From the Faculty of Pharmacy and Department of Pharmacology University of Toronto, Toronto, Ontario M5S 252, Canada
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ABSTRACT |
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The metabotropic glutamate receptor (mGluR) 4 subtype of metabotropic glutamate receptor is a presynaptic receptor
that modulates neurotransmitter release. We have characterized the
properties of a truncated, epitope-tagged construct containing part of
the extracellular amino-terminal domain of mGluR4. The truncated
receptor was secreted into the cell culture medium of transfected human embryonic kidney cells. The oligomeric structure of the soluble truncated receptor was assessed by gel electrophoresis. In the presence
of high concentrations of a reducing agent, the truncated receptor
migrated as a monomer; at lower concentrations of the reducing agent,
only higher molecular weight oligomers were observed. Competition
binding experiments using the radiolabeled agonist [3H]L-2-amino-4-phosphonobutyric acid
revealed that the rank order of potency of metabotropic ligands at the
truncated receptor was similar to that of the full-length
membrane-bound receptor. However, the truncated receptor displayed
higher affinities for agonists and lower affinities for antagonists
compared with the full-length receptor. Deglycosylation produced a
shift in the relative molecular weight of the soluble protein from
Mr = 71,000 to Mr = 63,000; deglycosylation had no effect on the binding of
[3H]L-2-amino-4-phosphonobutyric acid,
indicating that the asparagine-linked carbohydrates are not necessary
for agonist binding. These results demonstrate that although the
primary determinants of ligand binding to mGluR4 are contained within
the first 548 amino acids of the receptor, additional amino acids
located downstream of this region may influence the affinity of ligands
for the binding site.
The metabotropic glutamate receptors
(mGluRs)1 are a family of
neurotransmitter receptors that mediate a variety of physiological functions in the central nervous system including the modulation of
synaptic transmission. The mGluR family of receptors has been divided
into three subgroups based on sequence homology, signal transduction
properties, and pharmacological profiles (1, 2). Group I mGluRs include
mGluR1 and mGluR5 and are coupled to the stimulation of phosphoinositol
turnover. Group II receptors include mGluR2 and mGluR3, and group III
receptors include mGluR4, mGluR6, mGluR7, and mGluR8. In cell lines,
group II and III mGluRs couple to the inhibition of cAMP formation. The
mGluRs are homologous with the calcium-sensing receptors, the
GABAB receptors, and a class of mammalian pheromone
receptors (2). Within the family of mGluRs, the amino acid sequence
identity among members of a subgroup is approximately 70%, whereas the
homology between the different groups is about 45%.
The basic structural domains of mGluRs include a large extracellular
amino-terminal domain (ATD), a hydrophobic region containing seven
putative transmembrane domains, and an intracellular carboxyl terminus.
A molecular model of the tertiary structure of the ATD of the mGluR1
subtype of mGluR has been formulated based on the sequence similarity
between the mGluRs and the periplasmic-binding proteins in prokaryotes
(3). The periplasmic-binding proteins are a family of proteins that
transport nutrients into bacteria. Within the family of bacterial
proteins, the leucine, isoleucine, valine-binding protein is the most
homologous with a region within the ATD of mGluRs. The leucine,
isoleucine, valine-binding protein, like other periplasmic-binding
proteins in bacteria, is a soluble protein that forms the recognition
unit of a multimeric transport complex. In addition to the binding
proteins, the bacterial transport complexes also include membrane-bound
transport modules responsible for the translocation of amino acids and
other nutrients across the membrane (see Ref. 4 for a review).
Previous studies using site-directed mutagenesis and chimeric receptors
have suggested that the ligand binding pockets of mGluRs are located in
the ATDs (3, 5, 6). It remains unclear, however, whether all of the
determinants for ligand binding to mGluRs are contained within the ATD,
or whether there are additional amino acids in other regions of
receptor protein located extracellularly that may contribute directly
or indirectly to ligand binding affinity in mGluRs. Recently, Okamoto
et al. (7) have shown that a construct containing the entire
ATD of the mGluR1 receptor forms a soluble protein that retains the
ability to bind the group I mGluR agonist [3H]quisqualic
acid. In the present study, we demonstrate that a fragment of the ATD
of mGluR4 forms a soluble protein that is secreted from cells. This
truncated receptor retains the ability to bind the group III mGluR
agonist [3H]L-AP4 and displays the basic pharmacological
profile of the full-length receptor. However, the truncated receptor
has a higher affinity for agonists and a lower affinity for antagonists
compared to the membrane-bound receptor, indicating that additional
structural determinants contribute to ligand binding affinity in mGluR4.
