Promiscuous Coupling at Receptor-Galpha Fusion Proteins

THE RECEPTOR OF ONE COVALENT COMPLEX INTERACTS WITH THE alpha -SUBUNIT OF ANOTHER*

Paola Molinari, Caterina Ambrosio, Daniela Riitano, Maria Sbraccia, Maria Cristina Grò, and Tommaso CostaDagger

From the Department of Pharmacology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy

Received for publication, January 22, 2003, and in revised form, February 12, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fusion proteins between heptahelical receptors (GPCR) and G protein alpha -subunits show enhanced signaling efficiency in transfected cells. This is believed to be the result of molecular proximity, because the interaction between linked modules of one protein chain, if not constrained by structure, should be strongly favored compared with the same in which partners react as free species. To test this assumption we made a series of fusion proteins (type 1 and 4 opioid receptors with Go and beta 2 adrenergic and dopamine 1 receptors with GsL) and some mutated analogs carrying different tags and defective GPCR or Galpha subunits. Using cotransfection experiments with readout protocols able to distinguish activation at fused and non-fused alpha -subunits, we found that both the GPCR and the Galpha limb of one fusion protein can freely interact with non-fused proteins and the tethered partners of a neighboring fusion complex. Moreover, a bulky polyanionic inhibitor can suppress with identical potency receptor-Galpha interaction, either when occurring between latched domains of a fused system or separate elements of distinct molecules, indicating that the binding surfaces are equally accessible in both cases. These data demonstrate that there is no entropy drive from the linked condition of fusion proteins and suggest that their signaling may result from the GPCR of one complex interacting with the alpha -subunit of another. Moreover, the enhanced coupling efficiency commonly observed for fusion proteins is not due to the receptor tether, but to the transmembrane helix that anchors Galpha to the membrane.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fusion genes between a G protein-coupled receptor (GPCR)1 and the corresponding G protein alpha -subunit are not found in nature, but have been generated in several laboratories over the past years. First shown by Bertin et al. (1), if a GPCR with the carboxyl terminus tied to the amino terminus of an alpha -subunit are expressed as a single polypeptide, their functional interactions and the consequent signaling activity are preserved and even enhanced. Following that observation, similar constructs were created for a variety of studies, as extensively reviewed in Refs. 2-4.

Often the 1:1 stoichiometry, presumably imposed by fission, was reported to facilitate the study of GPCR-mediated activation of alpha -subunits in transfected cells (5-12), or it was exploited to test the specificity of signals generated by GPCR and multiple alpha -subunits (13-20). However, the interpretation of experiments in which fusion proteins are used as investigational probes requires an understanding of how receptor and G proteins interact in such chimeric proteins.

The underlying assumption is that the proximal GPCR unit in each fused assembly can interact with the distal tethered alpha -subunit. Thus, what is normally an intermolecular binding reaction between separate partners is believed to become, by fusion, an intramolecular interaction between flanked domains of the same protein. This assumption comes from thermodynamic considerations. If the binding surfaces of the receptor and the Galpha subunit can establish contacts in the fusion construct, molecular proximity should make this intrinsic interaction strongly favored over any other.

The validity of such assumption was never tested however. Bacterial toxin inactivation of endogenous G proteins (3, 5, 10) or transfection in cells lacking functional alpha -subunits (1, 2, 7, 8) were used to show that receptors and Galpha subunits of fused proteins are functional and capable of signaling in host cells. However, this is not proof that interactions occur intramolecularly in the fusion protein, since it may simply mean that the receptor of one fusion protein can interact with the alpha -subunit of another. In fact, there are data suggesting, to our view, that intramolecular interactions may not be likely; for example, the finding that receptors of the fusion proteins interact with endogenous alpha -subunits (21) and that agonist binding curves in fusion proteins show multistate behavior as in non-fused receptors (1, 7, 8, 10, 12). Here we address the question of whether the intrinsic interaction between receptor and the alpha -subunit of a fusion protein is entropically favored over the extrinsic interaction with fused or non-fused partners.

We prepared a number of fusion proteins consisting of a receptor sequence (beta 2-adrenergic, D1-dopamine, delta -opioid, and nociceptin receptors) chained through a short 15-mer peptide to the amino-terminal of a G protein alpha -subunit (Galpha sL and Galpha o). Fusions between mutated analogues of either the receptor or the alpha -subunit were also engineered in a similar fashion. Using such constructs and cotransfection experiments to distinguish the activation of tethered and non-tethered forms of Galpha subunits, we find that both components of one fusion protein freely interact with non-fused units or the fused elements of another.

