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CELLULAR AND MOLECULAR

A Highly Conserved Glycine within Linker I and the Extreme C Terminus of G Protein Subunits Interact Cooperatively in Switching G Protein-Coupled Receptor-to-Effector Specificity

7TM Pharma, Hoersholm, Denmark (E.K., L.M., P.K.N., R.J.); Ernest Gallo Clinic and Research Center, University of California, San Francisco, Emeryville, California (L.M., M.W., J.L.W.); Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland (J.E., G.M.); and Laboratory for Molecular Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark (A.H., M.M.R.)

Received for publication November 9, 2004
Accepted December 17, 2004.

	Abstract

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Numerous studies have attested to the importance of the extreme C terminus of G protein subunits in determining their selectivity of receptor recognition. We have previously reported that a highly conserved glycine residue within linker I is important for constraining the fidelity of receptor recognition by G_q proteins. Herein, we explored whether both modules (linker I and extreme C terminus) interact cooperatively in switching G protein-coupled receptor (GPCR)-to-effector specificity and created as models mutant G_q proteins in which glycine was replaced with various amino acids and the C-terminal five G_q residues with the corresponding G_i or G_s sequence. Coupling properties of the mutated G_q proteins were determined after coexpression with a panel of 13 G_i-and G_s -selective receptors and compared with those of G proteins modified in only one module. G proteins modified in both modules are significantly more efficacious in channeling non-G_q -selective receptors to G_q-mediated signaling events compare with those containing each module alone. Additive effects of both modules were observed even if individual modules lacked an effect on GPCR-to-effector specificity. Dually modified G proteins were also superior in conferring high-affinity agonist sites onto a coexpressed GPCR in the absence, but not in the presence, of guanine nucleotides. Together, our data suggest that receptor-G protein coupling selectivity involves cooperative interactions between the extreme C terminus and linker I of G proteins and that distinct determinants of selectivity exist for individual receptors.

Progress has been made in defining the interface between GPCRs and G proteins, mainly aided by the availability of G protein crystal structures (for review, see Preininger and Hamm, 2004) as well as a wealth of information from site-directed mutagenesis studies (Conklin et al., 1993; Lee et al., 1995; Liu et al., 1995, 2002; Bae et al., 1997; Kostenis et al., 1997a; Blahos et al., 2001; Havlickova et al., 2003; for review, see Cabrera-Vera et al., 2003). On the receptor side, various intracellular portions are involved in selective recognition of G proteins. These encompass the second (i2) and third (i3) intracellular loop as well as the receptor's C-terminal tail (Kobilka et al., 1988; Liu et al., 1995, 2004; Gomeza et al., 1996; Kostenis et al., 1997a; Orcel et al., 2000; Perroy et al., 2001; Kim et al., 2002). Within the rhodopsin family of GPCRs (also referred to as family A or class I receptors), the i3 loop plays a decisive role in selective recognition of G proteins (Kobilka et al., 1988; Liu et al., 1995). Conversely, within family C or class III receptors, i3 loops are rather conserved, but i2 loops display remarkable sequence variability consistent with their decisive role in selective G recognition (Gomeza et al., 1996; Havlickova et al., 2003). On the level of the G protein, both G and the subunit complexes are likely to possess receptor contact sites (Conklin et al., 1993; Kisselev et al., 1995; Taylor et al., 1996). Within the G subunit, several key regions governing GPCR-G protein coupling specificity have been identified so far; these include 1) the extreme C terminus (Conklin et al., 1993; Kostenis et al., 1997a; Liu et al., 2002; Havlickova et al., 2003), 2) the extreme N terminus (Kostenis et al., 1997b; Slessareva and Graber, 2003), 3) a region between 4 and 5 helices (Bae et al., 1997; Cai et al., 2001; Slessareva et al., 2003), and 4) a region within the loop linking the N-terminal -helix to the 1 strand of the Ras-like domain (Blahos et al., 2001) and the linker I region connecting the ras like with the helical domain (Heydorn et al., 2004). Whereas all of these domains have been evaluated independently for their specific interaction with GPCRs, relatively little is known about whether these regions interact in an additive or cooperative manner. We have previously reported that a glycine residue within linker I (glycine 66 in G_q), which is highly conserved among G subunits across all species, is a key residue for constraining the fidelity of G protein recognition by ligand-activated GPCRs (Heydorn et al., 2004). Molecular modeling studies suggested that glycine 66 is located in a key position within linker I and is the only amino acid that can be accommodated in this location without interfering with the proper arrangement of both helical and GTPase domains of G. Perturbation of the structural arrangement of the helical and GTPase domains via mutagenesis of this key residue in linker I (glycine 66 within G_q) increased the ease with which receptors otherwise selective for G_i or G_s gained the ability to activate G_q. Previously, we have only assessed the coupling properties of G_q in which glycine 66 was replaced by various other amino acids upon coexpression with G_i-, G_s-, and G_q-selective receptors. The present study sought to clarify the contributions of glycine 66 in linker I and the sequence at the extreme C terminus of G, a well established receptor recognition site, individually and in combination, and to study potential cooperative effects between these modules in determining receptor recognition and coupling efficiency. Our data reveal that glycine 66 plays an exquisite role in GPCR-G protein specificity both alone and as a facilitator for G_q proteins modified at their extreme C termini to permit coupling to GPCRs otherwise linked to non G_q-mediated signaling events.

