fig. 4, B and
C), and the phosphorylation of R2 was more severely blocked by CaM than that of R1 (Fig. 4D). These results thus
suggested that the direct CaM association to the peptide prevents the
access of PKC.
Since the binding of CaM on mGluR5 inhibited the
phosphorylation of mGluR5, it seemed likely that the phosphorylation
would conversely inhibit the CaM binding. We thus examined whether the mGluR5 phosphorylation interferes with the ability of mGluR5 to associate with CaM. The fusion protein GST-MGR5site I and
GST-MGR5site II was incubated with PKC in the absence and
presence of ATP; the former condition served as a control reaction.
Both unphosphorylated and phosphorylated proteins were further
incubated with the CaM-agarose beads, and the elution pattern of each
was analyzed (Fig. 5). The
uppermost band in Fig. 5, A or B,
corresponds to the band shown in Fig. 5C. This could thus
represent the fusion protein in full-length whereas the extra bands in
Fig. 5, A or B, could indicate the incompletely
translated proteins or the degraded proteins which include the GST
backbone degraded from the GST-fusion. The unphosphorylated fusion
protein of site II bound the CaM-agarose resin quite tightly; it did
not flow through the resin nor was it washed off (Fig. 5A,
right). Significant amounts of GST-MGR5site I passed through the resin, and some of the retained protein was released
by washing, thus indicating that its binding on the immobilized CaM is
weaker than the site II binding (Fig. 5A, left).
Unphosphorylated fusion proteins of site I and site II which bound the
CaM-agarose beads were eluted with the buffer containing 5 mM EGTA (Fig. 5A). In contrast, the majority of
the phosphorylated proteins GST-MGR5site I and
GST-MGR5site II either flowed through or washed off the
CaM-agarose resin (Fig. 5, B and C).
To verify the effect of phosphorylation on the CaM binding, we
performed a gel-shift assay with the phosphorylated R1 or R2 peptide
(Fig. 2C). The lower arrowheads indicate free
CaM; much higher molecular weight proteins may be aggregated CaM as
mentioned before. The CaM-peptide complexes indicated by the
upper arrowheads were missing when the phosphorylated
peptides were added for both the R1 and R2 peptides. With peptide R2,
the mobility shift of the monomer or aggregated CaM was almost
completely blocked by the phosphorylation. With peptide R1, only a
small change in the mobility of both the CaM monomer and the aggregated
CaM occurred by incubation with the peptide which had been subjected to
PKC phosphorylation. The extent of the mobility shifts at the
phosphorylated R1 peptide:CaM ratio of 20:1 (Fig. 2C) was
similar to or less than that at the unphosphorylated peptide:CaM ratio
of 1:1 (Fig. 2B). There are two possible reasons for the
fact that the small shift still occurred with peptide R1 in Fig.
2C. 1) One possibility is that the unphosphorylated R1
peptide is responsible for the mobility shifted CaM. We changed the
phosphorylation condition to achieve the maximal phosphorylation of the
peptide. The ATP concentration and the amount of PKC was increased, and
the incubation period was elongated. Nevertheless, it may be possible
that a small fraction of the R1 peptide still remained
unphosphorylated. Even if this happens, the unphosphorylated peptide
could be less than one-twentieth of the peptide based on the results of
Fig. 2, B and C. 2) The other possibility is that
the phosphorylated peptide has a weak affinity for CaM. As shown in
Fig. 5C, the majority of the 32P-incorporated
fusion protein flowed through the CaM-agarose resin. A small amount of
the phosphorylated protein, however, was released by washing and EGTA
treatment. This indicates that some of the fusion protein was retained
on the CaM-agarose resin and suggests that the phosphorylated protein
had a weak interaction with CaM. It is thus conceivable that the
phosphorylated peptides may also have a weak affinity for CaM. In any
event, the primary effect of phosphorylation is thus to prevent CaM
binding. Therefore, the findings on both the fusion proteins and the
peptides provide convincing evidence that the phosphorylation of the
CaM binding site either prevents or hampers its interaction with
CaM.