This indicates that there is no preferential interaction between the tied units enclosed in one fused assembly and suggests that signaling at these chimeric proteins is more likely the result of cross-interactions between receptor and alpha -subunits located in neighboring molecular assemblies.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Materials came from the following sources: Cell culture media, G418, and fetal calf serum were from Invitrogen. Nucleotides (GDP, GTP, GTPgamma S, ATP), thymidine, phosphocreatine, creatine phosphokinase, chloroquine diphosphate, DEAE-dextran, dextran sulfate (500 kDa average mass), l-isoproterenol, anti-FLAG M1, free and agarose-bound, monoclonal antibody were from Sigma. Pindolol, UK14,304, N,N-diallyl-Tyr-Aib-Aib-Phe-Leu (ICI 174,864), and SKF 82958 were from Tocris. Nociceptin, [D-Ala2,D-Leu5]enkephalin and vasopressin were from Bachem. Pertussis and cholera (holo and A protomer) toxins were from List Biologicals. Antibodies to HA epitope and to G protein alpha -subunits were from Santa Cruz Biotechnology. Wheat germ agglutinin and anti-HA monoclonal antibody covalently linked to agarose, from Vector Labs and Roche Molecular Biochemicals, respectively. Ni-NTA agarose beads were from Qiagen. Receptor radioligands, [125I]iodopindolol, [3H]dihydroalprenolol, [125I-Tyr14]nociceptin, [3H]naltrindole, [3H]SCH-23390, [125I]vasopressin antagonist, [3H]RS-79948-197, and labeled nucleotides, [gamma -32P]GTP (6 Ci/µmol), [35S]GTPgamma S (1.2 Ci/µmol), [alpha -32P]NAD (0.9-1 Ci/µmol) were from PerkinElmer Life Sciences or Amersham Biosciences. Purified rat myristoylated Go and a bovine brain purified mix of Gi/o heterotrimers were from Calbiochem. Purified beta gamma -subunits (bovine brain) free of ADP-ribosylating alpha -subunits were a kind gift from Pat Casey.

Construction of cDNA Coding for Fusion Proteins-- We gratefully acknowledge the following cDNA gifts: human beta 2-adrenergic receptor, alpha 2A-adrenergic receptor, and dopamine D1 receptor (D1R) (Dr. S. Cotecchia), rat OP1R (Dr H. Akil), human OP4R (Dr. G. Calò), rat Galpha sL (Dr. O. Ugur), rat Galpha o, and V2TM1-Galpha o (Dr. M. Parenti), rat V2R (Dr. F. Naro). Full-length cDNAs encoding fusion proteins were all constructed by a similar strategy and subcloned into the pcDNA3 expression vector (Invitrogen). After removal of the 3'-end stop codon from the receptor cDNA by PCR-based mutagenesis, the fusion between each receptor (beta 2AR, OP4R, OP1R) and alpha -subunit (Galpha sL and Galpha o1) coding sequence was achieved by insertion of a linker encoding a 15-mer peptide containing the enterokinase cleavage site (LDPRSDYKDDDDKGS). The linker, (made by annealing two synthetic oligonucleotides, sense 5'-CTGGATCCTCTAGACCCCCGGTCAGACTACAAGGATGACGATGACAAGGGCTCCATGGGAATTCGA-3' and the corresponding antisense sequence), was ligated between the carboxyl-terminal of the receptor and the Met initiator codon of Galpha via XbaI and NcoI cloning sites, respectively (underlined in the oligo). The only exception to this general design is the fusion protein D1R-Galpha sL, in which the two coding sequences were directly linked through two residues Leu and Asp (encoded by a PCR-made XbaI site), which removed the initiator Met of Galpha sL.

Fusion protein mutants carrying modifications in the receptor or in Galpha were constructed by a PCR-based strategy using mismatched primers. These include: the pertussis toxin-resistant (3) OP1R-Galpha o[C350I] and V2TM1-Galpha o[C350I] (22), the receptor-defective (23) OP1R[R146E]-Galpha o, the G protein-defective (24) OP1R-Galpha o[C350R], the GTPase-deficient proteins, OP1R-Galpha o[Q205L] (25), OP1R-Galpha o[D273N] (26) and OP1R-Galpha o[Q205L,D273N], the latter of which contains a xanthine nucleotides binding alpha o (Galpha oX) (27). Tagged constructs of beta 2AR-Galpha sL and OP1R-Galpha o were obtained by placing at the amino terminus the cleavable prolactin signal peptide tethered to the FLAG epitope (DYKDDDDK). Upon cleavage, that exposes an amino-terminal M1 epitope immediately before Gly2 in beta 2AR, and upstream Glu2 (with an inserted extra Gly residue) in rat OP1R. Two diverse tagged versions of the fusion protein D1R-Galpha sL were prepared by extending the receptor amino terminus with either the sequence of the HA epitope (YPYDVPDYA) or with a hexahistidine tag. Both were placed before Ala2 of human D1R with an interposed extra Ala residue. All plasmids used in this study were verified by total sequencing of the inserted cDNA.

Cell Culture and Transfection-- COS-7 and HEK-293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10%(v/v) fetal calf serum, 100 units/ml penicillin G, and 100 µg/ml streptomycin sulfate, in a humidified atmosphere of 5% CO2 at 37 °C. CHO cells were maintained in a 1:1 mix of Dulbecco's modified Eagle's medium/Ham's F-12,10% fetal calf serum. Transient transfections of the cells were performed by DEAE dextran/chloroquine, calcium phosphate precipitation, and FuGENE, for COS-7, HEK-293, and CHO cells, respectively. In all cases the total amount of transfected DNA was maintained constant by addition of empty vector. Cells were allowed to express the transfected gene for 48-72 h before harvesting. When necessary, pertussis toxin was added to the medium (10-20 ng/ml) 18 h before membrane preparation. To generate stable expressing HEK-293 and CHO clones, cultures were transfected with FuGENE and selected under G418 (600 µg/ml active drug) for 3-6 weeks prior to ring cloning. 20-60 G418-resistant clones were selected and screened by the appropriate radioligand binding assay.