	Materials and Methods

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DNA Construction
Construction of G_q and G_qG66X (X = D, N, V, K) in the pcDNA1.1 expression vector was reported previously (Heydorn et al., 2004). The C-terminally modified G_q subunits G_qi5/s5 were created by introducing, in a two-piece ligation, a polymerase chain reaction-based 175-base pair BglII-NsiI fragment containing the desired codon changes. To create G_qG66Xi5/s5, a 175-base pair BglII-NsiI fragment of G_qG66X was replaced, in a two-piece ligation, with the corresponding BglII-NsiI fragment of G_qi5/s5. All wild-type and mutant G subunits contained an internal hemagglutinin epitope tag DVPDYA (Wedegaertner et al., 1993), replacing G_qwt residues 125 to 130 to monitor protein expression. Construction of expression plasmids encoding for the chemokine CCR5 and CXCR2 receptor, opioid receptor (KOR), adrenergic 2 receptor, glucagon like peptide 1 (GLP1) receptor, prostaglandin D2 DP receptor, and the gastric inhibitory peptide (GIP) receptor were described previously (Heydorn et al., 2004). The Chemerin receptor ChemR23 was cloned from a bone marrow cDNA library and inserted into the pcDNA3.1(+) expression vector using BamHI and EcoRI. GPR7 was cloned from brain cDNA and inserted via EcoRI/XhoI into pcDNA3.1(+). The high-affinity nicotinic acid receptor HM74a was cloned from adipose tissue and inserted into pcDNA3.1(+) via HindIII/EcoRI sites. The human neuropeptide Y4 (NPY4), MCH-1, and somatostatin type I receptor (SSTR1) were cloned from brain cDNA and inserted via HindIII/EcoRI into the pcDNA3.1(+) expression vector.

Cell Culture and Transfections
COS-7 cells were maintained in Dulbecco's modified Eagle's medium 1885 supplemented with 10% fetal bovine serum and 10 µg/ml gentamicin and kept at 37°C in a 10% CO₂-humidified atmosphere. The cells were plated at a density of 6 x 10⁶ cells/175-cm² flask and were transfected using a calcium phosphate-DNA coprecipitation method, where 40 µg of cDNA (equal amounts of receptor and G protein) was diluted in 480 µl of CaCl₂ solution (10 mM Tris base, 1 mM EDTA, pH 8, and 250 mM CaCl₂) and dropwise added to 480 µl of HBS [280 mM NaCl, 50 mM HEPES, and 1.5 mM sodium phosphate]. The precipitation was incubated for 45 min at room temperature and was added to the cells in 10 ml of fresh culture media supplemented with 0.1 mM chloroquine for 5 h at 37°C in a 10% CO₂-humidified atmosphere. One day after transfection, the cells were transferred to poly-D-lysine-treated 12-well culture plates at a density of 100,000 cells/well in growth media supplemented with 5 µCi of [³H]myo-inositol. HEK293 cells were maintained in minimal essential medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM Glutamax-I, 1% nonessential amino acids, 1% sodium pyruvate, and 10 µg/ml gentamicin. HEK293 cells were transfected with the FuGENE transfection reagent as per manufacturer's instructions. For all receptor G protein coexpression experiments, a ratio of 1:1 was used for the respective cDNAs.

Inositol Phosphate Turnover Assays
Forty-eight hours after transfection, the cells were washed twice in HBSS buffer (including CaCl₂ and MgCl₂; catalog no. 14025-050; Invitrogen, Carlsbad, CA) and stimulated with the respective agonists in HBSS buffer supplemented with 5 mM LiCl for 45 min at 37°C. The reactions were terminated by the aspiration and addition of 10 mM ice-cold formic acid and incubated for 30 min on ice. The lysate was applied to AG 1-X8 anion exchange resin (Bio-Rad, Hercules, CA) and washed twice with buffer containing 60 mM sodium formate and 5 mM borax. The [³H]inositol-phosphate fraction was then eluted by adding 1 M ammonium formate and 100 mM formic acid solution and counted after addition of HiSafe3 scintillation fluid (PerkinElmer Life and Analytical Sciences, Boston, MA).

Calcium Mobilization Assay
Agonist-mediated intracellular Ca²⁺ release was measured in HEK293 cells coexpressing the human KOR and the respective set of G protein subunits as indicated in Fig. 6. One day after transfection, cells were loaded for 60 min with the FLEX calcium assay kit (Molecular Devices, Sunnyvale, CA) and subsequently challenged with increasing concentrations of agonist ligand. Intracellular calcium mobilization was recorded in real time during 120 s in a FLEX station (Molecular Devices): relative light units = maximum Ca²⁺ peak/cell number x 1000.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Time-dependent generation of [3H]inositol phosphates in COS-7 cells cotransfected with the GLP1 receptor and the indicated G subunits. COS-7 cells coexpressing the GLP1 receptor and the indicated G subunits were incubated for different times (37°C) in the absence or presence of 100 nM GLP1. The resulting increases in intracellular inositol phosphate levels were determined as described under Materials and Methods. Data are means ± S.D. of three independent experiments each performed in duplicate.

ELISA
Determination of cell surface expression levels of the MCH-1 receptor was performed using an N-terminally HA-tagged MCH-1 receptor in an ELISA assay. Approximately 48 h after transfection, cells were fixed in 48-well tissue culture plates with 3.7% paraformaldehyde, washed three times with washing buffer (0.14 M NaCl, 25 mM Tris base, 2.7 mM KCl, and 0.1 mM CaCl₂·2H₂O, pH 7.4), and then blocked with blocking buffer (3% dry milk, 1 mM CaCl₂, and 50 mM Tris-HCl, pH 7.5). The primary monoclonal HA-11 antibody (Babco, Richmond, CA) was applied into the same buffer at a 1:300 dilution for 1 h at room temperature followed by three washes and a 1-h incubation with the secondary antibody (1:2500, goat anti-mouse coupled to alkaline phosphatase; Bio-Rad) in blocking buffer. The secondary antibody was detected and quantified after adding a colorimetric alkaline phosphatase substrate 3,3',5,5'-tetramethylbenzidine (Sigma-Aldrich, St. Louis, MO). When adequate color change was reached, the reaction was terminated by addition of 0.5 M H₂SO₄. Samples were then transferred to a 96-well plate and colorimetric readings were obtained at an optical density of 450 on a Tecan Sunrise absorbance reader (Tecan, Maennedorf, Switzerland). All experiments were performed in quadruplicate determinations.