We have shown in this study that CaM directly binds the COOH
terminus of the mGluR5 in vitro. We identified the two
binding sites which are separated by the mGluR5b-specific alternative exon. There is growing evidence that the ion channels can be regulated by CaM through direct binding. In some cases, the binding reduces the
channel activities; in others, it activates the channels. The binding
of CaM on the olfactory cyclic nucleotide-gated cation channel reduces
the channel activity (25), and in contrast, the Paramecium
Na+ channels are activated by CaM (26). Direct CaM binding
might thus be a general mechanism for the
Ca2+-dependent regulation of ion channel
properties. There has however been no previous report of direct CaM
binding on G protein-coupled receptors as far as we know. In a recent
study, a subunit of the N-methyl-D-aspartate
receptor, which belongs to the glutamate-gated ion channels, was also
reported to be a target of CaM binding (14). Two CaM binding sites of
the subunit NR1 were found which have different affinities for CaM. It
was demonstrated by patch-clamp recording techniques that the
interaction of CaM with the NR1 subunit causes an inactivation of the
channels. We showed in this paper that CaM also interacts with the
subtype 5 of the metabotropic glutamate receptors at two distinct
sites. It is interesting that direct CaM binding has been demonstrated
in members of both categories of glutamate receptors: namely,
ionotropic receptors and metabotropic receptors. The degree of
cooperativity between the two CaM binding sites is unknown in both the
case of NR1 and the case of mGluR5. Although both of the fusion
proteins derived from mGluR5a and mGluR5b associated with CaM in
vitro, the affinities for CaM might be different in the two splice
variants of the native form. It is also conceivable that a
conformational change of the receptor by agonist stimulation might also
affect the accessibility of CaM to the receptor. The functional
consequences of the direct binding of CaM on mGluR5 thus remain to be
elucidated.
The eight mGluR subtypes can be classified into three groups (27). The
Group I mGluRs, which comprise mGluR1 and mGluR5, are coupled to
phospholipase C, which catalyzes phosphoinositide hydrolysis. It
results in the production of both inositol trisphosphate, which induces
the release of Ca2+ from intracellular stores, and
diacylglycerol, which stimulates PKC. PKC is a serine/threonine kinase
that is thought to play some role in diverse cellular processes. There
are many serine and threonine residues at the COOH terminus of mGluR1
and mGluR5, which suggests that the residues could be targets of
kinases that could thus regulate receptor activity. We herein
demonstrated that the two CaM binding sites in mGluR5 are substrates
for PKC. The changes in the cellular response by direct stimulation of PKC using phorbol esters have been previously investigated in many
studies. The mGluR-mediated phosphoinositide hydrolysis was inhibited
by the PKC stimulation in cerebellar granule cells (28) and in rat
hippocampal slices (29). The diacylglycerol production by the agonist
stimulation of mGluRs was also inhibited by the direct stimulation of
PKC in cerebrocortical nerve terminals (30). The receptor-mediated
response was inhibited by the preactivation of PKC in baby hamster
kidney (BHK) cells transfected with mGluR1a (31) whereas receptor
phosphorylation was also observed in response to agonist activation of
the receptor in the same BHK cells (32). These findings have led to the
suggestion that the PKC phosphorylation of Group I receptor may result
in receptor desensitization. The phosphorylation site responsible for
the desensitization has yet to be identified, and this study thus
suggests some likely candidates. Preliminary results suggest that a
higher rate of phosphate incorporation appears in the fusion protein
derived from mGluR5b than in the fusion protein from mGluR5a under the
same reaction conditions.2
Although the fusion protein which has only the mGluR5b-specific region
is not phosphorylated, the region might be phosphorylated when linked
to site I and/or site II. It is also conceivable that the
mGluR5b-specific exon between site I and site II affects the accessibility of the PKC enzyme to the two sites. The alternative exon
might serve as a modulatory domain which affects the phosphorylation state of the receptor.
The CaM-dependent inhibition of the phosphorylation was
demonstrated at both sites in mGluR5. The phosphorylation of the site II protein, which showed a higher affinity for CaM, was more
sensitively suppressed by CaM. We observed similar results with
peptides R1 and R2, where the latter was more sensitive in
phosphorylation suppression by CaM. Thus the most likely reason for the
reduced phosphorylation is that CaM blocks access of PKC to the
phosphorylation site. We further showed that the phosphorylation of the
two sites prevents their interaction with CaM. These results thus
indicated that the CaM binding and the PKC phosphorylation are mutually antagonistic.
