A Novel Site on the Galpha -protein That Recognizes Heptahelical Receptors*

Jaroslav BlahosDagger §, Thierry Fischer||**, Isabelle BrabetDagger , Daniela StaufferDagger Dagger , Giorgio RovelliDagger Dagger , Joël BockaertDagger , and Jean-Philippe PinDagger

From the Dagger  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 Dagger Dagger  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



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Specific domains of the G-protein alpha  subunit have been shown to control coupling to heptahelical receptors. The extreme N and C termini and a region between alpha 4 and alpha 5 helices of the G-protein alpha  subunit are known to determine selective interaction with the receptors. The metabotropic glutamate receptor 2 activated both mouse Galpha 15 and its human homologue Galpha 16, whereas metabotropic glutamate receptor 8 activated Galpha 15 only. The extreme C-terminal 20 amino acid residues are identical between the Galpha 15 and Galpha 16 and are therefore unlikely to be involved in coupling selectivity. Our data reveal two regions on Galpha 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 Galpha 16. One module comprises alpha 4 helix, alpha 4-beta 6 loop (L9 Loop), beta 6 sheet, and alpha 5 helix. The other, not described previously, is located within the loop that links the N-terminal alpha  helix to the beta 1 strand of the Ras-like domain of the alpha  subunit. Coupling of Galpha 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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 alpha , beta , and gamma  subunits, probably both Galpha and beta gamma dimers contact the receptors (3). The Galpha 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).

Several regions along the sequence of the Galpha -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 Galpha proteins with specific receptors (1, 11-15). Residue -4 in Galpha i, Galpha o, and Galpha 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 Galpha -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 alpha 4 and alpha 5 helices that includes the L9 loop and beta 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).

Among the various G-proteins identified so far, the Galpha 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 Galpha q protein and activates PLCbeta 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). Galpha 15 is therefore a G-protein that is not able to discriminate between a variety of receptors (26). Functional characterization of the human homologue Galpha 16 revealed that this protein also couples to many GPCRs (27) but is not as promiscuous as Galpha 15. It is of interest to understand what determines the lack of selectivity of Galpha 15 and which sequences restrict promiscuity of the Galpha 16 subunit.

There are several examples of receptors that cannot activate Galpha 16 (31, 32). Among the family 3 receptors we reported that the group II metabotropic glutamate receptor mGlu2 activated both Galpha 15 and Galpha 16, whereas the group III receptors (mGlu4, -7, and -8 receptors) activated Galpha 15 only (29, 30). Galpha 15 and Galpha 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 Galpha 15/Galpha 16 chimeric G-proteins was analyzed. This study revealed that amino acids within alpha 4 helix, beta 6 strand, L9 loop, and alpha 5 helix constitute one of the determinants that allows G-protein alpha  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 alpha  subunit involved in the selective recognition of group II versus group III mGlu receptors. This new region is located in the loop that connects alpha N helix with beta 1 sheet.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 Galpha q was kindly provided by Dr. Bruce Conklin (The Gladstone Institute, San Francisco, CA). The plasmids pCIS-Galpha 15 and pCIS-Galpha 16 were kindly provided by Dr. M. Simon (Caltech, Los Angeles, CA).

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 Galpha 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.

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 - ymin)/1 + (x/EC50)n) + ymin using the kaleidagraph program (Abelbeck software).

Antibodies Characterization-- The anti-Galpha 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 Galpha 15 and Galpha 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).

The chimeric Galpha qX5 and Galpha qX6 were constructed using a unique XbaI site of the cDNA encoding the Galpha q-HA-tagged protein. The XbaI sites in the C-terminal part of Galpha 15 and Galpha 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-Galpha 15/16 C-terminal antibody.

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-Galpha 15/16 C-terminal antibodies. The His6-tagged receptors were detected using specific antibodies recognizing the MRGS-His epitope (Qiagen, Paris, France).