Receptor Binding Studies
Whole Cell Binding. Twenty-four hours after transfection, COS-7 cells were seeded into 96-well plates at a density of 30,000 cells/well. Competition binding experiments on whole cells were then performed about 18 to 24 h later using 0.1 nM ¹²⁵I-MCH (74 TBq/mmol, 2000 Ci/mmol; Amersham Biosciences Inc., Piscataway, NJ) in a binding buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl₂, 5 mM MnCl₂, 10 mM NaCl, 0.1% bovine serum albumin (BSA), and 100 µg/ml bacitracin. Total and nonspecific binding were determined in the absence and presence of 10 µM MCH. Binding reactions were routinely conducted for 3 h at room temperature and terminated by two washes (100 µl each) with ice-cold binding buffer and addition of 25 µl of 1 N NaOH. Radioactivity was determined by liquid scintillation counting in a TopCounter (Amersham Biosciences Inc.) after overnight incubation in Microscint 20. Determinations were made in duplicates.

Membrane Binding. Binding assays were carried out in a total volume of 200 µl, containing cell membranes (10 µg of protein), ¹²⁵I-MCH (0.1 nM), and varying concentrations of MCH or GTPS. Competitor drug dilutions, radioligand, and cell membranes were all prepared in assay buffer [25 mM HEPES, pH 7.4, 10 mM MgCl₂, 5 mM CaCl₂, 0.1% (w/v) bacitracin, and 0.1% (w/v) BSA (protease free)]. Each data point was performed in duplicate, and reactions were incubated at room temperature for 2 h to allow equilibration. Membrane-bound radioligand was separated from free radioligand by vacuum filtration and washing with assay buffer over glass fiber filters [pretreated with 0.1% (w/v) polyethleneimine in assay buffer; grade GF/C, Brandel Inc., Gaithersburg, MD] using a Brandel M-24 harvester. Bound ¹²⁵I-MCH was measured using a Cobra II gamma-counter (Amersham Biosciences Inc.).

Membrane Preparation and Western Blotting
Membranes (standard P2 membrane preparations) from transiently transfected COS-7 cells were prepared essentially as described previously (Heydorn et al., 2004). Membrane proteins were quantified with the Bio-Rad protein assay kit using BSA as a standard. Samples (20–40 µg of membrane protein) were resolved by SDS-polyacrylamide gel electrophoresis (13%), transferred to nitrocellulose membranes, and probed with the 12CA5-peroxidase linked monoclonal antibody (1:1000 dilution in 10 mM Tris, pH 8, 150 mM NaCl, 10% SDS, and 0.1% Tween 20). Immunoreactive proteins were visualized with the enhanced chemiluminescence kit from Amersham Biosciences Inc.

Receptor Ligands
¹²⁵I-Melanin-concentrating hormone (2000 Ci/mmol) was obtained from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK); Chemerin 9 was synthesized by UFPeptides (Ferrara, Italy); somatostatin 28 was obtained from Bachem (Bubendorf, Switzerland); neuropeptide W23 was obtained from Phoenix Pharmaceuticals, Inc. (Belmont, CA), regulated on activation normal T cell expressed and secreted (RANTES) and interleukin-8 were obtained from PreproTech (London, UK), prostaglandin D2 was from Cayman Chemical (Ann Arbor, MI), and human pancreatic polypeptide, GLP1, and GIP were obtained from Sigma-Aldrich (St. Louis, MO) as were all other chemicals that were of the highest grade possible.

Calculations
IC₅₀ and EC₅₀ values were determined by nonlinear regression using Prism 3.0 software (GraphPad Software Inc., San Diego, CA). Values of the dissociation and inhibition constants (K_d and K_i) were estimated from competition binding experiments using the equations K_d = IC₅₀ – L and K_i = IC₅₀/(1 + L/K_d), where L is the concentration of radioactive ligand and K_d is its dissociation constant.