Myristoylated alanine-rich protein kinase C substrate (MARCKS) is a
major cellular substrate of PKC, and it binds to both CaM and actin.
PKC phosphorylation inhibits CaM and actin binding, and CaM binding
prevents PKC phosphorylation and actin binding (33, 34). The CaM
binding domains of mGluR5 might thus be involved in cytoskeletal
association in a manner analogous to the MARCKS protein. The relative
timing of surges of internal Ca2+ transient (and the
Ca2+-CaM complexes it produces) and PKC activity thus
determines the extent of MARCKS protein phosphorylation (35). It is
thus conceivable that the different times of arrival of
Ca2+ and PKC signal to the membrane-associated mGluR5 might
thus affect the extent of the phosphorylation of mGluR5.
Whereas the two mGluR5 variants and mGluR1a possess a long cytoplasmic
domain, the splice variants of mGluR1 named mGluR1b and mGluR1c have a
smaller COOH terminus than the three receptors because of an in-frame
stop codon in the alternative exon (6, 7). The shorter proteins
elicited a small and slowly generated oscillatory current in
Xenopus oocytes (7) whereas the subcellular distribution of
mGluR1b was different from mGluR1a and the two mGluR5 isoforms when
expressed in cells (8). The COOH-terminal domain of mGluR1a contains
regions homologous to site I and site II of mGluR5, whereas the smaller
cytoplasmic regions in mGluR1b and mGluR1c also have a region
corresponding to site I but do not have a region corresponding to site
II because of the truncation by the in-frame stop codon. We could thus
speculate that a loss of a target sequence of CaM or PKC or possibly a
binding domain of actin in mGluR1b and mGluR1c might thus confer the
characteristics shown in the shorter receptor. Our findings in this
study may provide new leads for approaching the
Ca2+-mediated modulation of the mGluRs as well as the
biological function of the spliced cassette of the receptors.
School of Health Sciences,
Construction and Isolation of GST-fusion Proteins
-end of the fragment followed by blunting
the end if necessary. Each fragment was cloned in-frame into the GST
gene fusion vector pGEX-2T. The fusion protein was expressed in
Escherichia coli and was purified by glutathione-Sepharose beads (Pharmacia) according to the manufacturer's protocol. The experiments of Figs. 1 and 3A were done with the immobilized
fusion proteins on the beads whereas the experiments of Figs.
3B and 5 were done with the fusion proteins eluted from the
matrix.
Fig. 1.
Identification of the cellular protein
associated with the mGluR5 fusion protein. A, schematic
presentation of the constructs that were fused to GST. The structure of
COOH-terminal domain of human mGluR5b is shown on the top.
The black box represents the mGluR5b-specific insert; the
shaded box, the seventh transmembrane segment; the
arrowheads, the restriction enzyme sites of the cDNA used for the construction of GST-fusion proteins. The beginning and
ending amino acid residue is depicted using a single amino acid code
and its corresponding human mGluR5b residue number (Ref. 11). The
column on the right summarizes the binding activity as
determined by the experiments in B, C, and
D. B, detection of mGluR5-binding proteins.
Lysates of the 35S-labeled P19 cells, which
were either undifferentiated or differentiated, or lysates of the
35S-labeled NG108-15 cells were incubated with the
GST-fusion protein or GST backbone (GST) immobilized on
glutathione-Sepharose beads. The bound proteins were subjected to
12.5% SDS-PAGE. The positions of the protein standards and their sizes
(in kDa) are shown on the left. C and
D, Western blotting using an anti-CaM antibody reveals that
CaM could associate with the mGluR5 fusion protein (C) and
that the association of mGluR5 and CaM is direct (D). Homogenate from the adult mouse brain (C) or bovine CaM
(D) was incubated with the GST-fusion protein or GST
backbone (GST) on glutathione-Sepharose beads in the
presence or absence of Ca2+ as indicated. The bound
proteins resolved by 15% SDS-PAGE were further subjected to an
immunoblot analysis using an anti-CaM antibody. CaM (C, 20 ng; D, 200 ng) was run as a control (CaM control). The positions of the protein standards and their sizes (in kDa) are shown on the right, and the position of the CaM
is marked on the left by an arrowhead.