Construction of Plasmids Expressing Higher Levels of Galpha 16-- We have noticed considerable lower expression levels of the human Galpha 16 protein than those of Galpha 15 in transfected HEK 293 cells. To get higher expression levels of Galpha 16, we removed most of the 5'-untranslated region and introduced a Kozak sequence corresponding to that of Galpha 15 by polymerase chain reaction using the unique EcoRI-PstI sites. These modifications were sufficient to raise the Galpha 16 protein expression obtained with this new Galpha 16PLUS construct to levels comparable with those reached by Galpha 15. This construct was used through out the study and is referred as to Galpha 16 in this text.

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 Galpha 15 subunit was constructed by homology using the coordinates of different G-protein alpha  subunits in their GDP form obtained by x-ray crystallography. These include transducin in its GDP plus Mg2+ form, the alpha t/alpha i chimeric protein in its heterotrimeric form with beta tgamma t (35), the alpha i1 in its GDP plus Mg2+ form (36), and in its heterotrimeric complex with beta 1gamma 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 alpha  helix (the 24 N-terminal residues of the resolved structure) and the extreme C terminus (4 residues) of the alpha i1 in its GDP plus Mg2+ form. Some constraints were also imposed during the modeling process of Galpha 15: an alpha  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 Galpha 15 subunit was modeled manually according to the resolved structure of the 10 C-terminal residues of transducin bound with rhodopsin (40). The beta 1gamma 2 subunits were added after superposition of the Galpha 15 model with the Galpha i1 subunit in its heterotrimeric complex.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Characterization of Anti-Galpha 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 Galpha 15 and Galpha 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 Galpha 15 or Galpha 16. In contrast, glutamate activated IP formation in cells expressing mGlu8 receptors and Galpha 15 but not in those coexpressing this receptor with Galpha 16. To conclude that Galpha 16 can be activated by mGlu2 but not mGlu8 receptors, it was necessary to verify that both G-proteins were expressed at similar levels.

We generated a new polyclonal antibody, called anti- Galpha 15/16AB that recognizes equally well both Galpha 15 and Galpha 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-Galpha 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 Galpha 15 or Galpha 16 were separated. No additional major band was detected, and no bands were detected in lanes where extracts from cells expressing other Galpha -protein subunits were loaded (data not shown). To verify that anti-Galpha 15/16AB was recognizing both G-proteins at the same extent, we expressed chimeric proteins corresponding to the HA-tagged Galpha q protein with its last 51 C-terminal residues replaced by their corresponding 63 residues of either Galpha 15 or Galpha 16 (chimeras Galpha qX5 and Galpha 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-Galpha 15/16AB, the bands corresponding to chimeras bearing Galpha 15 and Galpha 16 C termini were stained with similar intensity (Fig. 1).



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Fig. 1.   Characterization of the anti- Galpha 15/16 antibodies. Protein extracts of cells transfected with a hemagglutinin epitope-tagged (HA) Galpha q subunit and chimeras between the Galpha q and Galpha 15 and Galpha 16 were resolved by SDS-polyacrylamide gel electrophoresis. The indicated antibodies were used for immunoblotting. Upper panel, immunoblot using the polyclonal anti-Galpha 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 Galpha q-HA, Galpha 15, and Galpha 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 Galpha 15 and Galpha 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 Galpha 15 and Galpha 16 to mGlu8 receptors are shown.

Optimization of Galpha 16 Expression-- Employing the anti- Galpha 15/16AB antibody on immunoblots, we noticed that the level of expression of Galpha 16 was lower than that of Galpha 15 in cells transfected with the original vectors (data not shown). We therefore replaced the 5'-untranslated region of Galpha 16 cDNA, including the Kozak sequence with corresponding sequence from of Galpha 15 coding cDNA (see "Experimental Procedures"). This modification was found to raise the expression of Galpha 16 in transiently transfected HEK 293 cells to levels comparable with those of Galpha 15 (Figs. 1 and 3b).