	Results

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Construction, Expression, and Functionality of Mutant G_q Proteins. Replacement of the five C-terminal amino acids of G_q with the corresponding G_i sequence (the resulting chimera is referred to as G_qi5) has been shown to confer onto G_i-linked GPCRs the ability to stimulate the G_q pathway in assays measuring intracellular inositol phosphate generation or intracellular calcium mobilization (Conklin et al., 1993; Kostenis et al., 1997a; Milligan and Rees, 1999). Similarly, G_s-coupled receptors can be forced to stimulate PLC upon coexpression with G_qs5 (the C-terminal five amino acids of G_q are replaced with the corresponding G_s sequence) (Conklin et al., 1996). We have previously shown that mutation of a highly conserved glycine residue in linker I of the G_q subunit to various other amino acids confers onto G_q the ability to channel G_i- and G_s-selective receptors to the PLC pathway. Here, we created G protein _q subunits that combine mutational replacement of the highly conserved glycine residue in linker I and harbor five amino acids of either G_i or G_s sequence at their extreme C termini. The mutants are referred to as G_qG66Xi5 and G_qG66Xs5 (X = D, N, V, K) and are schematically depicted in Fig. 1. The sequences of various wild-type G subunits as well as chimeric G_qi5 and G_qs5 are included in the alignment for comparison. Initially, expression levels of the mutant G_q subunits and functionality upon coexpression with two G_i- and two G_s-selective receptors were determined in transiently transfected COS-7 cells (Fig. 2). All G_qG66Xs5 mutants were coexpressed with the Gs-selective GLP1 and the adrenergic 2 receptor, and the corresponding G_qG66Xi5 proteins with the G_i-selective NPY4 and the chemokine-like ChemR23 receptor, and ligand-stimulated inositol phosphate production was determined as a measure of coupling efficiency. Although replacing the five C-terminal amino acids of G_q with G_i or G_s sequence, respectively, suffices to alter coupling specificity of the adrenergic 2, NPY4, and ChemR23 receptors, G_qs5 did not allow the GLP1 receptor to produce stimulation of the PLC pathway. Remarkably, however, all four GPCRs gained the ability to productively stimulate inositol phosphate hydrolysis when coexpressed with the dually modified G_q proteins (Fig. 2). Furthermore, measurement of PLC activation showed that the gain in coupling efficiency of the dually modified G proteins was independent of the nature of amino acid chosen to replace the highly conserved glycine residue in linker I. In parallel with the functional experiments, G subunit expression was monitored by immunodetection using membrane preparations of the respective transient receptor-G protein cotransfections (Fig. 2). G subunits were visualized with the monoclonal 12CA5-peroxidase linked antibody that recognizes an internal HA epitope tag (DVPDYA) that had been engineered into each G protein. SDS-polyacrylamide gel-electrophoresis and Western blot analysis indicated equal expression of wild-type and mutant G_q proteins in the presence of cotransfected GPCRs. Thus, the possibility that the superior coupling efficiency of the GPCRs in the presence of the dually modified G proteins was due to alterations in G expression levels can be eliminated. Additional control experiments were performed to test whether the panel of dually modified G proteins was equally capable of adopting the active conformation as wild-type G_q or the C-terminally modified G_qi5/s5 proteins. Toward this end, COS-7 cells transiently transfected with the various G proteins were challenged with fluoroaluminate (AlF ^–₄) and inositol phosphate production was determined as a measure of G activity (not shown). AlF ^–₄ typically activates G protein subunits by binding to the GDP-bound form of G. The resulting G-GDP-AlF ^–₄ complex assumes an active state conformation, which resembles the G-GTP complex (Coleman et al., 1994). Both fluoroaluminate-activated and receptor-activated G subunits are capable of stimulating intracellular effector molecules. All G proteins were found to be equally capable to stimulate inositol phosphate hydrolysis (2.5–3-fold above basal levels), indicating that the mutations do not interfere with the ability to adopt an active conformation.

View larger version (38K): [in this window] [in a new window]   Fig. 1. A, alignment of the N- and C-terminal amino acid sequences of selected mutant and wild-type G protein subunits. Gaps were introduced for optimum sequence alignment. Gqi5 and Gqs5 denote mutant Gq constructs in which C-terminal five acids are replaced with the corresponding Gi (Gqi5) or Gs (Gqs5) sequence, respectively. GqG66X denotes mutant q subunits in which the highly conserved glycine in linker I (boxed in yellow) is replaced with amino acids of different physicochemical properties (X = D, N, V, K). These single mutants have previously been shown to impart promiscuity onto Gq in that they allow Gi- and Gs-linked receptors to activate the PLC pathway (Heydorn et al., 2004). GqG66Xi5/s5 are mutant G subunits combining a C-terminal sequence switch and mutational replacement of glycine in the linker I region. The single-letter amino acid code is used. B, mapping of the highly conserved glycine residue and the extreme C terminus onto the crystal structure of a G subunit. Shown is the crystal structure of the Gi subunit in its GDP-Mg2+-bound form because it is assumed that this corresponds to the conformation of the G protein that interacts with the receptor (Coleman and Sprang, 1998). The guanine nucleotide GDP is buried between the helical and the GTPase-domain of the G subunit. Both, the highly conserved glycine residue in the center of the linker I region and the extreme C terminus are highlighted in yellow. The structure was prepared in WebLab ViewerPro and the PDB identifier for the G protein is 1BOF [PDB] .

View larger version (53K): [in this window] [in a new window]   Fig. 2. Concentration-response curves for stimulation of PLC by two Gi- and two Gs-selective receptors. COS-7 cells were cotransfected with expression plasmids coding for the Gs-selective GLP1 and adrenergic 2 receptor or the Gi-selective NPY4 and ChemR23 receptor and the indicated set of G protein subunits. About 48 h after transfection, cells were stimulated with increasing concentrations of the respective agonist ligands, and increases in intracellular inositol phosphates were determined as described under Materials and Methods. Data are expressed as means + S.D. of triplicate determinations of a single representative experiment; two additional experiments gave similar results. In parallel to each functional experiment, expression of the G subunits was monitored by immunodetection. Forty-eight hours after transfection, membranes were prepared and analyzed (20–40 µg each) by SDS-polyacrylamide gel electrophoresis (13%) and Western blotting using the 12CA5-peroxidase-linked monoclonal antibody. For the GLP1 and 2 receptor-G protein cotransfections, lane 1 corresponds to mock-transfected cells; lane 2 to Gqs5, lane 3 to GqG66Vs5, lane 4 to GqG66Ns5, lane 5 to GqG66Ds5, and lane 6 to GqG66Ks5. For the NPY4R and ChemR23 receptor cotransfections, lane 1 (mock-transfected cells), lane 2 (G), lane 3 (Gqi5 qG66Vi5), lane 4 (GqGssNi5), lane 5 (GqG66Ds5), and lane 6 (GqG66Ks5). Similar results were obtained in two additional experiments with different batches of membranes.