GTPgamma S Binding and GTPase-- Enriched plasma membranes from transfected cells were prepared by differential centrifugation (28) and stored (2 mg/ml) at -80 °C. [35S]GTPgamma S was determined either in a 100-µl or 1-ml reaction mixture containing 50 mM Hepes-Tris, pH 7.4, 1 mM EGTA, 1 mM DTT, 100 mM NaCl, 5 mM MgCl2, 1-2 nM [35S]GTPgamma S, 3 µM GDP (or concentrations varying between 0.1 nM and 100 µM) and 1-2 µg of membrane proteins, with or without the appropriate full agonist. Samples were incubated 90 min at 20 °C, filtered onto GF/B (Packard Filterplate) and washed (6-10 volumes ice-cold buffer) prior to scintillation counting on a Packard Top Count. Nonspecific binding was determined in the presence of 10 µM GTPgamma S. Incubations containing purified alpha -subunits were filtered onto polyvinylidene difluoride membrane plates (Millipore). GTPase assays were done under similar conditions using 5 µg of membrane protein and a 100-µl reaction containing 50 mM Hepes-Tris, pH 7.4, 1 mM EGTA, 1 mM DTT, 100 mM NaCl, 5 mM MgCl2, 1 mM AppNHp, 5 mM phosphocreatine, 5 units/tube creatine phosphokinase, 0.5 mM ATP, 0.2 mM GTP (spiked with 0.25-0.5 × 106 cpm of [gamma -32P]GTP). Reactions lasted 10 min at 37 °C, and 32Pi release was determined by charcoal separation as described (28).

cAMP Determination in Intact Cells-- The determination of intracellular cAMP levels in transfected cells was made by RIA following extraction in 0.1 N HCl as described (29).

Receptor Binding Studies-- Radioreceptor binding assays were made in 1-ml reactions containing 50 mM Hepes-Tris, pH 7.4, 0.2 mM EGTA, 0.2 mM DTT, 5 mM MgCl2, 10 µM leupeptin, 10 µM bestatin, 0.1 mg/ml bacitracin, 0.1% (w/v) bovine serum albumin, and suitable amounts of membrane proteins (0.5-20 µg). The following radiolabeled ligands were used: [125I]pindolol (50-100,000 cpm) or [3H]dihydroalprenolol (25,000 cpm) for beta 2-adrenergic receptor; [125I]d(CH2)5-[D-Ile2-Ile4-Tyr-NH29] Arg-vasopressin (100,000 cpm), for V2R; [3H]naltrindole (25-50,000 cpm) for OP1R; [125I-Tyr14]nociceptin (50-100,000 cpm) for OP4R; [3H]SCH 23390 (50,000 cpm) for D1R, [3H]RS-79948-197 (30,000 cpm) for alpha 2-adrenergic receptor. Reactions lasted 90 min at room temperature and were terminated by rapid filtration onto GF/B glass fiber microplates (Filtermate 196, Packard). Filters were washed three times with 1 ml of ice-cold buffer and allowed to dry a few hours. The plates were counted in a Top Count (Packard) following the addition (25 µl) of Microscint 20 (Packard) to each well. Binding isotherms where fitted by mass-action law models (31) to compute binding affinity and receptor number (Bmax).

Toxin-catalyzed ADP-ribosylation-- Bacterial toxin labeling of alpha -subunits was performed in 50-µl reactions. The buffer contained 100 mM Tris-HCl, pH 7.8, 5 mM thymidine, 1 mM ATP, 2.5 mM MgCl2, and 2-5 µCi of [alpha -32P]NAD (~1Ci/µmol). For cholera toxin, we further included 10 mM arginine, 250 ng of cholera toxin A-protomer, 50-100 µg of membrane proteins, and, depending on the experiment, the desired concentration of receptor agonist. For pertussis toxin, we added 25-50 µg of membrane proteins, 100 µM GTP, 10 mM DTT, 0.5 µg pertussis holotoxin. Reactions were started by membrane addition, lasted 30 min at 20 °C, and were arrested by dilution with 0.5-ml ice-cold Tris-HCl prior to centrifugation (40,000 × g, 10 min at 4 °C). Supernatants were discarded and pellets resuspended in Laemmli sample buffer at 95 °C. For the experiments carried out with purified alpha - and beta gamma -subunits the 25-µl reaction volume was stopped by the addition of sample buffer 4× and heating for 5 min at 95 °C. Proteins were separated by SDS-polyacrylamide gel electrophoresis (10%) using either regular sized (16 × 18 cm) or mini slabs (6 × 8 cm). Gels were stained and dried under vacuum at 60 °C, and the radioactivity incorporated into the separated protein bands was quantified with a microchannel array detector counter (Packard Instantimager) with at least 2-sigma counting error accuracy.

Immunoblots-- Proteins separated by SDS-PAGE as above were electrotranferred onto polyvinylidene difluoride membranes (Millipore Immobilon). Blots were incubated overnight at 4 °C with primary antibodies and washed three times with TBST before probing with alkaline phosphatase or 125I-labeled secondary antibody. Reactive bands were then visualized by phosphatase staining with Promega reagents or by phosphorimaging (Packard Cyclone), respectively.