Expression Vectors--
For the expression of wild-type mGluR4a
in human embryonic kidney (HEK) 293 cells, the
BglII-EcoRI fragment of rat mGluR4a cDNA in
the pBluescript SK Plasmid Preparations and Transfections--
cDNA constructs
were transformed into XL-1 blue competent cells (Stratagene, La Jolla,
CA). Ampicillin-resistant colonies were inoculated in 3 ml of
Luria-Bertaini medium containing ampicillin and grown at 37 °C for
16 h; the cells were collected, and plasmid DNA was prepared using
the QIAspin Miniprep Kit (Qiagen). The DNA from the plasmid preparation
was subjected to restriction enzyme digestion and gel electrophoresis
to check the orientation of the insert. The plasmids were transiently
transfected into HEK cells by calcium phosphate precipitation. The
cells were grown in 100-mm plates to 80% confluence; 10 ml of fresh
minimum essential medium containing 6% fetal bovine serum (Hyclone
Corp., Logan, UT) were added to the cells 3 h before transfection.
For transfections, 20 µg of plasmid DNA were added to 450 ml of 1:10
TE buffer (pH 7.0), followed by the addition of 50 µl of 2.5 M CaCl2. The reagents were mixed with 500 µl
of 2× HEPES-buffered saline solution and added to HEK cells. The cells
were incubated with the DNA at 37 °C for 4 h, the medium was
aspirated, and the cells were exposed to 15% glycerol in
phosphate-buffered saline, pH 7.2, for 30 s and then washed with
phosphate-buffered saline; fresh medium was subsequently added.
Protein Expression and Processing--
Twenty-four h after
transfection, the cell culture medium was replaced with OptiMEM (Life
Technologies, Inc.). Each 100-mm plate of transfected cells was
subcultured into two 100-mm plates containing 7 ml of OptiMEM.
Forty-eight h after transfection, the culture media (the soluble
fraction) and the cells (the total cell fraction) were collected
separately. The cells were collected by centrifugation (1,380 × g for 10 min), and the pellet was suspended in 20 ml of cold
lysis buffer (30 mM HEPES, 1 mM ethylene
glycol-bis-N,N,N',N'-tetraacetic acid, and 5 mM
MgCl2, pH 7.4) and homogenized with a Polytron. The pellet
was then centrifuged at 48,400 × g at 4 °C for 20 min, resuspended in 15 ml of lysis buffer with 0.1 mM
phenylmethylsulfonyl fluoride and 0.08% Triton X-100, and incubated at
37 °C for 10 min. An additional 12 ml of nonsupplemented lysis
buffer were added, and the sample was centrifuged at 48,400 × g at 4 °C for 20 min. The pellet was resuspended in 15 ml
of lysis buffer with 0.1 mM phenylmethylsulfonyl fluoride,
centrifuged, resuspended in 1.0-2.0 ml of assay buffer (30 mM HEPES, 100 mM NaCl, 1.2 mM MgCl2-6H2O, 5 mM KCl, and 2.5 mM CaCl2, pH 8.0), and then homogenized with a
glass homogenizer. The homogenized samples were stored at Radioligand Binding Assay--
The radioligand binding assay for
the soluble truncated mGluR4 receptor was conducted in a total volume
of 250 µl. [3H]L-AP4 (specific activity, 49 Ci/mol) was purchased from Tocris Cookson (Bristol, United Kingdom).
Reagents were added in the following order: 25 µl of 10× competing
drug or 25 µl of 3 mM L-SOP (300 µM final concentration for blanks) or 25 µl of assay buffer (for total binding), 100 µl of assay buffer, 100 µl of the
dialyzed sample containing 9 µg of total protein, and 25 µl of
[3H]L-AP4 (final concentration, 30 nM, except for autocompetition experiments with
L-AP4 in which 10 nM
[3H]L-AP4 was used). Preliminary experiments
in which different incubation times with the radioisotope were tested
demonstrated that under these conditions, equilibrium had been reached
after 40 min. Therefore, after a 40-min incubation on ice, 340 µg of Deglycosylation--
Three µl of 10× G7 Buffer (New England
BioLabs, Beverley, MA) and 1 µl of PNGase F (500,000 units/ml, New
England BioLabs) were added to 26 µl of the dialyzed sample of
soluble receptor. After a 1-h incubation at 37 °C, 4× SDS sample
buffer and 100 mM dithiothreitol (final concentration) were
added, and the samples were subjected to electrophoresis and
immunoblotting. For control experiments, the samples were treated in
the same way, except that the PNGase was omitted. For experiments on
the effects of deglycosylation on the binding of
[3H]L-AP4, the proportions of the reagents
were scaled up accordingly.
