From the Mécanismes Moléculaires des
Communications Cellulaires, CNRS-UPR9023, CCIPE, 141 rue de
la Cardonille, F-34094 Montpellier, France, the § Laboratory
of Molecular Physiology, Department of Physiology, Charles
University 3rd Faculty of Medicine and Institute of Physiology, Czech
Academy of Science, Ke Karlovu 4, Prague 2, Czech Republic, the
Novartis Pharma Research, CH-4002 Basel,
Switzerland, and the ** INSERM U431, Université Montpellier
II, F-34095 Montpellier, France
Received for publication, June 6, 2000, and in revised form, September 19, 2000
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ABSTRACT |
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Specific domains of the G-protein The specific signaling pathway of a given G-protein-coupled
receptor (GPCR)1 depends on
the subset of G-proteins it can activate. Transduction of signals by
activated GPCR requires the interaction of the receptors with
heterotrimeric G-proteins. Recent progress in defining the structure of
G-proteins and site-directed mutagenesis studies of receptors and
G-proteins bring increasingly more specific and precise descriptions of
the contact sites between these proteins (1, 2). From the G-protein
Several regions along the sequence of the G Among the various G-proteins identified so far, the G There are several examples of receptors that cannot activate
G Materials--
Chemicals including glutamate were obtained from
Sigma (L'Isle d'Abeau, France) unless otherwise indicated. Serum,
culture media, and other solutions used for cell culture were from Life Technologies, Inc. (Cergy Pontoise, France). The plasmids expressing mGlu receptors were as described previously (14) or modified (see
below). The hemagglutinin epitope-tagged G Culture and Transfection of Human Embryonic Kidney (HEK 293)
Cells--
HEK 293 cells were cultured in Dulbecco's modified
Eagle's medium (DMEM) (Life Technologies, Inc.) supplemented with 10%
fetal calf serum and antibiotics (penicillin and streptomycin, 100 units/ml final). Electroporation was performed in a total volume of 300 µl with 10 µg of carrier DNA, mGlu2, or mGlu8 receptor plasmid DNA
(2 and 4 µg, respectively), wild-type or mutated G Determination of Inositol Phosphate (IP) Accumulation--
The
procedure used for the determination of IP accumulation in transfected
cells was adapted from previously published methods (33, 34). Cells
were washed 2-3 h after electroporation and incubated for 14 h in
DMEM-glutamax-I (Life Technologies, Inc., Paris, France) containing 1 µCi/ml myo-[3H]inositol (23.4 Ci/mol),
(PerkinElmer Life Sciences, Paris, France). Cells were then
washed three times and incubated for 1-2 h at 37 °C in 1 ml of
Hepes buffer saline (NaCl, 146 mM; KCl, 4.2 mM; MgCl2, 0.5 mM; glucose, 0.1%; Hepes, 20 mM, pH 7.4) supplemented with 1 unit/ml glutamate pyruvate
transaminase (Roche Molecular Biochemicals, Meylan, France) and 2 mM pyruvate (Sigma, Lisle d'Abeau, France). Cells were
then washed again twice with the same buffer, and LiCl was added to a
final concentration of 10 mM. The agonist was applied 5 min
later and left for 30 min. The reaction was stopped by replacing the
incubation medium with 0.5 ml of perchloric acid (5%), on ice.
Supernatants were recovered, and the IPs were purified on Dowex
columns. Total radioactivity remaining in the membrane fraction was
counted after treatment with 10% Triton X-100, 0.1 N NaOH
for 30 min and used as standard. Results are expressed as the amount of
IP produced over the radioactivity present in the membranes. The
dose-response curves were fitted according to the equation
y = ((ymax Antibodies Characterization--
The
anti-G
The chimeric G Immunoblotting Analysis--
Cells were transfected and treated
as described above. Cells cultured in one well were harvested for
immunodetection, and the rest of the cells were used for IP assay.
Protein samples (10 µg per lane) were separated using
SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose
membrane. Prior to the immunoblotting, total protein on the membranes
was visualized with Ponceau S to confirm that the same amounts of
protein were loaded on the gel. The G-proteins were detected using
primary monoclonal antibodies against hemagglutinin-epitope (generous
gift of Dr. B. Mouillac, Montpellier, France) or the newly described
polyclonal anti-G Construction of Plasmids Expressing Higher Levels of
G Construction of Chimeric G-proteins--
Silent mutagenesis
approach using Pfu-based QuickChangeTM
technique (Stratagene) was used to introduce a BamHI, a
PstI, and a BamHI restriction site at the
positions indicated in Fig. 2 as sites 1, 2, and 4, respectively.
Together with the existing XbaI site (position 3 in Figs.