Affinity Pull-down Assays-- Membranes or [32P]ADP-ribosylated pelleted samples (50-100 µg of proteins) were solubilized and denatured in 100 µl 0.5% SDS, 10 mM DTT, 5 min at 95 °C. Samples were equilibrated to room temperature for 1 h and then diluted in 5 volumes of buffer A (50 mM Tris-HCl, pH 7.4, 300 mM NaCl, 5 mM CaCl2, 0.2% Triton X-100, 0.1% bovine serum albumin, containing protease inhibitor mixture "Complete" from Roche Molecular Diagnostics). Samples were clarified by centrifugation (40,000 × g, 30 min) and to the supernatants were added 25-50 µl of a gel slurry (1:1) consisting of agarose-linked primary antibodies (mouse M1 anti-FLAG, or rat anti-HA) or wheat-germ agglutinin. Incubation lasted 3 h or overnight under gentle stirring, at the end of which, agarose beads were pelleted (14,000 × g, 1 min) and resuspended six times in Buffer A, prior to discarding the supernatants. Bound proteins were eluted in 40 µl of Laemmli sample buffer 5 min at 95 °C. For the batch purification of His6-tagged D1R-Galpha s fusion protein, membranes were extracted and denatured in 0.5% SDS, containing 70 mM beta -mercaptoethanol instead of DTT, diluted in Buffer B (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2% Titron-X 100, protease inhibitors mixture EDTA-free) prior to the addition of 25 µl of Ni-NTA agarose and incubated 1 h at room temperature with gentle agitation. Beads were washed six times in Buffer B supplemented with 20 mM imidazole before elution in Laemmli sample buffer. Samples were separated by SDS-PAGE as described above.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Biochemical Properties of Receptor-Galpha Fusion Proteins-- The molecular nature of the R-G fusion proteins expressed in host cells was studied by Western blot analysis, bacterial toxin catalyzed ADP-ribosylation, and lectin-mediated affinity precipitation, using amino-terminal FLAG-tagged versions to facilitate immunoblotting and affinity precipitation.

Fusion proteins appeared as diffuse bands in immunoblots reacted with either the anti-FLAG or the anti-alpha -subunit antibody, exhibiting molecular masses consistent with the presence of an added alpha -subunit (Fig. 1, a-f). This microheterogeneity reflects variable glycosylation, because it was also observed in immunoblots of the proteins after affinity precipitation by agarose-linked WGA (Fig. 1, c and f). Minor M1-reacting bands of smaller molecular weights were also detected, especially in transiently transfected cells with opioid receptor fusion protein (Fig. 1d) and may represent proteolytic forms, although neither the inclusion of protease inhibitors during membrane preparation nor the prolonged incubation of the membranes at 37 °C did significantly change their abundance. Since the bands were not retained by WGA-agarose, we suspect that may represent aberrant deglycosylated and proteasome-degraded forms of the fusion protein, similar to those previously characterized for wild-type opioid receptors (32). Broad 90-110 kDa radioactive bands corresponding to those in Western blotting were labeled, in addition to the endogenous alpha -subunits, by cholera toxin in membranes expressing beta 2AR-Galpha s (Fig. 1g) and pertussis toxin in those expressing OP1-Galpha o (Fig. 1h), respectively. Previous treatment of the transfected cells with the corresponding toxin decreased (30-50%), but did not abolish, the membrane labeling of the fusion proteins (Fig. 1, g and h), suggesting that receptor-tethered alpha -subunits are less susceptible to toxin-mediated ADP-ribosylation. (See more on this below).


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Fig. 1.   Molecular size of fusion proteins in Western blot analysis and ADP-ribosylation. Upper panel, Western blot analysis of fusion proteins after SDS-PAGE separation of total membranes or affinity-precipitated glycoproteins by agarose-linked WGA. Membranes from control cells (CTR) or a HEK-293 clone permanently expressing FLAGbeta 2AR-Gs (RG) were immunoblotted and visualized with M1 monoclonal (a), anti-Galpha s antibody (b), or subjected to a WGA affinity pull-down procedure (c) (see "Experimental Procedures") before immunoblotting and staining with anti-Galpha s. The blots for FLAG-OP1R-Go are shown in a corresponding succession (d-f), and were obtained using membranes prepared from transiently transfected HEK-293 cells, with empty vector (CTR) or encoding the tagged fusion protein (RG), except in e where the control lane (alpha o) shows membranes from cells transfected with Galpha o. Lower panel, bacterial toxin-catalyzed incorporation of [32P]ADP-ribose (see "Experimental Procedures") in membrane from COS-7 transiently transfected with fusion proteins. g, cells were transfected with pcDNA3 (CTR) or vector encoding beta 2AR-Gs (RG) and exposed or not to cholera toxin (10 ng/ml, 18 h) prior to membrane preparations and ADP-ribosylation by A-protomer. h, COS-7 transfected with pcDNA3 (CTR) or vector encoding OP1R-Go (RG) were similarly exposed and ribosylated with pertussis toxin.

The most prominent biochemical feature of membranes transfected with fusion proteins was enhanced agonist stimulation of GTPgamma S binding in the presence of GDP. The effect is striking for beta 2AR-Galpha s, since very little adrenergic stimulation of nucleotide binding can be detected in membranes expressing even high levels of beta 2AR, whereas in membranes containing beta 2AR-Gs agonist-induced enhancement of nucleotide binding (Fig. 2, top) and GTPase activity (not shown) are evident. In addition, a mark of fusion protein-mediated nucleotide exchange is the biphasic shape of the binding isotherms of GDP in competition for [35S]GTPgamma S induced by the addition of agonist (Fig. 2, top).