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Fig. 3.   a, chimeric proteins between Galpha 15 and Galpha 16 at the alpha 4-alpha 5 region were constructed by swapping corresponding portions using silent restriction sites or by introducing point mutations. b, immunoblot analysis of Galpha 15 and Galpha 16 proteins and the chimeras and the point mutations expressed in HEK293 cells, stained with the anti-Galpha 16 antibodies. c, differential coupling of the Galpha 15, Galpha 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 alpha  subunits and their chimeras. The new constructs of the Galpha 16 proteins with augmented expression levels were used. Data represent the radioactivity in the IP fraction divided by the total radioactivity in the membranes.

In Contrast to mGlu2 Receptors, mGlu8 Receptors Do Not Efficiently Activate Galpha 16-- Using the expression plasmids described above, we examined the coupling of mGlu2 and mGlu8 receptors to both Galpha 15 and the modified Galpha 16. As shown in Fig. 3a, glutamate stimulated IP formation in cells coexpressing mGlu2 receptors with either Galpha 15 or Galpha 16 , an effect that is dose-dependent (Fig. 6). Using the new Galpha 16 vector, the maximal glutamate effect was found to be slightly lower than that obtained with Galpha 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 Galpha 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 Galpha 15, but no change was observed with Galpha 16. All the glutamate effects were dose-dependent and the calculated EC50 values (for mGlu2 receptor-Galpha 15 pair the EC50 for glutamate was 8.2 ± 0.8 µM; mGlu2 receptor with Galpha 16 results in EC50 8.0 ± 0.3 µM glutamate, and mGlu8 receptor coexpressed with Galpha 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 Galpha 16 to mGlu2 and mGlu8 receptors.

Two Regions on Galpha 15 and Galpha 16 Are Recognized by mGlu Receptors-- To identify the putative regions on the G-protein that cause the differential coupling of Galpha 15 and Galpha 16 to mGlu8 receptor, we constructed a series of Galpha 15/Galpha 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- Galpha 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).

Chimera Galpha 15/16C1, which corresponds to Galpha 15 with the C-terminal 44 residues from Galpha 16 (Fig. 3a), showed a smaller response to glutamate as compared with wild-type Galpha 15 (Fig. 3c). This indicates that the C-terminal portion of Galpha 16 contains a site that decreases G-protein coupling efficacy to mGlu8 receptors (Fig. 3c). Progressive exchange of Galpha 15 C terminus with corresponding sequences of Galpha 16, as in chimera Galpha 15/16C2, resulted in a more pronounced decrease in coupling efficiency to mGluR8 (Fig. 3c). The converse chimera Galpha 15/16C3, which corresponds to Galpha 16 with the C-terminal 44 residues of Galpha 15, was activated by mGlu8 receptors, although the glutamate response was smaller than that obtained with Galpha 15 (Fig. 3b). The module in Galpha 16 responsible for discriminating between the two receptors is most likely a region that includes alpha 4 helix, L9 loop, the beta 6 sheet, and the alpha 5 (residues 331-354). Residues that are different between Galpha 15 and Galpha 16 is these regions are: 10 in the alpha 4 helix and N-terminal part of L9 loop, 3 in the C-terminal part of L9, 3 in beta 6, and 3 in alpha 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 Galpha 15/16 chimera suggests that the region between position 3 and 4 (Fig. 3a) decreases coupling efficacy of Galpha 16. However, swapping this region of Galpha 16 for that of Galpha 15 in the chimera C4 did not improve the coupling compared with chimera C3, but rather diminished it. Also in chimera Galpha 15/16C5, the exchange of the 3 residues of alpha 5 in Galpha 15 by those of Galpha 16 lead to a decrease of functional responses obtained with both mGlu2 and mGlu8 receptors. The most likely explanation is that the region including alpha 4, L9 loop, beta 6, and alpha 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-Galpha -protein coupling, there had to be another discriminatory region.