[View Larger Version of this Image (41K GIF file)]
Fig. 3.
Autoradiographs showing the effect of CaM on
PKC phosphorylation of the mGluR5 fusion proteins. A, the
GST-fusion protein or GST backbone on glutathione-Sepharose beads was
preincubated with either CaM (CaM:fusion protein ratio is 10; denoted
by +) or the appropriate volume of buffer (denoted by ) as described under "Experimental Procedures." The components necessary for phosphorylation were then added including [
-32P]ATP
and 0.005 milliunit of PKC, and the mixture was incubated at 30 °C
for the times indicated. The reactions were stopped by adding SDS-PAGE
sample buffer. The samples were processed by 12.5% SDS-PAGE. The
positions of the protein standards and their sizes (in kDa)
are shown on the right. The prolonged incubation up to 1 h did not reveal the phosphorylation of the
GST-MGR5bb-specific nor the GST backbone (data not shown).
B, effect of CaM on phosphorylation of the site I and site
II. The GST-fusion proteins were preincubated with varying molar
amounts of CaM. The CaM:fusion protein ratios are indicated in the
figure. The components necessary for phosphorylation including
[
-32P]ATP and 0.005 milliunit of PKC were then added.
Phosphorylated bands of the GST-fusion proteins which were resolved on
12.5% SDS-PAGE are indicated by arrows.
[View Larger Version of this Image (29K GIF file)]
Fig. 5.
Effect of mGluR5 phosphorylation on its
interaction with CaM. A-C, the GST-fusion protein
(GST-MGR5site I or GST-MGR5site
II) was incubated with PKC (0.005 milliunit) either in the
absence (A) or presence of ATP (B, ATP;
C, [-32P]ATP). Samples were then incubated
with CaM-agarose resin, and the resin was sequentially subjected to the
indicated treatments as described under "Experimental Procedures."
Each fraction was analyzed by immunoblotting with an anti-GST antibody
(A, B) and by image-analyzing the radioactivities
(C). The position of the fusion protein is marked on the
right by an arrowhead.
[View Larger Version of this Image (52K GIF file)]
-32P]ATP, and PKC (Boehringer Mannheim). Reactions
were incubated at 30 °C. For quantification of
32P-labeled fusion proteins, the reaction was stopped by
the addition of the SDS-PAGE sample buffer, and 12.5% SDS-PAGE was
performed. The gels were fixed with 10% glacial acetic acid and 30%
methanol for 20 min and dried. The radioactivities were then
image-analyzed (FUJIX BAS 1000). For quantification of the
phosphorylated peptide, aliquots were spotted from the reaction mixture
onto P81 phosphocellulose paper (Whatman) and then were subjected to
washing and Cerenkov counting (16). In some experiments, we performed
SDS-PAGE with Tricine buffer (17) and quantified
32P-labeled peptides to confirm the propriety of the
procedure with P81 papers (data not shown). To estimate the maximal
levels of 32P incorporation into the peptides, the
incubation was done with 0.005 milliunit of PKC for 20-25 h for the
peptide R1 and 2-4 h for the peptide R2. No further increase in the
phosphorylation of the peptides was seen after incubation for the
additional 1 h.
-32P]ATP for 1 h at 30 °C. The solution (20 µl) was then mixed with a 37.5-µl bed volume of CaM-agarose resin
(Sigma) in a total volume of 200 µl of buffer B. After a 1-h
incubation at room temperature, the resin was washed with 7.7 volumes
of the same buffer. This fraction was denoted by Unbound in
Fig. 5. The resin was then washed twice with 12 volumes of buffer B
(CaCl2 concentration in buffer B was reduced to 0.01 mM) and eluted with 12 volumes of buffer C. Each fraction
was concentrated by Ultrafree-MC, UFC3LGC00 (Millipore) and then 25%
of the unbound, wash, and eluted fractions as well as 2.5% of the
input were resolved by 12.5% SDS-PAGE. An immunoblot analysis was done
with an anti-GST antibody (Pharmacia Biotech Inc.). Image analyzing was
performed for the phosphorylated fusion protein.