Immunoblotting--
SDS-polyacrylamide gel electrophoresis was
conducted on samples of the soluble and total cellular fractions.
The samples were suspended in SDS sample buffer containing
100 mM dithiothreitol (unless indicated otherwise) and
separated on 8% polyacrylamide gels, and the proteins were transferred
onto a nitrocellulose membrane (pore size, 0.45 µm; Schleicher & Schuell) by transblotting at 225 mA for 2 h at 4 °C. The blots
were incubated in washing buffer (10 mM Tris, 150 mM NaCl, and 0.2% Tween 20, pH 8.0) containing 5%
powdered milk for 16 h, followed by three incubations with washing
buffer for 5 min each. The blots were incubated with the anti-c-myc
mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA)
for 2 h, followed by three 5-min washes, and then incubated in
anti-mouse IgG conjugated to horseradish peroxidase (Amersham Canada,
Oakville, Ontario, Canada) for 2 h, followed by three 5-min
washes. After an additional 30-min washing period, the membrane was
soaked in ECL Western blotting detection reagents for 2 min and exposed
to Hyperfilm ECL (Amersham).
Expression of the Truncated Receptor--
A truncated cDNA
expression construct was produced that contained a sequence encoding
the mGluR4 signal peptide, a c-myc epitope tag, and a portion of the
amino-terminal domain of mGluR4 up to and including Tyr-548. The
carboxyl terminus of the truncated protein was 39 amino acids upstream
from first putative transmembrane domain (Fig.
1). Previous experiments in our
laboratory have shown that the insertion of the c-myc epitope at this
position in mGluR4 does not alter the pharmacological properties of the
full-length receptor (data not shown).
The full-length c-myc-tagged mGluR4a or the truncated c-myc-tagged
expression plasmids were transiently expressed in HEK cells, and
aliquots of the cell culture media (the soluble fraction) and the
transfected cells (the cellular fraction) were collected 48 h
after transfection and examined by immunoblotting using an anti-c-myc
antibody. Cells transfected with full-length c-myc-tagged mGluR4a
expressed immunoreactive proteins (Mr 93,000 and
Mr 109,000) that likely correspond to the
unglycoslylated and glycosylated mGluR4a monomers. An additional higher
molecular weight band at approximately Mr
224,000 was also observed; this protein is likely a dimer of mGluR4
(Fig. 2). The soluble fraction from cells
transfected with the truncated mGluR4 construct contained a c-myc
immunoreactive protein that migrated at Mr
71,000 (Fig. 2, lane 2). Samples of the soluble fraction
collected from untransfected HEK cells contained low levels of a
protein that cross-reacted with the c-myc antibody; this protein
migrated at a slightly slower rate than the Mr
71,000 protein present in the soluble fraction from transfected cells (Fig. 2, lane 1).
Oligomeric Structure of Truncated mGluR4--
The effects of
the reducing agent DTT on the truncated receptor were examined using
SDS-polyacrylamide gel electrophoresis and immunoblotting. In the
presence of high concentrations of DTT (1-100 mM), the
receptor migrated as a monomer with a Mr of 71,000 (Fig. 3). Lowering the
concentration of DTT from 1 mM to 0.1 mM
produced an abrupt shift in the pattern of the immunoreactive bands;
the band corresponding to the monomer disappeared, and two higher
immunoreactive bands appeared. The lower molecular weight band migrated
at Mr 145,000, whereas the upper band migrated with an estimated Mr of 202,000; these likely
correspond to dimers and trimers of the truncated receptor. The pattern
of bands observed in the presence of 0.01 mM DTT was
identical to that seen with 0.1 mM DTT. These results
indicate that oligomers of the truncated receptor are formed by
intermolecular disulfide bonds.