2-4) and the HindIII site in the 3'-untranslated region,
these sites were used to construct the chimeric proteins described in
Figs. 3 and 4 using conventional subcloning techniques. The same
approach was used for making the point mutations.
Construction of Epitope-tagged Receptors--
The mGlu2 and
mGlu8 receptors were tagged with MRGS-His6 epitope at the C
termini as in our previous studies (14). Briefly, the pRK5 plasmid
containing the sequence encoding the epitope MRGS-His6
flanked by a unique NheI site at its 5' end, and an in-frame
TAA stop codon at its 3' end was used. The receptors coding sequences
were then introduced by polymerase chain reaction using NheI
restriction site so that the stop codon was replaced by alanine
followed by serine and the MRGS-His6 epitope (where MRGS
indicates: methionine, arginine, glycine, serine).
Molecular Modeling--
The three-dimensional model of the
G Characterization of Anti-G
We generated a new polyclonal antibody,
called anti- G Optimization of G In Contrast to mGlu2 Receptors, mGlu8 Receptors Do Not Efficiently
Activate G Two Regions on G
Chimera G
Swapping the N-terminal 32 residues of G The Molecular Modeling of G In this study we showed that two distinct regions along the
sequences of G To better characterize the regions in G-proteins involved in GPCR
recognition, we took advantage of the differential coupling of
G We were expecting that the residues responsible for the differential
coupling of G Taken together, our data indicate that at least two sets of residues in
G subunit
have been shown to control coupling to heptahelical receptors. The
extreme N and C termini and a region between
4 and
5 helices of
the G-protein
subunit are known to determine selective interaction
with the receptors. The metabotropic glutamate receptor 2 activated
both mouse G
15 and its human homologue
G
16, whereas metabotropic glutamate receptor 8 activated
G
15 only. The extreme C-terminal 20 amino acid residues
are identical between the G
15 and G
16 and
are therefore unlikely to be involved in coupling selectivity. Our data
reveal two regions on G
16 that inhibit its coupling to
metabotropic glutamate receptor 8. On a three-dimensional model, both
regions are found in a close proximity to the extreme C terminus of
G
16. One module comprises
4 helix,
4
6 loop
(L9 Loop),
6 sheet, and
5 helix. The other, not described
previously, is located within the loop that links the N-terminal
helix to the
1 strand of the Ras-like domain of the
subunit. Coupling of G
16 protein to the metabotropic
glutamate receptor 8 is partially modulated by each module alone,
whereas both modules are needed to eliminate the coupling fully.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
subunits, probably both G
and
dimers
contact the receptors (3). The G
subunit is likely to play a
decisive role in discriminating between different receptor subtypes
(4-6) and also between different functional states of receptors (7,
8).
-protein are involved in
the selectivity of its activation by GPCR (1, 9, 10). The best
characterized is the extreme C terminus, where residues at position
3
and
4 (the residue
1 being the last one) are decisive for coupling
of G
proteins with specific receptors (1, 11-15). Residue
4 in
G
i, G
o, and G
t is the
cysteine residue that is ADP-ribosylated by pertussis toxin, a covalent modification that prevents the interaction of the G-protein with the
receptors. In the Gq family, the residue
4 is a tyrosine residue that has to be phosphorylated for an efficient coupling to the
PLC-activating receptors (16). Conformational changes in the C-terminal
structures upon coupling to the GPCR probably cause activation of the
G
protein. Another region determining coupling selectivity is the
extreme N terminus. In the case of Gq/11-proteins, this N
terminus has been shown to restrict coupling of these G-proteins to a
subset of GPCRs, namely to those known to activate PLC (17). Finally,
the region between
4 and
5 helices that includes the L9 loop and
6 sheet (18) is also involved in coupling selectivity probably by
directly interacting with the receptors of the rhodopsin-like family
(family 1 GPCRs) (10, 19-22). The overall surface charge of a region
that comprises the structures close to the C terminus is probably
defining either selectivity or coupling properties in general (23).
15
subunit has unique properties. This G-protein that is found exclusively in the murine hematopoietic cell lineage shares the closest sequence similarity with the G
q protein and activates PLC
s
(24, 25). In cells normally expressing this G-protein as well as in
heterologous expression systems it was found that it can couple to many
GPCRs, including those that naturally do not stimulate PLC. This was observed with the members of family 1 GPCRs (26-28) and the mGlu receptor family (family 3 GPCRs) (29, 30). G
15 is
therefore a G-protein that is not able to discriminate between a
variety of receptors (26). Functional characterization of the human homologue G
16 revealed that this protein also couples to
many GPCRs (27) but is not as promiscuous as G
15. It is
of interest to understand what determines the lack of selectivity of
G
15 and which sequences restrict promiscuity of the
G
16 subunit.