Detection of a Cellular Protein That Interacts with the mGluR5
Fusion Protein
) and the 54-amino acid sequence (at residue numbers 917-970 in human mGluR5b; denoted by site II) have
been shown to be responsible for the binding (Fig. 1, A and
B). The amounts of the 17-kDa protein associated with the
fusion protein containing site II were larger than those with the site
I
fusion protein (Fig. 1B). The mGluR5b-specific exon is
located between site I
and site II in the mGluR5b (Fig.
1A).
Ca2+
lanes of Fig. 1, C and D, because the CaM
antibody binding to CaM is Ca2+-dependent. The
slower migrating bands seen in Fig. 1D may represent aggregated CaM (20). CaM tends to wash off the blotted matrix (15)
probably because the protein is small and highly acidic. The aggregated
CaM may be retained on the filter matrix and thus be detected by the
immunoblotting more efficiently than the CaM monomer. We also observed
the extra bands by Coomassie Blue staining, which showed a similar
mobility to the upper bands in Fig. 1D but were much fainter
than the band corresponding to the CaM monomer (data not shown).
than site II in the experiments
with either the brain homogenate (Fig. 1C) or CaM (Fig. 1D), and these findings coincided with the results by the
35S-labeled cell lysate in Fig. 1B. Since site
I
begins at the amino acid residue 846 and thus lacks the
NH2-terminal region of the cytoplasmic domain, we
constructed and expressed the GST-MGR5site I containing the
amino acid sequence at positions 827-876 (Fig. 1A). The
amounts of the associated CaM to the GST-MGR5site I
and
those to the GST-MGR5site I were similar (Fig.
1C). This indicates that the lower affinity between the
GST-MGR5site I
and CaM is not due to the lack of the
NH2-terminal region of the intracellular domain. We would
mainly use the GST-MGR5site I afterward since site I
lacks a threonine residue at position 841 which is reported to be
responsible for the generation of calcium oscillations (Ref. 22;
see below). The mGluR5b-specific amino acid region was connected to the
COOH terminus of the GST backbone and was then subjected to a
protein-binding assay, in which CaM did not bind to it (Fig. 1D).
-helix (19). The amino acid sequence of both peptides has a stretch
of basic and hydrophobic amino acids such as a possible CaM binding
site. The incubation of CaM with either peptide in the presence of
Ca2+ shifted the mobility of CaM on nondenaturing
polyacrylamide gel (Fig. 2B). As the peptide:CaM molar ratio
increased, the intensity of the band corresponding to free CaM
decreased. The upper bands appearing at high peptide:CaM ratios may
correspond to the stable peptide-CaM complexes. For peptide R2, free
CaM disappeared, and a complete shift of CaM occurred to the upper band
at a peptide:CaM ratio of 2:1. In contrast, some amounts of protein
remained stained at the position of the free CaM on the gel even at the
R1:CaM ratio of 20:1. The mobility-shifted CaM was reproducibly smeared in the experiments with R1, and thus the structure of the R1-CaM complexes may be more relaxed and/or unstable in the polyacrylamide gel
than that of the R2-CaM complexes. These results indicated that the two
peptides are able to bind CaM directly and suggested that the R2
peptide has a higher affinity for CaM than the R1 peptide even though
the observation was limited in the gel matrix. The removal of
Ca2+ and the addition of EGTA in the incubation mixture and
the gel abolished the gel shift (data not shown), hence the formation of the complexes is Ca2+-dependent.
Fig. 2.
CaM binding to peptides R1 and R2.