Pharmacological Profile of the Soluble Receptor--
High affinity
binding of L-AP4 and L-SOP are unique
pharmacological features of group III mGluRs. No specific
[3H]L-AP4 binding was detected in samples of
the soluble fraction from untransfected HEK cells. However, in the
soluble fraction of cells transfected with the truncated mGluR4, a high
level of specific binding was consistently observed. Typically, 100 µl of the soluble preparation in a 250-µl assay yielded
approximately 12,770 specific d.p.m.s (0.12 pmol) bound at 30 nM [3H]L-AP4, and specific
binding represented 66 ± 3% of the total binding. The inhibitory
potency of the agonists L-AP4, L-SOP, L-glutamate, and cyclobutylene AP5 and the group III mGluR
antagonists MAP4, CPPG, and MPPG for the truncated soluble mGluR4
receptor were compared with the full-length membrane-bound receptor.
The rank order of potency of the agonists at the soluble truncated receptor (L-AP4 > L-SOP > L-glutamate > cyclobutylene AP5) was identical to
that of the full-length membrane-bound receptor. The affinities of all
four agonists were higher for the truncated receptor compared with the
full-length receptor. The affinity for the endogenous ligand
L-glutamate at the truncated receptor was about 2-fold
higher than that at the membrane-bound receptor (Fig.
4; Table
I). The rank order of the antagonists at
the truncated receptor (MAP4 > MPPG > CPPG) was similar to
that at the membrane-bound receptor (CPPG = MAP4 > MPPG)
except for CPPG, which was the most potent of the three compounds for
the full-length receptor but was the least potent at the soluble
receptor (Table I; Fig. 4). In contrast to the agonists, the
antagonists displayed lower affinities for the soluble truncated
receptor compared with the membrane-bound receptor.
The binding properties of the soluble truncated mGluR4 were also
compared with the membrane-bound receptor using other glutamate receptor ligands including the nonselective mGluR agonist
(1S,3R)-1-amino cyclopentane-1,3-dicarboxylic
acid, the group I mGluR antagonist (R,S)
Deglycosylation of the Truncated Receptor--
The soluble
truncated receptor migrated on SDS-polyacrylamide gel electrophoresis
with a Mr of 71,000; this is approximately Mr 8,000 larger than the predicted molecular
weight based on the amino acid sequence, suggesting that the truncated
receptor was glycosylated. Treatment of the soluble fraction containing
the truncated mGluR4 receptor with PNGase F, which cleaves all
asparagine-linked carbohydrates, shifted the immunoreactive band from
Mr 71,000 to Mr 63,000 (Fig. 6); the lower molecular weight
estimate was very close to the predicted molecular mass of the
nonglycosylated protein (62,700 daltons). The binding of
[3H]L-AP4 to the deglycosylated receptor was
also assessed. [3H]L-AP4 binding to samples
treated with PNGase was 99% of control samples (average of two
independent determinations) that were not treated with the enzyme,
indicating that agonist binding to truncated mGluR4 is not dependent
upon asparagine-linked carbohydrates.
The truncated receptor analyzed in this study included the first
548 amino acids of the 912 amino acids of mGluR4a. The expression construct contained a part of the ATD that extended from the amino terminus to Tyr-548, which is located 39 amino acids upstream from the
junction of the ATD and the first putative transmembrane domain. This
portion of the ATD includes the entire leucine, isoleucine, valine-binding protein homology region. The expression construct incorporated a c-myc epitope tag inserted immediately downstream of the
signal peptide. The truncated receptor was secreted into the cell
culture medium, indicating that the protein did not possess regions of
sufficient hydrophobicity to cause retention in the endoplasmic
reticulum or plasma membrane of the cell and that it was soluble in an
aqueous environment.
The soluble truncated mGluR4 receptor exists as a monomer in the
presence of high concentrations of a reducing reagent and as a dimer
and trimer in the presence of low concentrations of reducing agents.