16 (31, 32). Among the family 3 receptors we reported
that the group II metabotropic glutamate receptor mGlu2 activated both G
15 and G
16, whereas the group III
receptors (mGlu4, -7, and -8 receptors) activated G
15
only (29, 30). G
15 and G
16 proteins share
85% sequence identity. Interestingly, they share identical C termini,
indicating this sequence element is not responsible for their
differential coupling. Such a situation appears ideal to identify other
regions in these G-proteins involved in their specific interaction with
GPCRs. In the present study, the coupling of both mGlu2 and mGlu8
receptors to a series of G
15/G
16 chimeric G-proteins was analyzed. This study revealed that amino acids within
4 helix,
6 strand, L9 loop, and
5 helix constitute one of the
determinants that allows G-protein
subunit to discriminate between
members of family 3 receptors, a situation corresponding to data
reported for family 1 GPCRs. In addition we identified a new region
within the G-protein
subunit involved in the selective recognition
of group II versus group III mGlu receptors. This new region
is located in the loop that connects
N helix with
1 sheet.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q was kindly
provided by Dr. Bruce Conklin (The Gladstone Institute, San Francisco, CA). The plasmids pCIS-G
15 and pCIS-G
16
were kindly provided by Dr. M. Simon (Caltech, Los Angeles, CA).
subunit plasmid
DNA (1 µg), and 10 million cells in electroporation buffer (K2HPO4, 50 mM;
CH3COOK, 20 mM; KOH, 20 mM). After
electroporation (260 V, 960 microfarads, Bio-Rad gene pulser
electroporator), cells were resuspended in DMEM supplemented with 10%
fetal calf serum and antibiotics and split in 12-well clusters (Falcon,
Paris, France) (10 million cells per cluster) previously coated with poly-L-ornithine (15 µg/ml; Mr
40,000), (Sigma, Paris, France) to favor adhesion of the cells.
ymin)/1 + (x/EC50)n) + ymin
using the kaleidagraph program (Abelbeck software).
15/16 antibodies were raised in rabbits
against a synthetic peptide corresponding to the last 10 amino acid
residues that are common for both the G
15 and
G
16. The antiserum was tested in several dilutions (data
not shown). The 1:2000 dilution gave optimal results. ECL
chemiluminescence system was used to stain the secondary antibodies
(Amersham Pharmacia Biotech, Paris, France).
qX5 and G
qX6 were
constructed using a unique XbaI site of the cDNA
encoding the G
q-HA-tagged protein. The XbaI
sites in the C-terminal part of G
15 and
G
16 were introduced by silent mutagenesis into the
consensus sequence Met-Asp-Leu. This resulted in the expression of
chimeric proteins that were detectable by both the HA antibodies and
our anti-G
15/16 C-terminal antibody.
15/16 C-terminal antibodies. The
His6-tagged receptors were detected using specific
antibodies recognizing the MRGS-His epitope (Qiagen, Paris, France).
16--
We have noticed considerable lower expression
levels of the human G
16 protein than those of
G
15 in transfected HEK 293 cells. To get higher
expression levels of G
16, we removed most of the 5'-untranslated region and introduced a Kozak sequence corresponding to
that of G
15 by polymerase chain reaction using
the unique EcoRI-PstI sites. These modifications
were sufficient to raise the G
16 protein expression
obtained with this new G
16PLUS construct to levels
comparable with those reached by G
15. This construct was
used through out the study and is referred as to G
16 in
this text.
15 subunit was constructed by homology using the
coordinates of different G-protein
subunits in their GDP form
obtained by x-ray crystallography. These include transducin in its GDP
plus Mg2+ form, the
t/
i chimeric protein in its
heterotrimeric form with
t
t (35), the
i1 in its GDP plus
Mg2+ form (36), and in its heterotrimeric complex with
1
2 (37) (Protein Data Bank accession numbers 1TAG, 1GOT, 1GDD, and 1GP2, respectively). Some structural elements were deleted due to their inappropriate folding in the expected heterotrimeric form
bound to the receptor. These deletions include the N-terminal
helix
(the 24 N-terminal residues of the resolved structure) and the extreme
C terminus (4 residues) of the
i1 in its GDP plus Mg2+
form. Some constraints were also imposed during the modeling process of
G
15: an
helical secondary structure was imposed to
residues 7-39, 305-325, 330-334, and 349-370. The sequence alignment and three-dimensional models were generated using the program
Modeler (38) in the Insight-II environment (Molecular Simulation Inc.,
San Diego, CA) on a Silicon Graphic R10000 O2 work station.