Functional Characterization of G_qG66Di5/s5 in Inositol Phosphate Hydrolysis Assays. Because we were interested to examine the generality of the observed cooperative effects between the linker I region and the extreme C terminus in altering GPCR-to-effector specificity, we then chose to compare coupling properties of a selected dually modified G protein (G_qG66Di5 was chosen as a model) with G proteins containing the individual mutations (G_qG66D and G_qi5) upon coexpression of a broad range of Gi-linked receptors. To this end, the panel of G proteins was transiently coexpressed in COS-7 cells with each of the ChemR23, chemokine CCR5, and CXCR2, KOR, GPR7, MCH-1 receptor, HM74a, SSTR1, and neuropeptide Y NPY4 receptors, and agonist-induced mobilization of inositol phosphates was determined (Fig. 3). Of these nine Gi/o-coupled receptors, eight did not affect PLC even when wild-type G_q was overexpressed. In contrast, the MCH-1 receptor, which preferentially activates G_i/o proteins, is also capable of activating G_q (Saito et al., 1999) and therefore, gave rise to inositol phosphate production in the absence of any cotransfected G protein. Coexpression of the ChemR23, CCR5, CXCR2, and MCH-1 receptors with G_qG66D resulted in a significantly increased inositol phosphate response (compared with wild-type G_q). By contrast, no inositol phosphate generation was observed for the KOR, GPR7, HM74a, SSTR1, and NPY4 receptors. However, all these GPCRs produced significant increases in inositol phosphate levels after cotransfection with G_qi5. Remarkably, coexpression of all nine GPCRs with G_qG66Di5 induced robust inositol phosphate increases that clearly exceeded those observed upon coexpression with the individual C-terminal or the G66D mutants. It should be noted that basal inositol phosphate levels in cells transfected with G protein alone were not significantly different from receptor-G protein-cotransfected cells (not shown). Inositol phosphate production was also determined in COS-7 or HEK293 cells cotransfected with the G_s-linked GLP1, GIP, prostaglandin D2 DP, and adrenergic 2 receptor and vector DNA as a control or the various mutant G_q and G_q/s chimeric constructs (Fig. 4). In agreement with the results obtained with G_i-selective receptors, combination of the G66D mutation with the C-terminal G_qà G_s sequence switch gave rise to a mutant G superior to the individual domain modified G proteins with respect to stimulation of PLC. Furthermore, it seems as if distinct determinants of selectivity exist for receptors from different coupling classes. Although G_i-selective receptors interacted more productively with C-terminally compared with linker-modified G proteins, G_s-selective receptors displayed the opposite phenotype and interacted more productively with the linker-modified G proteins. Collectively, the data provided evidence that both the linker I region and the extreme C terminus of G determine receptor-G protein coupling specificity and that both regions act in concert in a cooperative manner to achieve maximal PLC stimulation by non-G_q-selective GPCRs.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Functional interaction of selected Gi-coupled receptors with wild-type Gq, GqG66D, Gqi5, and GqG66Di5. COS-7 cells coexpressing pcDNA1.1 vector DNA or the indicated G subunits and different Gi -linked receptors were incubated for 45 min (37°C) in the absence or presence of increasing concentrations of the appropriate agonist ligands. The resulting increases in intracellular inositol phosphate levels were determined as described under Materials and Methods. Data are means ± S.E. of three to five experiments each performed in duplicate.

View larger version (30K): [in this window] [in a new window]   Fig. 4. Functional interaction of selected Gs-coupled receptors with wild-type Gq, GqG66D, Gqs5, and GqG66Ds5. COS-7 cells coexpressing the GLP1, GIP, prostaglandin D2 DP, and adrenergic 2 receptor and pcDNA1.1 vector DNA or the indicated G subunits were incubated for 45 min (37°C) in the absence or presence of increasing concentrations of the appropriate agonist ligands. The resulting increases in intracellular inositol phosphate levels were determined as described under Materials and Methods. Data are means ± S.D. of two to five independent experiments each performed in duplicate.

Functional Characterization of G_qG66Di5 in Calcium Mobilization Assays. We next tested whether the functional superiority of G_qG66Di5 over the other mutant G proteins was also visible in calcium assays where more signal amplification exists and used the KOR as a model. Figure 5A shows concentration-response curves of HEK293 cells cotransfected with the KOR and the set of G proteins and challenged with the KOR-selective agonist U50,488. As was the case in inositol phosphate assays (Fig. 3), G_qG66Di5 was clearly superior to G_qi5 in channelling the G_i-selective KOR to intracellular calcium mobilization, whereas no detectable calcium mobilization was visible in the presence of the other G proteins. Importantly, mock- or G subunit transfected cells did not respond to U50,488, whereas they responded equally to 10 nM ATP that stimulates an endogenously expressed purinoceptor (not shown). We were interested in calcium mobilization assays for a second reason and tested the hypothesis that functional superiority of G_qG66Di5 may be due to the fact that it confers onto G_i-selective GPCRs the ability to maintain the active conformation for a longer period. The inability of a GPCR to convert from a high- to a low-affinity agonist state is detectable when monitoring real time calcium dynamics. Typically, GPCRs induce rapid increases of intracellular calcium that decrease rapidly. In contrast, GPCRs that are "locked" in the high-affinity conformation generate calcium peaks that increase quickly but decrease very slowly (Okuno et al., 2003). Real-time calcium peaks are depicted in Fig. 5, B and C, for the KOR coexpressed with either G_qi5 (B) or G_qG66Di5 (C), respectively, and show that they only differ in magnitude but not in their kinetic profiles. These data suggest that the functional superiority of G_qG66Di5 over G_qi5 is not due to interference with the ability of the GPCR to convert from the high- to the low-affinity conformation and thus its signal termination.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Agonist-dependent mobilization of intracellular calcium in cells cotransfected with the opioid receptor and the indicated G subunits. HEK293 cells were transiently cotransfected with the KOR and the indicated G subunits. Agonist-mediated changes in intracellular calcium were measured by the FLEX-station as described under Materials and Methods. A, transformation of real-time calcium peaks into dose response curves. B, real-time calcium peaks for the KOR/Gqi5 cotransfection. C, real-time calcium peaks for the KOR/GqG66Di5 cotransfection. The arrow in B and C indicates the addition of agonist. Shown is one of three representative experiments, each performed in triplicate determinations.