Swapping the N-terminal 32 residues of Galpha 16 by those of Galpha 15 resulted in chimera Galpha 15/16N1, which showed a feeble coupling to mGlu8 receptors (Fig. 4). When the first 101 residues of Galpha 16 were exchanged by the corresponding region in Galpha 15, chimera Galpha 15/16N2, a significant increase in the capability of the chimeric G-protein to couple to mGlu8 receptors was observed. Interestingly, Galpha 15/16N2 did not perform any better than the Galpha 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 Galpha 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 Galpha 15/16N1C4 that is characterized by residues 331-354 of Galpha 15 and residues 32-101 of Galpha 16 was activated by mGlu8 receptors but not to the extent showed by wild type Galpha 15. Chimera Galpha 15/16N2C4, which is constituted by the backbone of Galpha 16 with both critical regions corresponding to residues 32-101 and 331-354 replaced by the respective Galpha 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 Galpha 15 and Galpha 16.



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Fig. 4.   a, chimeric proteins between Galpha 15 and Galpha 16 at the alpha N-beta 1 region were constructed by swapping corresponding portions using silent restriction sites. b, immunoblot analysis of Galpha 15 and Galpha 16 proteins and the chimeras and the point mutations expressed in HEK293 cells, stained with the anti-Galpha 16 antibodies. c, differential coupling of the Galpha 15, Galpha 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 alpha  subunits and their chimeras. The new constructs of the Galpha 16 proteins with augmented expression levels were used. Data represent the radioactivity in the IP fraction divided by the total radioactivity in the membranes.

The alpha N-beta 1 Loop of the Galpha -protein Selectively Modifies Coupling to mGlu Receptors-- Within region 32-101, several amino acid residues differ between Galpha 15 and Galpha 16 (Fig. 2). Two of these nonconserved residues are located in the loop that links the N-terminal alpha  helix and the first beta  strand of the Ras-like domain (the alpha N-beta 1 loop), the other residues are located in the alpha A helix of the helix-rich domain. Among this set of residues, those located in the alpha N-beta 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 beta 1 sheet is highly conserved within the Galpha -protein family, whereas the contiguous alpha N-beta 1 loop is extremely variable. As mentioned above, two residues in the alpha N-beta 1 loop are different between Galpha 15 and Galpha 16: glutamic acid 39 and 41 in Galpha 15 are replaced by an aspartic acid and a glycine in Galpha 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 Galpha 16 have to be replaced by the corresponding residues of Galpha 15 (chimera Galpha 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 Galpha 15D-G showed a significant decrease of coupling performance with mGlu8 receptor as compared with wild-type Galpha 15. All mutants exhibited comparable good coupling to mGlu2 receptor, even though with slightly lower efficacy than Galpha 15. Accordingly, basal IP formation induced by mGlu2 receptor was lower with these mutants than with Galpha 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.



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Fig. 5.   a, point mutations of and Galpha 16 at the alpha 4-alpha 5 region were constructed by swapping corresponding residues. b, immunoblot analysis of Galpha 15 and Galpha 16 proteins and the chimeras and the point mutations expressed in HEK293 cells, stained with the anti-Galpha 16 antibodies. c, differential coupling of the Galpha 15, Galpha 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 alpha  subunits and their chimeras. The new constructs of the Galpha 16 proteins with augmented expression levels were used. Data represent the radioactivity in the IP fraction divided by the total radioactivity in the membranes.



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Fig. 6.   Glutamate-induced IP formation in cells coexpressing mGlu2 or mGlu8 receptors with either Galpha 15 or Galpha 16 or the Galpha 16EE chimera. Cells coexpressing mGlu2 receptors with Galpha 15 (open circles) and the modified construct of Galpha 16 (closed circles) were stimulated with increasing concentrations of glutamate. b, same as in a with mGlu8 receptor and the newly constructed vectors encoding Galpha 15, Galpha 16, and Galpha 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.