A statistical evaluation of the three-dimensional model was performed
using the Verify 3D algorithm (39) and the Verify 3D
Structure Evaluation Server. The model giving the best one-dimensional/three-dimensional scores was selected and subjected to
energy minimization using the program Discover 2.9.7 (Molecular Simulation Inc.) and the CVFF force field. The extreme C
terminus of the G
15 subunit was modeled manually
according to the resolved structure of the 10 C-terminal residues of
transducin bound with rhodopsin (40). The
1
2 subunits were added
after superposition of the G
15 model with the
G
i1 subunit in its heterotrimeric complex.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
15/16 Antibodies--
The
mGlu2 and mGlu8 receptors couple to the Gi type of
G-proteins and do not activate PLC pathway when expressed alone in HEK
293 cells. Because G
15 and G
16 activate
PLCs, their efficient coupling to these mGlu receptors can easily be
assayed by measuring the capability of glutamate to activate the PLC
pathway in cells expressing both the receptor and one of these
G-proteins. As mentioned in the introduction, we reported previously
that glutamate activated PLC in HEK 293 cells coexpressing mGlu2
receptors and either G
15 or G
16. In
contrast, glutamate activated IP formation in cells expressing mGlu8
receptors and G
15 but not in those coexpressing this
receptor with G
16. To conclude that G
16
can be activated by mGlu2 but not mGlu8 receptors, it was necessary to
verify that both G-proteins were expressed at similar levels.
15/16AB that recognizes equally well
both G
15 and G
16 (Fig. 1). The antiserum was raised in rabbits immunized with a peptide corresponding to the extreme C-terminal region (10 amino acid residues) common to
both proteins (Figs. 1 and 2). On Western
blots, anti-G
15/16AB recognized a single major
band that migrated at a velocity appropriate for these G-proteins (Fig.
1). This band was detected only in lanes where membrane proteins from
HEK 293 cells expressing proteins possessing C termini of
G
15 or G
16 were separated. No additional major band was detected, and no bands were detected in lanes where extracts from cells expressing other G
-protein subunits were loaded
(data not shown). To verify that anti-G
15/16AB was
recognizing both G-proteins at the same extent, we expressed chimeric
proteins corresponding to the HA-tagged G
q protein with
its last 51 C-terminal residues replaced by their corresponding 63 residues of either G
15 or G
16 (chimeras
G
qX5 and G
qX6, see Fig. 1). Those were expressed in HEK 293 cells at the same levels, as shown with the HA
specific monoclonal antibody. When blots were reprobed with the new
antibody anti-G
15/16AB, the bands corresponding to
chimeras bearing G
15 and G
16 C
termini were stained with similar intensity (Fig. 1).
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Fig. 1.
Characterization of the anti-
G 15/16 antibodies. Protein
extracts of cells transfected with a hemagglutinin epitope-tagged
(HA) G
q subunit and chimeras between the
G
q and G
15 and G
16 were
resolved by SDS-polyacrylamide gel electrophoresis. The indicated
antibodies were used for immunoblotting. Upper panel,
immunoblot using the polyclonal anti-G
15/16 antibodies.
In the lower part, a portion of the same blot developed with
the monoclonal anti-HA antibody. The bands corresponding to the
G-proteins migrated with expected velocity. On the right, a
schematic representation of the chimeric proteins between
G
q-HA, G
15, and G
16. The
bars indicate prestained molecular mass standards
migrating at molecular mass (from the top): 200, 116, 97, 66, 45, 31, and 21 kDa. The arrowhead indicates position of the
G-protein.
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Fig. 2.
Alignment of the sequences of the
G 15 and
G
16. The predicted secondary
structures are marked. The silent restriction sites used to swap
portions of the G-proteins to generate the chimeras used in this study
are indicated by numbers in black circles (1-4). The
empty bar at the C terminus indicates the sequence used to
rise the polyclonal antibodies. In b the position of the
secondary structures and the two coupling restrictive regions
(hatched bars) responsible for the differential coupling of
G
15 and G
16 to mGlu8 receptors are
shown.
16 Expression--
Employing the
anti- G
15/16AB antibody on immunoblots, we noticed
that the level of expression of G
16 was lower than that of G
15 in cells transfected with the original vectors
(data not shown). We therefore replaced the 5'-untranslated region of
G
16 cDNA, including the Kozak sequence with
corresponding sequence from of G
15 coding cDNA (see
"Experimental Procedures"). This modification was found to raise
the expression of G
16 in transiently transfected HEK 293 cells to levels comparable with those of G
15 (Figs. 1
and 3b).