Comparison of Wild-Type and Mutant G_q Proteins in Their Temporal Response to Receptor Stimulation. To investigate whether the series of G_q proteins differ in their mechanism of interaction with the ligand-activated GPCRs, we coexpressed the G_s-selective GLP1 receptor with wild-type G_q or the different mutant G_q proteins and measured inositol phosphate production in response to 100 nM GLP1 over time (Fig. 6). In agreement with the observed coupling profile of the GLP1 receptor (Fig. 4), no detectable inositol phosphate accumulation was measured at any time in the presence of coexpressed wild-type G_q. Conversely, linker as well as C-terminally modified subunits gave rise to significant increases in inositol phosphates over time. Temporal resolution of inositol phosphate production clearly showed that the various subunits differ only in the quantity of second messenger production but not in their quality of interaction with the ligand-stimulated GLP1 receptor.

Induction of High-Affinity Agonist Sites onto a GPCR upon Coexpression of Different G Proteins. G proteins are known to affect the structure and the ligand binding properties of GPCRs. Likewise, agonists demonstrate high affinity for receptor states due to the promotion of G protein coupling. It is well accepted that high-affinity agonist binding depends on the formation of a ternary complex between agonist, receptor and G protein (Hepler and Gilman, 1992; Samama et al., 1993). We therefore hypothesized that the different G proteins described in this study might be distinguished by their ability to confer high-affinity agonist sites onto a given GPCR and used the MCH-1 receptor as a model. Figure 7A depicts that high-affinity agonist binding of the MCH-1 receptor is indeed increased in the presence of various G proteins in the following rank order: G_qG66Di5 > G_qi5 = G_qG66D > wild-type G_q (note that wild-type G_q exerts a "dominant negative" effect on ¹²⁵I-MCH binding, most likely via competing with endogeneous G_i, whereas cotransfection of the MCH-1 receptor with G_i leads to a further increase in high-affinity agonist sites (Fig. 7A, inset). We would have preferred to perform these binding experiments after treatment with pertussis toxin (PTX) to prevent the receptor from interacting with the G_i proteins endogeneous to the membranes; however the five C-terminal residues of G_i are sufficient to confer PTX sensitivity onto the chimeric G_qi proteins and therefore precluded use of PTX in this experimental setup. To eliminate that the increase in MCH agonist binding sites reflected a nonspecific increase of the GPCR at the cell surface as opposed to a higher percentage of receptors residing in the high-affinity conformation, the total number of receptors was monitored with an ELISA using an N-terminally HA-tagged MCH-1 receptor. Results of the ELISA (Fig. 7B) clearly proved that, when coexpressed with the MCH-1 receptor, the various G proteins selectively influence the number of receptors in the high-affinity state as opposed to exerting nonspecific effects on receptor expression. In agreement with functional data on inositol phosphate production (cf. Fig. 3), G_qG66Di5 was superior to both G_qi5 and G_qG66D in conferring high-affinity sites onto the MCH-1 receptor. Collectively, the binding and intracellular calcium elevation data suggest that the different G proteins can be distinguished by their ability to confer high-affinity signaling competent binding sites to a given receptor rather than interfere with its ability to convert from the high- to the low-affinity conformation and thus signal termination after ligand activation.

View larger version (29K): [in this window] [in a new window]   Fig. 7. A, induction of high-affinity agonist binding to MCH-1 receptors in whole cells by cotransfection of G subunits. COS-7 cells were cotransfected with the MCH-1 receptor and pcDNA1.1 vector DNA or the indicated G subunits. After 48 h, whole cell binding experiments were performed for 3 h at room temperature as described under Materials and Methods. Receptor-bound radioactivity was measured by liquid scintillation counting. Results are means ± S.D. of one representative experiment; three additional experiments gave similar results. B, comparison of the surface expression level of the MCH-1 receptor cotransfected with the indicated G proteins in an ELISA assay. Cotransfection of the MCH-1 receptor with the various G proteins does not alter its surface expression level as verified by ELISA using an HA-antibody. Mock represents the signal obtained with mock-transfected cells using both the HA and the secondary antibody. Values are means ± S.D. of triplicate determinations of a representative experiment, two additional experiments gave similar results. C, disruption of high-affinity agonist binding by GTPS. COS-7 cells were cotransfected with the MCH-1 receptor and Gqi5 (diamonds) or GqG66Di5 (circles). Forty-eight hours after transfection, cells were harvested and membranes were prepared. Membrane protein (10 µg) was incubated in binding buffer (for composition, see Materials and Methods) with 0.1 nM 125I-MCH and the indicated concentrations of GTPS for 2 h at room temperature. Radioactivity bound to the membranes was measured by liquid scintillation counting. Results are means + S.D. of one of three representative experiments. Inset, verification of G subunit expression in the membrane preparations used for determination of high affinity agonist binding. G proteins were detected with a primary anti C-terminal antiserum and a secondary horseradish peroxidase-linked anti-rabbit IgG (1:5000 dilution; Amersham Biosciences Inc.). The band in the vector lane represents Gi endogeneous to the membrane.