Dimeric forms of mGluR1 (12, 13), mGluR2 and mGluR3 (14), mGluR4 (15),
mGluR5 (16), mGluR6 (17), and mGluR7 and mGluR8 (18) have been observed
on immunoblots of brain tissue and transfected cells. Romano et
al. (16) have shown that mGluR5 migrates on SDS-polyacrylamide gel
electrophoresis as a dimer under nonreducing conditions and as a
monomer under reducing conditions, indicating that the dimers are
formed by intermolecular disulfide bonds. Analysis of the
electrophoretic mobility of a truncated construct of mGluR5 indicated
that the intermolecular disulfide bonds are formed between cysteine
residues located in the ATD of mGluR5 (16). Modulation of monomer and dimeric forms by reducing agents was also seen with the soluble truncated form of the mGluR1 receptor (7).
Despite the studies outlined above providing evidence for dimers of
mGluRs, no data exist demonstrating a direct link between the ability
to bind ligands or activate signal transduction pathways and a
particular oligomeric configuration of an mGluR. However, in the
experiments described here, the [3H]L-AP4
radioligand binding assay was carried out in the absence of reducing
agents, whereas the addition of reducing agents decreased binding
activity in a concentration-dependent
fashion.2 It is likely that
in the typical oxidation/reduction environment of the extracellular
space in nerve tissue, mGluRs exist as oligomeric complexes. Although
the oligomeric configurations of the mGluRs as they exist in nerve
cells in vivo are not known, our observations on the
amino-terminal domain of mGluR4 together with the results on mGluR1 and
mGluR5 noted above all suggest that intermolecular disulfide bonds are
a general structural feature of the mGluR family of receptors and that
the active form of the receptor is likely to be a multimeric complex.
The homologous calcium-sensing receptor is also present in tissues and
transfected cells in a dimeric configuration (19). The oligomeric
structures of mGluRs may resemble the structures of the insulin and
insulin-like growth factor receptors. The insulin receptors exist as
preformed disulfide-linked dimers, and unlike other tyrosine
kinase receptors, monomer-to-dimer transitions are not modulated by the
presence of ligand (20).
The truncated mGluR4 receptor contains four consensus sequences for
asparagine-linked glycoslylation. The decrease in the molecular weight
of the truncated protein by about Mr 8,000 after complete deglycoslation indicates that one or more of these sites are
glycosylated. An analysis of the effects of deglycosylation on the
binding of [3H]L-AP4 to the soluble receptor
demonstrated that the bound carbohydrate is not required for agonist
binding. Similarly, asparagine-linked oligosaccharides also do not
appear to be obligatory for the activation of ionotropic glutamate
receptors (21). Although our data show that asparagine-linked
oligosaccharides are not required for ligand binding to mGluR4, it is
possible that the bound carbohydrates may be important in the
subcellular targeting of this receptor in the nervous system. In the
case of mGluR4, the targeting is to presynaptic nerve terminals (18,
22) where the receptor acts to inhibit glutamate release and thereby
modulates synaptic transmission (15).
Many G-protein-coupled receptors display biphasic binding curves
indicative of the presence of high and low affinity binding sites. The
commonly accepted explanation for high and low affinity binding sites
is that they reflect receptors with bound and unbound G-protein,
respectively. However, some G-protein-coupled receptors, such as
muscarinic acetylcholine receptors, show multiple states of affinity in
the absence of G-proteins (23, 24). Conversely, other G-protein-coupled
receptors such as the cloned mGluR4 receptor display only a single
class of sites in radioligand binding assays (9, 10); it is possible
that lower affinity sites may exist in mGluR4 but cannot be detected in
the binding assay because of the very low affinity. Alternatively, it
is possible that the unitary nature of the binding site of the cloned
membrane-bound mGluR4 receptor reflects only a low affinity site due to
the absence of the appropriate G-proteins in the host cell lines (baby
hamster kidney cells, Ref. 9; sf9 insect cells, Ref. 10; HEK-293 cells, the present study). However, we have observed that co-expression of full-length mGluR4 with various mammalian G-protein subunits including Gi1, Gi2, Gi3, and
Go in insect sf9 cells does not significantly increase the affinity or the capacity (Bmax) of
[3H]L-AP4 for
mGluR4.3 Moreover,
[3H]L-AP4 binding experiments conducted in
rat (25) and mouse (26) brain have detected only a single class of
sites with a KD value very similar to that seen with
the cloned receptor. Thus, the binding site in the full-length receptor
with an affinity for [3H]L-AP4 in the
400-500 nM range may represent the G-protein-coupled form
of mGluR4.