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Fig. 3.
a, chimeric proteins between
G 15 and G
16 at the
4-
5 region were
constructed by swapping corresponding portions using silent restriction
sites or by introducing point mutations. b, immunoblot
analysis of G
15 and G
16 proteins and the
chimeras and the point mutations expressed in HEK293 cells, stained
with the anti-G
16 antibodies. c, differential
coupling of the G
15, G
16, and their
reciprocal chimeras to mGlu2 and mGlu8 receptors. Basal (open
bars) and 1 mM glutamate-induced (dark
bars) IP formations were determined in HEK 293 cells
coexpressing the mGlu2 (a) or mGlu8 (b)
receptors with the G-protein
subunits and their chimeras. The new
constructs of the G
16 proteins with augmented expression
levels were used. Data represent the radioactivity in the IP fraction
divided by the total radioactivity in the membranes.
16--
Using the expression plasmids
described above, we examined the coupling of mGlu2 and mGlu8 receptors
to both G
15 and the modified G
16. As
shown in Fig. 3a, glutamate stimulated IP formation in cells coexpressing mGlu2 receptors with either G
15 or
G
16 , an effect that is dose-dependent (Fig.
6). Using the new G
16 vector, the maximal
glutamate effect was found to be slightly lower than that obtained with
G
15 in cells expressing mGlu2 receptors, whereas it was
50% smaller when the original plasmid was used (29, 30). This is in
agreement with the increased protein expression levels of
G
16 that resulted from the modification of the coding
plasmid. In cells expressing mGlu8 receptors, a large increase in basal
and glutamate-induced IP formation was detected when coexpressed with
G
15, but no change was observed with G
16.
All the glutamate effects were dose-dependent and the calculated EC50 values (for mGlu2
receptor-G
15 pair the EC50 for glutamate was
8.2 ± 0.8 µM; mGlu2 receptor with
G
16 results in EC50 8.0 ± 0.3 µM glutamate, and mGlu8 receptor coexpressed with
G
15 determined EC50 for glutamate was
9.3 ± 1.6 µM) were close to those determined with
these receptors using other assays (41). It was important to establish
that the expression levels of both the receptors and the G-proteins are
similar at their different combinations. At the receptor level this was
confirmed by using His6-tagged receptors (data not shown).
These data further confirm the discriminative coupling of
G
16 to mGlu2 and mGlu8 receptors.
15 and G
16 Are
Recognized by mGlu Receptors--
To identify the putative regions on
the G-protein that cause the differential coupling of
G
15 and G
16 to mGlu8 receptor, we
constructed a series of G
15/G
16 chimeras
(see panel a in Figs. 3-5). When transiently transfected
into HEK 293 cells, all chimeras were expressed at levels similar to
those of the original proteins as revealed by the antiserum
anti- G
15/16AB (panel b in Figs. 3-5).
Moreover, all constructs did couple well to mGlu2 receptor, as they all
allowed an effective coupling of this receptor to IP formation upon
glutamate application, showing that they were all functional. Coupling
of each single constructs with the receptors are shown in Figs. 3-5
(panel c).
15/16C1, which corresponds to
G
15 with the C-terminal 44 residues from
G
16 (Fig. 3a), showed a smaller response to
glutamate as compared with wild-type G
15 (Fig.
3c). This indicates that the C-terminal portion of
G
16 contains a site that decreases G-protein coupling
efficacy to mGlu8 receptors (Fig. 3c). Progressive exchange
of G
15 C terminus with corresponding sequences of
G
16, as in chimera G
15/16C2, resulted in
a more pronounced decrease in coupling efficiency to mGluR8 (Fig.
3c). The converse chimera G
15/16C3, which
corresponds to G
16 with the C-terminal 44 residues of
G
15, was activated by mGlu8 receptors, although the
glutamate response was smaller than that obtained with
G
15 (Fig. 3b). The module in
G
16 responsible for discriminating between the two receptors is most likely a region that includes
4 helix, L9 loop, the
6 sheet, and the
5 (residues 331-354). Residues that are different between G
15 and G
16 is these
regions are: 10 in the
4 helix and N-terminal part of L9 loop, 3 in
the C-terminal part of L9, 3 in
6, and 3 in
5 (Fig. 2). Attempts
to further characterize the critical residues in this C-terminal region
failed. For example, the comparison of the IP formation obtained with
mGlu8 receptors expressed with the C1 and C2
G
15/16 chimera suggests that the region between
position 3 and 4 (Fig. 3a) decreases coupling efficacy of
G
16. However, swapping this region of G
16
for that of G
15 in the chimera C4 did not improve the
coupling compared with chimera C3, but rather diminished it. Also in
chimera G
15/16C5, the exchange of the 3 residues of
5
in G
15 by those of G
16 lead to a decrease of functional responses obtained with both mGlu2 and mGlu8 receptors. The most likely explanation is that the region including
4, L9 loop,
6, and
5 discriminates the GPCRs as a net of many forces, a
situation where a given charge of a side chain of an amino acid residue
is less important than the outcome of additions of neighboring forces.