Molecular Modeling of Galpha 15 Reveals Clusters of Residues Involved in Receptor Recognition in a Close Proximity to the C Terminus-- A three-dimensional model of Galpha 15 subunit was constructed to determine the exact spatial location of the two critical regions, which allow coupling of Galpha 15 to mGlu8 receptors. This model was generated using the coordinates of the alpha  subunits transducin and Galpha 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 alpha 4 and alpha 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 Galpha 15 to mGlu8 receptors (Glu39 and Glu41) are in close proximity to the C terminus (Fig. 7). The majority of the other residues of Galpha 15 that are not conserved in Galpha 16 are in our model far away from the C terminus of the alpha  subunit (the only exception are Lys200 and Lys203 in Galpha 15 replaced by Gln and Asn in Galpha 16, respectively) and as such are unlikely to affect coupling selectivity of the heterotrimeric G-protein.



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Fig. 7.   Model of the Galpha 15 protein in an heterotrimeric form with beta 1gamma 2. A three-dimensional structure of the Galpha 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 alpha  helix and the first beta  sheet of the Ras-like domain residues that are not conserved in Galpha 16 and that are compatible with the coupling of Galpha 15, but not of Galpha 16 to mGlu8 receptor. The orange residues are trhose that are different between Galpha 15 and Galpha 16 at the alpha 4 helix, L9 loop, beta 6 sheet, and alpha 5 helix region. The yellow residues are those that are different between Galpha 15 and Galpha 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 beta  subunit is in light blue, and the gamma  subunit is in green.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study we showed that two distinct regions along the sequences of Galpha 15 and Galpha 16 control their coupling to heptahelical receptors. One such region is between residues 311 and 354 that includes the C-terminal part of the alpha 4 helix, L9 loop, the beta 6 strand, and the alpha 5 helix. The second region comprises residues 39 and 41, which lie within the loop that links the N-terminal alpha  helix with the first beta  strand of the Ras-like domain of the G-protein alpha  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 Galpha -protein in controlling coupling to heptahelical receptors and extent the putative surface on Galpha -protein that modulates the interaction with this class of receptors.

To better characterize the regions in G-proteins involved in GPCR recognition, we took advantage of the differential coupling of Galpha 15 and Galpha 16 to mGlu2 and mGlu8 receptors. The mGlu receptors and Galpha 15/16 are unlikely to naturally interact. The mouse Galpha 15 and human Galpha 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 Galpha -protein, the best characterized being the extreme C terminus. Since Galpha 15 and Galpha 16 share identical C termini, this study allowed us to identify additional regions with coupling-restrictive abilities. All Galpha subunits share high sequence similarity and therefore have highly conserved structures; thus, identification of additional regions involved in coupling selectivity of Galpha 15/16 would help us to better map residues in all the alpha  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 alpha  subunit involved in specific interaction with these receptors should allow a comparison of the coupling mechanisms between family 1 and family 3 receptors. Galpha 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 Galpha 15/16 that influence their promiscuous coupling could constitute an helpful information for the construction of modified G-proteins with even lower selectivity.

We were expecting that the residues responsible for the differential coupling of Galpha 15 and Galpha 16 toward mGlu8 receptors would be located in the L9 (alpha 4-beta 6) loop. This region was described as a key site in G-protein alpha  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 alpha  subunits, is the most variable region between of Galpha 15 and Galpha 16 proteins. Our results revealed that, as observed with family 1 GPCRs, alpha 4 helix, L9 loop, beta 6 sheet, and alpha 5 helix together play an important role in controlling their coupling of Galpha -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 alpha  subunits. Our results also indicate that differences in amino acid residues in the region comprised by alpha 4 and alpha 5 helices are not sufficient to explain the differential coupling sensitivity of Galpha 15 and Galpha 16 to mGlu8 receptors. Indeed, we identified a second region in Galpha 16 that, once replaced by the equivalent Galpha 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 Galpha 15 and Galpha 16, where Glu39 and Glu41 in Galpha 15 are replaced by Asp and Gly in Galpha 16, respectively. These two residues are located in the loop linking the N-terminal alpha  helix and the first beta  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 Galpha 15 and Galpha 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 Galpha -protein contacts the beta  subunit, this might influence beta -gamma coupling and so coupling to receptors (43).

Taken together, our data indicate that at least two sets of residues in Galpha 15 and Galpha 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 alpha  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.


    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


    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.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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