Effects of GTPS on High-Affinity Agonist Binding. We next examined the effects of GTPS, a hydrolysis-resistant GTP analog, on ¹²⁵I-MCH binding to membrane preparations from COS-7 cells transiently cotransfected with the MCH-1 receptor and the two selected G proteins G_qi5 and G_qG66Di5. Excess GTPS is known to lower the affinity of receptor agonists through disassembly of ternary complexes, and this approach can be applied to estimate the affinity of a GPCR for a given G protein. Figure 7C shows that ¹²⁵I-MCH binding to the MCH-1 receptor was decreased by increasing concentrations of GTPS in the G_qi5 and G_qG66Di5 cotransfections. The fraction of ¹²⁵I-MCH displaced with high affinity by GTPS was substantially greater after cotransfection of the MCH-1 receptor with G_qG66Di5 (85.8 ± 2.3%, mean ± S.E.M., n = 3) than with G_qi5 (44.6 ± 3.6%). Surprisingly, though, we could not detect a substantial difference in potency of GTPS to reduce ¹²⁵I-MCH binding to the MCH-1 receptor cotransfected with either G protein. Together, these results suggest that the two G proteins differ in their ability to confer high-affinity binding sites to the MCH-1 receptor and may form ternary complexes with different stability that would reflect different affinities of the receptor for the individual G proteins.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Interactions between GPCRs and their cognate G proteins are known to involve several different domains on both the receptor and the G protein heterotrimer. Within G, the best characterized GPCR contact site is the extreme C terminus where residues at positions –3 and –4 are particularly important for specific receptor recognition (Conklin et al., 1993, 1996; Kostenis et al., 1997c; Bahia et al., 1998; Blahos et al., 1998; Liu et al., 2002). We have recently demonstrated the importance of the linker I region of G_q proteins in constraining the fidelity of receptor recognition. A highly conserved glycine residue in linker I (glycine 66) regulates coupling selectivity indirectly by playing a role in the specificity of nucleotide exchange within G_q induced by ligand-activated GPCRs (Heydorn et al., 2004). Here, we analyzed 1) the relationship between the linker I region and the extreme C terminus of G in determining selective GPCR coupling and 2) whether different GPCRs use different G domains to achieve specific coupling. To this end, we generated G proteins carrying a glycine-to-aspartate mutation within linker I and five amino acids of either G_i or G_s sequence at their extreme C termini (G_qG66Di5 and G_qG66Ds5). Coupling properties of the mutant G proteins were compared with G proteins individually modified within linker I (G_qG66D) or the C terminus (G_qi5 and G_qs5) upon coexpression with G_i- or G_s-selective receptors. Our coexpression studies confirmed the importance of the extreme C terminus of G in selective GPCR recognition because 12 of 13 tested non-G_q-linked receptors gained the ability to generate inositol phosphates when coexpressed with G_qi5 or G_qs5, respectively. Although almost all GPCRs use the last five amino acids for specific G activation, eight GPCRs (ChemR23, CCR5, CXCR2, MCH-1-R, GLP1R, GIPR, DPR, and 2AR) stimulated robust inositol phosphate production upon coexpression of G_qG66D and thus bypassed the requirement of the extreme C terminus for specific G activation. Remarkably, all GPCRs coexpressed with either G_qG66Di5 or G_qG66Ds5 and stimulated with the appropriate agonist ligands gave rise to inositol phosphate increases exceeding those observed upon coexpression with the G proteins modified in only one of these two domains, even if they were unable to signal when coexpressed with G_qG66D (KOR, GPR7, HM74a, SSTR1, and NPY4R) or G_qs5 (GLP1R). Thus, we demonstrated that 1) the relative importance of G domains for specific GPCR coupling varies for different receptors and that 2) participation of certain G domains may first be uncovered in the context of additional modifications. Our data are in agreement with a study from Slessareva et al. (2003) who have shown that four G_i-coupled receptors use slightly different domains on G subunits to achieve selective coupling. It will be interesting to determine whether linker I of G proteins interacts in a similarly cooperative manner with the 4-helix-4/6 loop, another region shown to be critical for specific GPCR-G protein recognition (Bae et al., 1997).