One can imagine this situation also as a mosaic, where the whole
picture is the decisive one. This is in agreement with results obtained
previously by other researchers (23). Since the result of swapping this
part of the G-proteins had only partial effect on the mGlu8
receptor-G
-protein coupling, there had to be another
discriminatory region.
16 by those of
G
15 resulted in chimera G
15/16N1, which
showed a feeble coupling to mGlu8 receptors (Fig.
4). When the first 101 residues of
G
16 were exchanged by the corresponding region in
G
15, chimera G
15/16N2, a significant
increase in the capability of the chimeric G-protein to couple to mGlu8
receptors was observed. Interestingly, G
15/16N2 did not
perform any better than the G
15/16C4 chimera
(compare Figs. 3 and 4),suggesting that the region between position 101 and 313 did not play an important role in controlling coupling selectivity to mGlu2 and mGlu8 receptors. Taken together, these results
indicate that two distinct regions in G
16, encompassing residues 32-101 and 331-354, respectively, are responsible for the
observed decrease in coupling efficacy to mGlu8 receptors (see Fig. 2).
In agreement with this hypothesis, chimera G
15/16N1C4 that is characterized by residues 331-354 of G
15 and
residues 32-101 of G
16 was activated by mGlu8 receptors
but not to the extent showed by wild type G
15. Chimera
G
15/16N2C4, which is constituted by the backbone of
G
16 with both critical regions corresponding to residues
32-101 and 331-354 replaced by the respective G
15
sequences, allowed a more efficient coupling of mGlu8 receptors to PLC.
Considering the critical role played by residues 32-101 in conferring
G-protein coupling specificity, we decided to make point mutations
within this region in both G
15 and
G
16.
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Fig. 4.
a, chimeric proteins between
G 15 and G
16 at the
N
1 region were
constructed by swapping corresponding portions using silent restriction
sites. b, immunoblot analysis of G
15 and
G
16 proteins and the chimeras and the point mutations
expressed in HEK293 cells, stained with the anti-G
16
antibodies. c, differential coupling of the
G
15, G
16, and their reciprocal chimeras
to mGlu2 and mGlu8 receptors. Basal (open bars) and 1 mM glutamate-induced (dark bars) IP formations
were determined in HEK 293 cells coexpressing the mGlu2 (a)
or mGlu8 (b) receptors with the G-protein
subunits and
their chimeras. The new constructs of the G
16 proteins
with augmented expression levels were used. Data represent the
radioactivity in the IP fraction divided by the total radioactivity in
the membranes.
N-
1 Loop of the G
-protein Selectively Modifies
Coupling to mGlu Receptors--
Within region 32-101, several amino
acid residues differ between G
15 and G
16
(Fig. 2). Two of these nonconserved residues are located in the loop
that links the N-terminal
helix and the first
strand of the
Ras-like domain (the
N-
1 loop), the other residues are
located in the
A helix of the helix-rich domain. Among this set of
residues, those located in the
N-
1 loop are the most likely to
affect G-protein coupling selectivity, since they are located in close
proximity to the known G-protein-mGlu receptors interacting site, the
extreme C terminus, and are exposed on the surface of the protein.
Interestingly, the
1 sheet is highly conserved within the
G
-protein family, whereas the contiguous
N-
1 loop is extremely
variable. As mentioned above, two residues in the
N-
1 loop are
different between G
15 and G
16: glutamic acid 39 and 41 in G
15 are replaced by an aspartic acid
and a glycine in G
16, respectively (Fig. 2). Our results
indicate that both residues at positions 39 and 41 are involved in
determining coupling selectivity to mGlu receptors. To reach maximal
mGlu8 receptor coupling activity, both positions in G
16
have to be replaced by the corresponding
residues of G
15 (chimera G
16 E-E; Figs. 5
and 6); this is in agreement with what
has been observed with chimera N2 (Fig. 5). Moreover, the
reverse mutant G
15D-G showed a significant decrease of
coupling performance with mGlu8 receptor as compared with wild-type
G
15. All mutants exhibited comparable good coupling to
mGlu2 receptor, even though with slightly lower efficacy than
G
15. Accordingly, basal IP formation induced by mGlu2
receptor was lower with these mutants than with
G
15 (Figs. 5 and 6). It is important to mention
again that the expression levels of the G-proteins and their mutants
and the receptor levels were similar in all experiments, even when
cDNAs coding for different G-protein constructs were used.