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Fig. 2.   Enhancement of agonist-stimulated nucleotide exchange in receptor-Galpha fusion proteins. Binding of [35S]GTPgamma S in the presence of increasing concentrations of GDP (abscissa) measured in membranes prepared from COS-7 cells transfected with wild-type receptors or the corresponding receptor-G protein fusion protein. Upper panels, membranes (1 µg of protein) expressing beta 2AR (45 ± 6 pmol/mg), left, or beta 2AR-Galpha s (31 ± 4 pmol/mg), right were tested in the absence (BAS) or presence of 10 µM isoprorerenol (ISO). Lower panels, membranes (2 µg of protein) expressing OP1R (23 ± 7 pmol/mg) left, or OP1R-Galpha o (19 ± 3 pmol/mg) right were tested in the absence (BAS) or presence of 1 µM DDL. Binding is expressed as B/T, i.e. ratio of bound versus total (~1 nM) radioligand. Data are representative of at least four experiments for beta 2AR-Galpha s and more than six for OP1R-Galpha o.

Similar enhancements were observed in the OP1R-Go system, although in this case wild-type receptor stimulation of nucleotide exchange is measurable. Strongly biphasic GDP competition curves in the presence of agonist are also evident for Go fusion proteins and not observable in membranes expressing wild-type receptors (Fig. 2, bottom), even if cotransfected with alpha o (not shown). Thus, they seem to be a feature of the receptor-tied G protein and indicate that agonist-induced decrease of GDP affinity is far more noticeable on the fused than on the non-fused alpha -subunit.

Interaction of Fusion Proteins with Gbeta gamma Subunits-- Reduced sensitivity to toxin ADP-ribosylation may result from an impaired ability of the amino-terminal modified alpha -subunit to interact with beta gamma . This point was investigated in more detail using OP1R-linked Go, since toxin-mediated ADP-ribosylation of this alpha -subunit strictly depends on beta gamma interaction. As a control to evaluate the role of the amino-terminal extension in the alpha o sequence (which blocks amino terminus myristoylation), we used a fusion protein consisting of the first transmembrane portion of the vasopressin 2 receptor (amino-terminal plus TM1) and Galpha o (V2TM1-Galpha o). Such TM-anchored alpha -subunits were previously found localized in the plasma membrane, despite the lack of fatty acid acylation (22). Thus, V2TM1-Galpha o closely mimics the structural modification of the GPCR-tethered alpha -subunit, but in the absence of a functional receptor. Cells transfected with wild-type Galpha o, V2TM1-Galpha o, or OP1R-Galpha o, were exposed or not to pertussis toxin and compared. The level of toxin-catalyzed incorporation of [32P]ADP-ribose detected in membranes indicates that when Galpha o is N-tethered either to a full receptor or to a single transmembrane domain, it is less ribosylated in vivo than native resident Gi or exogenously transfected Go (Fig. 3A). This implies that the presence of the amino-terminal transmembrane anchor reduces the accessibility of alpha o to toxin-mediated modification.


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Fig. 3.   Pertussis toxin resistance of OP1R-Galpha o and interaction with beta gamma -subunits. A, duplicate T75 flasks of COS-7 cells transfected with empty plasmid (CTR) or vectors expressing wild-type Galpha o (Go), or the fusion proteins V2TM1-Galpha o (TM-Go) and OP1R-Galpha o (R-Go), were exposed or not to pertussis toxin (20 ng/ml, 18 h) before harvesting. Membranes were [32P]ADP-ribosylated with pertussis toxin and separated by SDS-PAGE. Histograms indicate the radioactivity determined (Instantimager) in the 40-kDa bands for CTR and Go, in the 50-55-kDa range for TM-Go, and in the 80-110-kDa range for R-Go (inset). The percent of ribosylable protein that survives toxin exposure of intact cells is indicated on top of each bar. The experiment was repeated twice with similar results. B, membranes (25 µg) from COS-7 cells expressing the fusion protein OP1R-Galpha o, and membranes from pertussis toxin-treated control cells supplemented with 5 ng of purified Galpha o (alpha o) were ADP-ribosylated in the presence of increasing concentrations of purified bovine beta gamma (x-axis), separated by SDS-PAGE, and counted in a Packard Instantimager. The radioactivity incorporated into purified alpha o-subunit (open circle ) or fusion protein () is plotted as a function of beta gamma concentrations. The experiment was repeated two additional times, (using V2TM1-Galpha o in one case), with similar results.

To test the competence of tethered alpha o to interact with beta gamma , we measured the ability of increasing concentrations of purified beta gamma -subunits to support pertussis toxin-catalyzed incorporation of [32P]ADP-ribose into OP1R-Galpha o (Fig. 3B). Purified myristoylated Galpha o added to membranes of pertussis toxin-treated COS-7 cells (to suppress background labeling of endogenous Gi) served as a reference. Gbeta gamma was equally effective in enhancing ADP-ribosylation of the two proteins (Fig. 3B), and similar results were obtained when beta gamma was added to V2TM1-Galpha o (not shown). Thus, the presence of the amino-terminal tethered sequence does not disrupt the interaction of the alpha -subunit with beta gamma in isolated membranes.