A pharmacological analysis of the truncated receptor was conducted by
assessing the ability of various mGluR4 ligands to compete with the
binding of the radiolabeled agonist
[3H]L-AP4. Our results demonstrate that the
binding properties displayed by the soluble truncated mGluR4 reflect
the unique pharmacological profile of the group III mGluRs. Except for
the antagonist CPPG, the rank order of potency for a series of group
III mGluR ligands at the truncated receptor was the same as at the
full-length membrane-bound receptor. Moreover, ligands at group I
mGluRs or ionotropic glutamate receptors showed little or no affinity
for either receptor. These observations suggest that the primary
determinants of [3H]L-AP4 binding are
conferred by residues present in the ATD of mGluR4. However, the
inhibition constants for L-glutamate, L-AP4, L-SOP, and cyclobutylene AP5 were lower than those of the
full-length receptor, indicating that the truncated soluble receptor
displayed higher affinities for agonists compared with the
membrane-bound receptor. The differences in affinity of the ligands
could have been caused by differences in assay conditions for the
soluble and membrane-bound receptors. However, if differences in assay conditions were responsible for the differences in the affinities, it
is likely that the direction of change would have been the same for all
ligands tested. This explanation seems unlikely because the affinity of
the truncated receptor for antagonists was lower compared with the
full-length receptor whereas the affinity for agonists was higher.
An alternative explanation for the differences in affinity is that
ligand affinity might be influenced by regions of mGluR4 that were
excluded from the soluble construct that was examined in the present
study; these regions include the transmembrane domains, the
extracellular loops between the transmembrane domains, and the 40 amino
acids immediately upstream from the first putative transmembrane
domain. It is possible that the ATD may interact with one or more of
these regions to induce an increase in affinity for antagonists and a
decrease in affinity of the receptor for agonists. Whether or not
ligand binding in other mGluRs is affected by regions outside the ATDs
remains to determined. Although the pharmacological results reported by
Okamoto et al. (7) on the binding of
[3H]quisqualic acid to the soluble mGluR1 receptor are
difficult to compare with ours because they did not report
IC50 values for L-glutamate or other compounds
that were assessed in the present study, it appears that the affinity
for [3H]quisqualic acid for the soluble receptor was very
similar to the affinity measured for the full-length receptor. One
difference between the truncated mGluR1 receptor and the truncated
mGluR4 receptor is that the former contained the entire ATD of mGluR1, whereas the latter did not include the 39 amino acids immediately upstream from the first putative transmembrane domain. Our results demonstrate that this part of the receptor is not required for high
affinity binding of [3H]L-AP4 to mGluR4. This
observation is consistent with mutational and chimeric studies
conducted on mGluR1 that showed that several residues located in the
more amino-terminal regions of the ATD of mGluR1 are required for
ligand binding (3, 5). However, we cannot rule out the possibility that
this 39-amino acid segment of mGluR4 may affect the affinity of ligands
for the binding site.
Of interest in this regard are studies on the receptor for the
glycoprotein hormones luteinizing hormone and choriogonadotropin. The
luteinizing hormone/choriogonadotropin receptor is a member of the
G-protein-coupled receptor family and, like mGluRs, it has a large ATD
that can be separated from the seven transmembrane domains and still
retain ligand binding activity. Radioligand binding studies using
I125-labeled human choriogonadotropin have shown that the
exodomain (equivalent to the ATD) alone has a higher affinity for the
glycoprotein hormone than the full-length receptor (27, 28). A
structural analysis of this receptor using site-directed mutagenesis
has shown that several amino acids in the second extracellular loop within the transmembrane domain region affect the affinity of the
ligand for the receptor; it was suggested that residues within this
loop constrain the affinity of the hormone for the receptor (28).