View larger version (33K):
[in a new window]
Fig. 5.
a, point mutations of and
G 16 at the
4-
5 region were constructed by swapping
corresponding residues. b, immunoblot analysis of
G
15 and G
16 proteins and the chimeras and
the point mutations expressed in HEK293 cells, stained with the
anti-G
16 antibodies. c, differential coupling
of the G
15, G
16, and their reciprocal
chimeras to mGlu2 and mGlu8 receptors. Basal (open bars) and
1 mM glutamate-induced (dark bars) IP formations
were determined in HEK 293 cells coexpressing the mGlu2 (a)
or mGlu8 (b) receptors with the G-protein
subunits and
their chimeras. The new constructs of the G
16 proteins
with augmented expression levels were used. Data represent the
radioactivity in the IP fraction divided by the total radioactivity in
the membranes.
View larger version (18K):
[in a new window]
Fig. 6.
Glutamate-induced IP formation in cells
coexpressing mGlu2 or mGlu8 receptors with either
G 15 or
G
16 or the
G
16EE chimera. Cells
coexpressing mGlu2 receptors with G
15 (open
circles) and the modified construct of G
16
(closed circles) were stimulated with increasing
concentrations of glutamate. b, same as in a with
mGlu8 receptor and the newly constructed vectors encoding
G
15, G
16, and G
16EE. Data
represent the radioactivity in the IP fraction divided by the total
radioactivity in the membranes. Values are means ± S.E. of three
independent experiments performed in triplicates.
15 Reveals Clusters of
Residues Involved in Receptor Recognition in a Close Proximity to the C
Terminus--
A three-dimensional model of G
15 subunit
was constructed to determine the exact spatial location of the two
critical regions, which allow coupling of G
15 to mGlu8
receptors. This model was generated using the coordinates of the
subunits transducin and G
i in their GDP-Mg2+
form (18, 42), since these are expected to correspond to the
conformation of the G-protein that interacts with the receptor. Within
the region between helices
4 and
5 (residues 313-354), these
residues (except Val352) are on the surface of the
protein and are found in a relative close proximity to the C terminus
(Fig. 7). Within the N-terminal region
(residues 32-101), the two residues that we identified as being
important for the efficient coupling of G
15 to mGlu8 receptors (Glu39 and Glu41) are in close
proximity to the C terminus (Fig. 7). The majority of the other
residues of G
15 that are not conserved in
G
16 are in our model far away from the C terminus of the
subunit (the only exception are Lys200 and
Lys203 in G
15 replaced by Gln and Asn in
G
16, respectively) and as such are unlikely to affect
coupling selectivity of the heterotrimeric G-protein.
View larger version (60K):
[in a new window]
Fig. 7.
Model of the
G 15 protein in an heterotrimeric
form with
1
2. A
three-dimensional structure of the G
15 molecule is
presented in light gray with the five extreme C-terminal
residues important for coupling in purple. In red
are the 2 residues that are located in the loop linking the N-terminal
helix and the first
sheet of the Ras-like domain
residues that are not conserved in G
16 and that are
compatible with the coupling of G
15, but not of
G
16 to mGlu8 receptor. The orange residues
are trhose that are different between G
15 and
G
16 at the
4 helix, L9 loop,
6 sheet, and
5
helix region. The yellow residues are those that are
different between G
15 and G
16 but had no
effect on the differential coupling. On the left is the view
of the G-protein trimer from the receptor-oriented side. On the
right is the same model rotated 90° (side view). The
subunit is in light blue, and the
subunit is in
green.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
15 and G
16 control their
coupling to heptahelical receptors. One such region is between
residues 311 and 354 that includes the C-terminal part of the
4
helix, L9 loop, the
6 strand, and the
5 helix. The second region
comprises residues 39 and 41, which lie within the loop that links the
N-terminal
helix with the first
strand of the Ras-like
domain of the G-protein
subunit. Both regions are in close
proximity to the extreme C terminus in the spatial arrangement of the
proteins. These data further strengthen the role of the area located in close proximity to the C terminus of G
protein in controlling coupling to heptahelical receptors and extent the putative surface on
G
protein that modulates the interaction with this class of receptors.
15 and G
16 to mGlu2 and mGlu8 receptors.
The mGlu receptors and G
15/16 are unlikely to naturally
interact. The mouse G
15 and human G
16 are
known to be exclusively expressed in the hematopoietic cell lineage,
whereas mGlu receptors are mostly expressed in neurons and/or in
astrocytes in the central nervous system. However, several consideration prompted us to perform this study. The interaction between GPCR and the heterotrimeric G-protein is known to involve several regions on the G
-protein, the best characterized being the
extreme C terminus. Since G
15 and G
16
share identical C termini, this study allowed us to identify additional
regions with coupling-restrictive abilities. All G
subunits share
high sequence similarity and therefore have highly conserved
structures; thus, identification of additional regions involved in
coupling selectivity of G
15/16 would help us to better
map residues in all the
subunits that control the coupling
selectivity and/or efficacy to their partner receptors. The mGlu
receptors are members of a subfamily of GPCRs (family 3) that is
distinct from the rhodopsin-like family 1 GPCRs. Thus, identification
of regions on a G-protein
subunit involved in specific interaction
with these receptors should allow a comparison of the coupling
mechanisms between family 1 and family 3 receptors.