Interaction of Fused Receptors with Endogenous alpha -Subunits-- Fusion proteins between alpha 2-adrenergic receptors and Gi were found to interact with endogenous G proteins in the membrane of host cells (21). We wondered whether there was any preference in the interaction of fused receptors for tethered and non-tethered alpha -subunits. To distinguish the two forms we used agonist-enhancement of cholera toxin-catalyzed ADP-ribosylation, which reflects receptor-mediated activation at several G proteins, such as Gi/o, Gs, and Gt (33-36), and gives the molecular mass of the activated alpha -subunit.

Isoproterenol caused a concentration-dependent increase of toxin-induced ADP-ribosylation of Galpha s (Fig. 4A, left) in membranes of CHO cells expressing wild-type beta 2-adrenergic receptor. At the same concentrations, it enhanced the labeling of both the endogenous Gs and of the 110-kDa bands corresponding to the fusion protein in membranes expressing beta 2AR-Gs (Fig. 4A, right). This means that the tethered beta 2-adrenergic receptor can freely interact with both fused and non-fused Galpha s subunits and implies little preference for the two types of interactions. Analogous results were observed in experiments performed by transient expression in COS cells (not shown).


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Fig. 4.   Interaction of receptor-Galpha fusion proteins with endogenous alpha -subunits. A, membranes prepared from CHO clones permanently expressing wild-type beta 2-adrenergic receptor (left) or beta 2AR-Gs fusion protein (right) were ADP-ribosylated with [32P]NAD and cholera toxin A-protomer in the presence of increasing concentrations of isoproterenol (graph x-axis). The radioactivity (120 min counting time) incorporated into endogenous Gs (46 and 52-kDa bands,open circle ) and fusion protein (97-115 kDa,) is plotted as a function of the log of isoproterenol (ISO) concentration beneath. The curves (solid lines) were fitted with a 4-parameter logistic function (30) to compute the EC50 for agonist-mediated enhancement of ADP-ribosylation: (in beta 2AR CHO, 7 ± 2 nM; in beta 2AR-Galpha s CHO, 16 ± 6 nM for alpha s stimulation and 8 ± 3.2 nM for beta 2AR-alpha s stimulation, respectively). The experiment is representative of two independently made membrane preparations of the same CHO clone. An additional experiment made on transiently transfected COS-7 cells also gave comparable results. B, membranes from HEK-293 clones permanently expressing Go fused to two different opioid receptors, OP1R-Galpha o or OP4R-Galpha o, were [32P]ADP-ribosylated as above under basal conditions (B) or in the presence of the respective agonists, [D-Ala2,D-Leu5]enkephalin (D) 1 µM and nociceptin (N) 1 µM for OP4R-Galpha o. Left, SDS-PAGE separation of total membranes (tot); right, samples were first solubilized and affinity-precipitated with WGA before electrophoresis. Note that after affinity purification of the fusion protein agonist enhancement of ADP-ribose incorporation in tethered alpha o is evident.

Similarly, in membranes from permanent HEK-293 clones expressing OP1-Galpha o or OP4-Galpha o, the addition of agonist strongly enhanced cholera toxin-mediated ADP-ribosylation of endogenous 40/41-kDa bands, demonstrating interaction of the fused receptor with endogenous Gi (Fig. 4B). The extent of stimulation was comparable to that observed in cells transfected with non-fused receptors. The concurrent incorporation of ADP-ribose into the fusion protein was not detectable in this experimental protocol, because the lower level of labeling of the tethered alpha o-subunit by cholera toxin was obscured by an endogenous ADP-ribosylated band of similar molecular mass. This band, particularly abundant in HEK-293 cells, does not correspond to a bacterial toxin substrate, since it was also ribosylated in its absence (not shown). However, agonist enhancement of cholera toxin ADP-ribosylation of the fused Galpha o becomes clearly detectable if the fusion protein is separated from the bulk of membrane proteins by WGA-agarose (Fig. 4B). Thus, both Gs- and Go-linked receptors can liberally and efficiently interact with endogenous free alpha -subunits of the membrane.

Interaction of Tethered alpha -Subunits with Non-fused Receptors-- To determine the accessibility of the alpha -subunit of a fusion protein to an external receptor we used two approaches. In the first approach, we evaluated how efficiently a wild-type GPCR can activate a cotransfected alpha -subunit that is linked to a transmembrane domain peptide.

For the Go system, we used a pertussis toxin-resistant version (37) of V2TM1-Galpha o (TM-Galpha o[C350I]) (22). This was cotransfected with wild-type OP1R, and cells were exposed to pertussis toxin to eliminate the contribution of endogenous Gi. Agonist-induced nucleotide exchange was abolished by pertussis toxin treatment in membranes expressing only OP1 receptors, but not in those co-expressing OP1R and TM1-Galpha o[C350I] (Fig. 5). Moreover, the curve for GDP inhibition of [35S]GTPgamma S binding induced by agonist in OP1R/V2TM-Galpha o[C350I] co-expressing membranes was biphasic as that typically observed for transfected OP1R-Go fusion protein (compare with Fig. 2). Thus, transmembrane-anchored Go interacting with a non-fused receptor can emulate the enhancement of receptor-mediated nucleotide exchange observed for fusion proteins. Cotransfection of OP1R with wild-type Go (which was expressed at levels 2-3-fold greater than V2TM-Galpha o as judged by Western blots) could not reproduce the same type of agonist-induced GDP shift (data not shown), indicating that a transmembrane tether is important for such an effect.