Although the amino acid sequences of the mGluRs are not homologous with
the glycoprotein hormone receptors, our observations suggest that
residues in the extracellular loops or residues located within or
immediately upstream of the membrane domains may influence the affinity
of ligands for the binding site located in the ATD of mGluR4.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phagemid (8) was subcloned into the pcDNA3
mammalian expression vector (Invitrogen Corp.) at the BamHI
site. For the construction of c-myc-tagged mGluR4a, the mGluR4a-pcDNA3 plasmid was cut with XhoI, and the larger
fragment containing the pcDNA3 backbone was ligated to itself (the
5'-mGluR4a-pcDNA3 plasmid). The oligonucleotides encoding the c-myc
epitope s-BstEII-c-myc (5'-GTCACGAACAAAAGCTTATTTCTGAAGAAGACTTGGATCCAG) and
rev-BstEII-c-myc (5'-GTGACCTGGATCCAAGTCTTCTTCAGAAATAAGCTTTTGTTC) were phosphorylated, annealed to each other, and inserted into the 5'-mGluR4a-pcDNA3 plasmid at the BstEII site to produce
5'-mGluR4a-c-myc-pcDNA3. The 931-bp NdeI-XhoI
fragment from 5'-mGluR4a-c-myc-pcDNA3 and a 3335-bp
XhoI-NotI fragment of mGluR4a-pcDNA3 were
subcloned into pcDNA3 at the NdeI-NotI sites
using a three-piece ligation to produce c-myc-mGluR4-pcDNA3. The
construction of an expression vector containing a segment of the
ATD of mGluR4 was made by digesting c-myc-mGluR4 with KpnI.
The 1,686-bp KpnI fragment contained a sequence encoding the
mGluR4 signal peptide, the c-myc epitope, and the amino-terminal domain
of mGluR4 truncated immediately after Tyr-548 (numbering is
based on the untagged rat receptor as indicated by Tanabe et
al. (8)). The KpnI fragment from the c-myc mGluR4
construct was subcloned into the pcDNA3 vector.
75 °C.
For the processing of the proteins secreted into the medium, the cell
culture medium was centrifuged at 48,400 × g for 30 min at 4 °C, and the supernatant was dialyzed at 4 °C in 800 ml
of assay buffer containing 0.1 mM phenylmethylsulfonyl fluoride and 1 mM ethylene glycol-bis-N,N,N',N'-tetraacetic
acid. The dialysis buffer was changed three times over a 24-h period; the samples were pooled and stored at
75 °C.
-globulin were added to each tube, followed by the addition of 200 µl of 30% polyethylene glycol. The mixture was incubated on ice for
an additional 3 min and centrifuged at 14,000 × g for 2 min, and the supernatant fraction was aspirated. The pellet was
washed with 500 µl of cold 15% polyethylene glycol and dissolved in
500 µl of 1 M NaOH. After 24 h, the samples were
transferred to scintillation vials containing 4.5 ml of scintillation
solution (Ultima Gold; Packard Instrument Co., Meriden, CT). After a
2-h equilibration time, the samples were counted on a Tri-Carb 2100TR liquid scintillation analyzer (Packard Instruments). The assay for
membrane-bound receptors was carried out as described by Eriksen and
Thomsen (9) and Thomsen et al. (10), except that 300 µM L-SOP was used for blanks. The inhibition
constants were obtained using Prism software from GraphPad, Inc. The
equilibrium dissociation constant (KD) of
[3H]L-AP4 binding to the membrane-bound and
truncated mGluR4 receptors was calculated from the IC50
values from autocompetition experiments using labeled and unlabeled
L-AP4 and the equation IC50 = KD (1 + [P]/Kp)
where [P] is the concentration of the labeled probe ([3H]L-AP4, 10 nM), and
Kp is the KD of the labeled probe.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic diagram of the expression
constructs of mGluR4. The upper diagram depicts the
full-length wild type (WT) mGluR4a receptor, and the
lower diagram depicts the truncated receptor (KpnI
ATD-mGluR4). The seven black boxes represent the
putative transmembrane domains (TMD) and the
carboxyl-terminal domain (CTa) of the full-length receptor;
the open box depicts the segment of the ATD that is
homologous with the leucine, isoleucine, valine-binding protein. The
vertical arrow indicates the position of the c-myc epitope
tag. The dotted line depicts the 5' untranslated DNA.
SP, signal peptide.
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Fig. 2.
Immunoblotting analyses of full-length
membrane-bound mGluR4a and the soluble truncated mGluR4. Samples
of the soluble preparation (2 µg of protein) or the whole cell
preparation (16 µg of protein) from untransfected or transfected HEK
cells were separated on 8% SDS-polyacrylamide gels, transferred to
nitrocellulose membranes, and detected with an anti-c-myc monoclonal
antibody. Lane 1, cell culture (soluble) fraction from
mock-transfected HEK cells; lane 2, cell culture (soluble)
fraction from cells transfected with truncated mGluR4; lane
3, total cell extract from mock-transfected HEK cells; lane
4, total cell extract from HEK cells transfected with
c-myc-mGluR4a.