G
15/16 are known to couple to a wide variety of GPCRs,
making these G-proteins a very useful tool for the establishment of
functional tests for receptors for which no information on the
G-protein coupling selectivity is available (28). Identification of the
specific epitopes on G
15/16 that influence their
promiscuous coupling could constitute an helpful information for the
construction of modified G-proteins with even lower selectivity.
15 and G
16 toward mGlu8
receptors would be located in the L9 (
4-
6) loop. This region was
described as a key site in G-protein
subunits discriminating
between members of family 1 GPCRs (9, 10, 19-21). This loop, which is
longer than the corresponding sequences of other G-protein
subunits, is the most variable region between of G
15 and
G
16 proteins. Our results revealed that, as observed
with family 1 GPCRs,
4 helix, L9 loop,
6 sheet, and
5 helix
together play an important role in controlling their coupling of
G
-protein to family 3 receptors. This strengthens the hypothesis
that these two distant heptahelical receptor types are recognized by
similar molecular determinants on the G-protein
subunits. Our
results also indicate that differences in amino acid residues in the
region comprised by
4 and
5 helices are not sufficient to explain
the differential coupling sensitivity of G
15 and
G
16 to mGlu8 receptors. Indeed, we identified a second region in G
16 that, once replaced by the equivalent
G
15 sequence, lead to a chimera showing distinctive and
measurable coupling to mGlu8 receptors. This novel region is
characterized by a 2-amino acid residue exchange between
G
15 and G
16, where Glu39 and
Glu41 in G
15 are replaced by Asp and Gly in
G
16, respectively. These two residues are located in the
loop linking the N-terminal
helix and the first
sheet of the
Ras-like domain. Our three-dimensional model indicates that
side chains of these residues are likely to be facing the surface of
the protein in close proximity to the C terminus. As such they are
likely to constitute a major determinant in defining the differential
coupling observed for G
15 and G
16 to
mGlu8 receptor. This region has never been previously reported as being
involved in the control of receptor G-protein coupling, and it is
therefore a novel possible contact site between the G-proteins and
family 3 GPCRs. An alternative that could not be ruled out in the
present study is that these residues may affect flexibility of the
N-terminal helix. Since the N terminus of G
-protein contacts the
subunit, this might influence
-
coupling and so coupling to
receptors (43).
15 and G
16 contribute to coupling
restriction of these G-proteins to the mGlu8 receptors. The residues
comprised in these two regions are located on both sides of the extreme C-terminal domain known to interact with the intracellular loops of the
heptahelical receptors. This cooperativity between different portions
of the G-protein
subunit, that leads to differential coupling to
heptahelical receptors, is reminiscent of the cooperativity between
different intracellular portions of the receptors in controlling their
coupling to G-proteins. This strengthens the concept that coupling
efficacy and/or selectivity between heptahelical receptors and the
G-proteins results from a multiplicity of contact zones. It remains to
be clarified which portion of a G-protein is recognized by a given
intracellular region on the receptors.
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FOOTNOTES |
---|
* This work was supported by grants from the action incitative Physique et Chimie du Vivant (PCV00), the European Community Biotech2 (BIO4-CT96-0049) programs, the Fondation pour la Recherche Médicale, and the Fonds de Recherche Hoechst Marion Roussel (FRHMR1/9702).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.
¶ Supported by the Fondation pour la Recherche Médicale and the Fonds de Recherche Hoechst Marion Roussel. To whom correspondence should be addressed: CNRS-UPR9023, CCIPE, 141 rue de la Cardonille, F-34094 Montpellier Cedex 5, France. Tel.: 33-467-14-2933; Fax: 33-467-54-2432; E-mail: blahos@seznam.com.
Present address: University of California San Diego, Cellular
and Molecular Medicine West, Rm. 218, 9500 Gilman Dr., La Jolla, CA
92093-0651.
Published, JBC Papers in Press, October 10, 2000, DOI 10.1074/jbc.M004880200
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ABBREVIATIONS |
---|
The abbreviations used are: GPCR, G-protein coupled receptor; mGlu receptors, metabotropic glutamate receptors; PLC, phospholipase C; DMEM, Dulbecco's modified Eagle's medium; HEK, human embryonic kidney; IP, inositol phosphate; HA, hemagglutinin.
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