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Fig. 5.   Single transmembrane domain (V2TM1)-tethered alpha o cotransfected with opioid receptor emulates the enhanced nucleotide exchange observed with the corresponding fusion protein. Binding of [35S]GTPgamma S in the presence of increasing concentrations of GDP measured in membranes prepared from transiently expressing HEK-293 cells, individually transfected with wild-type OP1R (top) or cotransfected (1:1) with OP1R and the pertussis toxin-resistant fusion protein V2-TM1-Galpha o[C350I]. Cells were treated with pertussis toxin (20 ng/ml, 18 h) before membrane preparation. Binding was measured in the absence of ligand (Bas) or in the presence of either DDL 1 µM, or the negative antagonist ICI 174,864 1 µM. Data are representative of three independent transfections.

For the Gs system, we employed Galpha s fused to OP1R (OP1R-Galpha s), acting as transmembrane carrier of the appended alpha -subunit. In fact, in membranes expressing OP1R-Gs, pertussis toxin suppressed opioid agonist-stimulated GTPase activity (Fig. 6A, left), indicating that it entirely result from the interaction of the Gs-fused receptor with endogenous Gi proteins. Moreover, transfection of OP1R-Gs in intact cells caused enhancement of basal cAMP levels, which were inhibited by opioid agonist, just like in cells cotransfected with OP1R and Gs as separate proteins (Fig. 6A, right). Therefore Gs attached to the opioid receptor neither interacts with it nor disturbs its interaction with endogenous Gi, and this fusion protein can thus be used as a form of Gs linked to an inert transmembrane sequence, exactly like V2TM-Go described above. In membranes co-expressing OP1R-Galpha s and wild-type beta 2-adrenergic receptor, isoproterenol promoted at similar concentrations cholera toxin-catalyzed incorporation of ADP-ribose in both endogenous Gs and OP1R-linked alpha s (Fig. 6B, right), as observed for the beta 2AR-Galpha s fusion protein. In addition, in the same membranes isoproterenol induced GDP inhibition curves very similar to those measured for beta 2AR-Galpha s (data not shown). This again indicates that enhanced nucleotide exchange can be generated when a wild-type receptor interacts with the transmembrane-anchored alpha -subunit.


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Fig. 6.   Interaction of opioid receptor-tethered Gs (OP1R-Galpha s) with non-fused beta 2AR. A, left, GTPase activity in membranes prepared from COS-7 cells transfected with OP1R (R) or the fusion protein OP1R-Galpha s (RG) that were treated or not with Cholera (CT) or pertussis toxins (PT) (both, 10 ng/ml 18 h). Activity was measured in the absence (bas) or presence of opioid agonist (DDL) and negative antagonist (ICI) (both 1 µM). Data are means (± S.E.) of three experiments. Right, HEK-293 plated in 24-well dishes, were individually transfected with control vector (CTR), OP1R (R), OP1R-Galpha s (RG), Galpha sL (Gs), or cotransfected with both OP1R and Gs (R+Gs). Cells were incubated (30 min) 48 h later in the absence (bas) or presence of 1 µM DDL, and the intracellular levels of cAMP were measured by RIA. Data are means (± S.E.) of four experiments. B, COS-7 cells membranes cotransfected with beta 2AR and OP1R-Galpha s were ADP-rybosylated with [32P]NAD and A-protomer of cholera toxin in the presence of increasing concentrations of isoproterenol as in Fig. 4. The gel shown is representative of two independent experiments, both of which were quantified (microarray detector), and the incorporation of ADP-ribose into endogenous Gs (open circle ) and OP1R-Gs () is plotted as a function of the log of isoproterenol (ISO) concentration. Points are means (± range) of c.p.m. values normalized as ratios over the values in the absence of agonist.

In the second approach the cross-interaction was studied more directly in letting a non-fused wild-type receptor react with the alpha -subunit linked to another functional GPCR. To do so, we co-expressed pairs of pharmacologically distinct receptors, both able to couple, but only one of which covalently docked to the same G protein.

For the Go system, the toxin-resistant fusion protein OP1R-Galpha o[C350I] was co-expressed with wild-type alpha 2-adrenergic receptors. In pertussis toxin-treated membranes, the OP1R-tethered Galpha o displayed enhanced nucleotide exchange in response to either alpha 2-adrenergic and opioid agonists (Fig. 7A). The effects of the two agonists were not additive, indicating that there was no apparent co-partitioning of Galpha between the two types of receptors (Fig. 7A).