A, schematic representation of the COOH-terminal domain of mGluR5b
and the synthesized peptides R1 and R2. The black box
represents the mGluR5b-specific insert; the shaded box, the
seventh transmembrane segment. The numbers correspond to the
human mGluR5b residue number. B, detection of CaM-peptide
complexes by nondenaturing polyacrylamide gel-shift assays. CaM was
incubated with varying molar amounts of R1 and R2 peptides and was
resolved on 12.5% nondenaturing polyacrylamide gel. The upper
arrowheads indicate CaM-peptide complexes, and the lower
arrowheads indicate free CaM. C, the inhibition of the CaM-peptide complex formation by the peptide phosphorylation. CaM was
incubated with varying molar amounts of the R1 and R2 peptides which
had been subjected to PKC phosphorylation as described under
"Experimental Procedures" and were resolved on 12.5% nondenaturing polyacrylamide gel. The lower arrowheads indicate free CaM.
The CaM-peptide complexes which are expected at the position indicated by the upper arrowheads were not detected.
[View Larger Version of this Image (68K GIF file)]
lacks the threonine residue at position 841 of human mGluR5
that corresponds to position 840 of rat mGluR5, we tested whether
GST-MGR5site I
could be phosphorylated. Its
phosphorylation rate was similar to that of GST-MGR5site I
(Fig. 3A), which indicated that the PKC phosphorylation of
GST-MGR5site I
could occur at other serine/threonine
residue(s). The phosphorylation rate of site II was much faster than
site I or site I
(Fig. 3, A and B).
, and site II is almost fully
inhibited at the CaM:fusion protein ratio of 10:1. We did not observe
any inhibition of phosphorylation by CaM in a control experiment using
histone H1 as a substrate of PKC (data not shown). As shown in Fig.
3B, site II phosphorylation was affected by CaM more readily
than site I phosphorylation. The different sensitivity to CaM between
site I and site II could thus be interpreted by their different
affinity for CaM.
Fig. 4.
Effect of CaM on phosphorylation of peptides
R1 and R2. A, time course of phosphorylation. R1 (open
circles) or R2 (filled circles) was incubated with
[-32P]ATP and PKC (0.0005 milliunit, n = 1; 0.0009 milliunit, n = 3) at 30 °C. The
incubations were terminated at the times indicated, and the
radioactivity incorporated into the peptide was measured as described
under "Experimental Procedures." The radioactivity for the
phosphorylated R2 peptide after incubation with PKC for 40 min was
arbitrarily assigned a value of 100, and the radioactivities of the
other samples are expressed as a percentage of this value. The results
represent the mean ± S.E. (n = 4). B
and C, R1 (B) or R2 (C) was
preincubated with varying amounts of CaM as described under
"Experimental Procedures." The CaM:peptide ratios are as follows:
0, open circles; 0.2, filled circles; 0.5, open triangles; 1, filled triangles; 2, open squares. The components necessary for the
phosphorylation were then added including PKC (0.005 milliunit for the
R1 and 0.0005 milliunit for the R2), and the mixture was incubated at
30 °C for the times indicated. The values were normalized against
the quantity of the phosphorylated peptide after incubation with PKC in
the absence of CaM for 40 min. The results represent the mean ± S.E. (R1, n = 7; R2, n = 6).
D, the quantity of the phosphorylated peptide R1 (open
circles) or R2 (filled circles) after incubation with
PKC for 20 min in the experiments shown in B or C
is plotted against the molar ratio of CaM/peptide. The radioactivity in
the absence of CaM was taken as 100. The results represent the
mean ± S.E. (R1, n = 7; R2, n = 6). *, p < 0.01, significance by t test
comparing the R1 phosphorylation to the R2 phosphorylation under the
same peptide:CaM ratio.
[View Larger Version of this Image (29K GIF file)]
*
This study was supported by grants from the Ministry of
Education, Science, Sports and Culture of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
81-92-642-2630; Fax: 81-92-642-2645; E-mail:
hsugiscb{at}mbox.nc.kyushu-u.ac.jp.
1
The abbreviations used are: mGluR, metabotropic
glutamate receptor; GST, glutathione S-transferase; CaM,
calmodulin; PKC, protein kinase C; PAGE, polyacrylamide gel
electrophoresis; BHK cells, baby hamster kidney cells; MARCKS,
myristoylated alanine-rich protein kinase C substrate; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine.
2
R. Minakami, unpublished observations.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.