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Fig. 3.
The effects of the reducing agent DTT on the
soluble truncated mGluR4. The electrophoresis samples were treated
with various concentrations of DTT as indicated, separated on a 10%
polyacrylamide gel, and transferred to nitrocellulose. The immunoblot
was labeled with the anti-c-myc antibody.
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Fig. 4.
Competition curves for the inhibition of
[3H]L-AP4 binding to soluble truncated mGluR4
by the agonists L-glutamate, L-AP4,
L-SOP, and cyclobutylene-AP5 (A) and the
antagonists MAP4, MPPG, and CPPG (B). The assays were
conducted in triplicate using 30 nM
[3H]L-AP4 (except for L-AP4, for
which 10 nM [3H]L-AP4 was used).
Each point represents the mean ± S.E. of three determinations on
samples obtained from two to three different preparations
(transfections). The Ki values are listed in Table
I.
Binding constants for agonists and antagonists at the truncated and
full-length mGluR4a receptors
-methyl-4-carboxyphenylglycine, and the ionotropic glutamate receptor agonists kainic acid and N-methyl-D-aspartate
(Fig. 5). Each drug was tested at a
concentration of 100 µM. At the truncated receptor,
kainic acid, N-methyl-D-aspartate, and
(R,S)
-methyl-4-carboxyphenylglycine produced
less than 10% inhibition of [3H]L-AP4
binding; a similar low level of inhibition was observed with the
membrane-bound receptor. The very low potency of
(R,S)
-methyl-4-carboxyphenylglycine for
mGluR4 has also been reported for experiments on human mGluR4 expressed
in Chinese hamster ovary cells (11).
(1S,3R)-1-Amino cyclopentane-1,3-dicarboxylic
acid showed a greater degree of inhibition at the truncated mGluR4 receptor (27% of control binding) compared with the full-length receptor (61% of control binding), indicating that as seen with the
other agonists tested, (1S,3R)-1-amino
cyclopentane-1,3-dicarboxylic acid may be more potent at the truncated
receptor. The relatively low potency of
(1S,3R)-1-amino cyclopentane-1,3-dicarboxylic
acid compared with group III mGluR ligands such as L-AP4
and L-SOP is consistent with previous binding data from
mGluR4 expressed in sf9 insect cells (10).
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Fig. 5.
Inhibition of
[3H]L-AP4 binding to the full-length
membrane-bound mGluR4a receptor ( ) and the soluble truncated mGluR4
receptor (
) by various ionotropic and metabotropic glutamate
receptor ligands. Each drug was tested at a concentration of 100 µM. Each column is the mean ± S.E. of three
experiments.
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Fig. 6.
Enzymatic deglycosylation of truncated
mGluR4. Left panel: lane 1, cell culture medium
from HEK cells transfected with truncated mGluR4 and incubated at
37 °C for 1 h; lane 2, cell culture medium from HEK
cells transfected with truncated mGluR4 and treated with PNGase F at
37 °C for 1 h. Right panel, histogram summarizing
the results of [3H]L-AP4 binding (30 nM) to the soluble truncated receptor treated with
PNGase.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. S. Nakanishi for the mGluR4a cDNA and J. W. Wells for helpful comments.
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FOOTNOTES |
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* This work was supported by a grant from the Medical Research Council of Canada and by a studentship (to G. H.) from the Medical Research Council and the Pharmaceutical Manufacturers Association of Canada.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: Faculty of Pharmacy,
University of Toronto, 19 Russell St., Toronto, Ontario, Canada M5S
2S2. Tel.: 416-978-4494; Fax: 416-978-8511; E-mail: d.hampson{at}utoronto.ca.
2 G. Han and D. R. Hampson, unpublished observation.
3 L. Blythe and D. R. Hampson, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are:
mGluR, metabotropic
glutamate receptor;
ATD, amino-terminal domain;
CPPG, (R,S)--cyclopropyl-4-phosphonophenylglycine;
DTT, dithiothreitol;
HEK, human embryonic kidney;
L-AP4, L-2-amino-4-phosphonobutyric acid;
L-SOP, serine-O-phosphate;
MAP4, (S)-2-amino-2-methyl-4-phosphonobutyric acid;
MPPG, (R, S)-
-methyl-4-phosphonophenylglycine.
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REFERENCES |
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