To differentiate between whether increased inositol phosphate or calcium levels generated upon coexpression of GPCRs with G_qG66Di5 compared with G_qG66D or G_qi5 was due to different abilities of the G proteins to induce/stabilize high-affinity agonist binding sites as opposed to inhibit conversion of the GPCR from the high- to the low-affinity state after ligand activation, agonist radioligand binding studies were performed as well as experiments monitoring real-time calcium dynamics. According to the ternary complex model, high-affinity agonist binding requires a complex between agonist, receptor and G protein (Hepler and Gilman, 1992; Samama et al., 1993). We therefore tested whether the G proteins differed in their ability to induce a high-affinity agonist state of the MCH-1 receptor. We found that G_qG66Di5 was superior to each of G_qi5, G_qG66D, and wild type G_q in increasing the fraction of high-affinity agonist binding sites of this receptor (Fig. 7A). Because G_qG66Di5 allowed detection of a larger fraction of MCH-1 receptors in the high-affinity state compared with G_qi5 or G_qG66D, it is tempting to speculate that the extent of functional inositol phosphate production correlates with the number of high-affinity receptor sites induced by the respective G proteins. To test whether the G proteins also interfered with the ability of the receptor to terminate intracellular signaling upon ligand activation, they were cotransfected with the KOR as a selected example, and calcium dynamics was monitored as a measure of G activation. Calcium dynamics are particularly suitable to detect an altered conversion of receptors from the high- to the low-affinity state (Okuno et al., 2003). Typically, ligand-activated GPCRs mobilize intracellular calcium displaying peaks that rapidly increase and decrease within about 120 s. Receptors deficient in shutdown of intracellular signaling due to the inability to convert to the low-affinity state after activation, display calcium peaks that increase rapidly but decrease very slowly. We noted that the calcium peaks observed upon cotransfection of the KOR with G_qG66Di5 or G_qi5 differed only in magnitude but not in their kinetic profile, suggesting that increased functional signals of the KOR in inositol phosphate and calcium assays is not due to prolonged intracellular signaling of the receptors. This is most likely the case for the other GPCRs examined in this study because, consistent with this notion is the inability of the receptors to signal in a ligand-independent fashion in the presence of the respective G proteins. Receptors deficient in shutdown of an intracellular signal because they are trapped in the active conformation by a G protein could in fact be expected to display intrinsic signaling, as has been shown for the adrenergic 2 receptor, which signals constitutively in the presence but not in the absence of a G16/z chimera (Mody et al., 2000). However, none of the 13 GPCRs tested in our study showed significant increases in second messenger production in the absence of an activating ligand. To gain more insight into the molecular mechanism underlying the induction of high-affinity agonist sites and to confer robust functional signaling characteristics onto non-Gq linked GPCRs, we sought to determine the affinity of receptors for two selected G proteins G_qi5 and G_qG66Di5 using the MCH-1 receptor as a selected example. We found that GTPS was able to disassemble ternary complexes in a concentration-dependent manner (Waldhoer et al., 1998; Welsby et al., 2002; Alves et al., 2004) as evidenced by a decrease in the binding of a concentration of ¹²⁵I-MCH that is expected to occupy only high-affinity sites. Although the fraction of ¹²⁵I-MCH binding that was abolished by the G protein uncoupling agent GTPS with high affinity was greater when the receptor was coexpressed with G_qG66Di5 than with G_qi5, the absolute potency of GTPS for the high-affinity site was not substantially different. A similar lack of correlation between receptor-G protein affinity and GTPS potency was recently reported by Alves et al., (2004) who investigated interaction of the human opioid receptor with the different G protein subtypes G_i1, G_i2, G_i3, and G_o. Thus, the superiority in functional and binding assays of G_qG66Di5 over G_qi5 is reflected by its ability to induce significantly more high-affinity agonist sites; at present, however, it cannot be stated unequivocally that this arises as a consequence of receptor G protein affinity. Alternative methods, such as plasmonwaveguide resonance spectroscopy will be required to clarify the question of receptor-G protein affinity.

It should also be of interest to determine whether the activation by GPCRs of the engineered G proteins differentially affects GDP release of G and hence their ability to reside in the very short-lived empty pocket conformation. In fact, the G66D mutation in the linker I region is ideally placed to generate a G protein with a slightly "opened" nucleotide binding pocket, a condition that may well facilitate receptor-mediated nucleotide exchange. Since the free energy required for GDP release upon receptor stimulation is related to the stability of the ternary complex (Roberts and Waelbroeck, 2004), association and dissociation kinetics of agonist tracers may help to explore this issue in more detail. Such studies are currently underway in our laboratory.

Together, our data indicate that the extreme C terminus that is part of the receptor G protein interface and the linker I region outside the receptor G protein interface of G subunits interact cooperatively in switching GPCR-to-effector specificity of GPCRs. The observed cooperativity is reminiscent of the cooperativity between different intracellular regions of the GPCRs in controlling their coupling to G proteins and underscores the hypothesis that specificity of GPCR-G protein interaction results from a multiplicity of contact zones between both proteins and cooperative interactions among various G domains within and outside the GPCR-G protein interface. We expect that the novel G proteins presented here will prove valuable for linking an even wider range of receptors than those examined here to efficient stimulation of the G_q-PLC-inositol phosphate-calcium pathway and may aid in the deorphanization process of orphan GPCRs as well as allow G_i-and G_s-selective GPCRs to be screened alongside G_q-selective receptors in assays recording inositol phosphate or calcium mobilization.

	Footnotes

 
doi:10.1124/jpet.104.080424.

ABBREVIATIONS: GPCR, G protein-coupled receptor; KOR, opioid receptor; GLP1, glucagon like peptide 1; GIP, gastric inhibitory peptide; NPY4, neuropeptide Y4; MCH-1R, melanin concentrating hormone-1 receptor; SSTR1, somatostatin type I receptor; HBSS, Hank's balanced salt solution; HEK, human embryonic kidney; ELISA, enzyme-linked immunosorbent assay; HA, hemagglutinin; BSA, bovine serum albumin; GTPS, guanosine 5'-O-(3-thio)triphosphate; PLC, phospholipase C; AlF ^–₄, fluoroaluminate; PTX, pertussis toxin; U50,488, (trans)-3,4-dichloro-N-methyl-N[lqsb]2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide.

Address correspondence to: Dr. Evi Kostenis, 7TM Pharma, Fremtidsvej 3, 2970 Hoersholm, Denmark. E-mail: ek{at}7tm.com

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