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Fig. 7.   Interaction of a non-fused receptor with the alpha -subunit tethered to a different GPCR. A, binding of [35S]GTPgamma S in the presence of increasing concentrations of GDP in membranes prepared from transiently expressing HEK-293, either transfected individually with wild-type alpha 2BAR (left) or cotransfected (1:1) with alpha 2BAR and the fusion protein OP1R-Galpha o[C350I]. Cells were treated with pertussis toxin (10 ng/ml, 18 h) before membrane preparation. The binding was measured in the absence of ligand (Bas), and in the presence of 1 µM DDL, 1 µM alpha 2-adrenergic agonist UK14,304 (UK), or both. The Bmax of expressed receptors was 13 pmol/mg for alpha 2AR and 15.8 pmol/mg for OP1R-Galpha o[C350I]. Data are representative of three independent experiments. B, agonists effect on cholera toxin-catalyzed ADP-ribosylation of COS-7 membranes cotransfected with vasopressin receptors (V2R) (Bmax, 1.7 pmol/mg) and the fusion protein beta 2AR-Gs (Bmax, 9.8 pmol/mg). Experiments were performed and quantified as in Figs. 4A and 6B. The inset shows a representative autoradiogram displaying the effect of varying concentration of arginine vasopressin (AVP). The graph shows concentration-response curves for AVP-induced enhancement of the ribosylation of the beta 2AR-Gs band in the absence () or presence () of isoproterenol (1 µM). Data are averaged (± range) from two independent experiments normalized as ratios of cpm in the presence of ligand versus cpm in the absence. C, a, Stimulation of cholera toxin ADP-ribosylation in membranes from COS-7 cells cotransfected with D1R (Bmax, 6.4 pmol/mg) and beta 2AR-Gs (10.2 pmol/mg). Histograms are the radioactivity incorporated into endogenous Gs (Gs band) or the fusion protein (beta 2AR-Gs band). bas, no ligand; skf, 100 nM SKF 82958; iso, 1 µM isoproterenol; skf+iso, both ligands. Data are representative of two independent experiments. b, stimulation of [35S]GTPgamma S binding in the presence of 3 µM GDP determined in COS-7 membranes individually transfected with D1R or cotransfected with either D1R and Galpha s (D1R + Gs) or D1R and beta 2AR-Gs. Agonists are indicated as in a. Data are means (± S.E.) of three experiments.

For the Gs system, we used two experimental schemes, where in both cases one fusion protein, beta 2AR-Gs, was co-expressed with a second Gs-competent receptor. Vasopressin V2 receptor (VP2R) was used in one protocol and D1R in the other. As shown in Fig. 7B vasopressin enhanced in a concentration-dependent manner cholera toxin-catalyzed labeling of the fusion protein band in membranes expressing V2R and beta 2AR-Gs, with an effect non-additive to that of isoproterenol. Similarly, in membranes co-expressing D1R and beta 2AR-Gs, both isoproterenol and the dopamine selective agonist SKF-82958 increased ADP-ribosylation of endogenous and beta 2-adrenergic receptor-tethered Gs (Fig. 7C, a), and produced non-additive enhancements in GTPgamma S/GDP binding exchange (Fig. 7C, b).

Cross-interaction between Receptor and Galpha Linked to Distinct Fusion Proteins-- Finally, we evaluated the existence of interactions between the receptor unit of one fusion protein and the Galpha unit of the other. First, we examined a couple of fusion proteins in which the receptor of one and the alpha -subunit of the other carried inactivating mutations. An inactive OP1R mutant (Arg146 in the highly conserved DRY motif replaced with Glu), was fused to wild-type Galpha o (OP1R[R146E]-Galpha o). The matching G protein-defective counterpart was made by tethering intact OP1R to a Galpha o subunit that was inactive because of the substitution of Cys350 by Arg (OP1R-Galpha o[C350R]) (24). Since, as shown before, a large portion of receptor-tethered Galpha o can survive ADP-ribosylation in vivo, cells cotransfected with these two constructs were exposed to pertussis toxin to eliminate endogenous Gi response. As shown in Fig. 8A, neither the receptor-defective nor the Galpha -defective fusion proteins displayed agonist-mediated enhancement of GTPgamma S binding when they were individually expressed in membranes of pertussis toxin-treated cells. However, co-expression of the two proteins restored efficient agonist effect, indicating that the intact receptor of one protein can easily interact with the functional Galpha of the other.


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Fig. 8.   Interaction of non-fused receptors with alpha -subunits fused to a different GPCR. A, HEK-293 were individually transfected or cotransfected (as indicated) with two mutants of the opioid receptor-Go fusion protein: OP1R[R146E]-Galpha o (carrying an inactive opioid receptor) and OP1R-Go[C350I] (carrying an inactive Galpha o). Control cells were transfected with empty plasmid (CTR) or wild-type opioid receptor (OP1R). Cells were treated with pertussis toxin, and the stimulation of [35S]GTPgamma S binding in the presence of agonist (DDL, 1 µM)or negative antagonist (ICI, 1 µM) was studied in the presence of GDP 3 µM. Data are means (± S.E.) of three experiments. B, binding of [35S]GTPgamma S in the presence of increasing concentrations of GDP in membranes prepared from HEK-293 cells individually transfected with a fusion protein between delta opioid receptor and a constitutive active mutant of alpha o (OP1R-Galpha o[Q205L]) (a), a fusion protein between nociceptin receptor and the pertussis-resistant mutant of alpha o (OP4R-Galpha o[C350I]) (b), or cotransfected with both fusion proteins (c). Cells were exposed to pertussis toxin (10 ng/ml, 18 h) before membrane preparation. Binding is shown in basal conditions (Bas), and in the presence of agonists, DDL or nociceptin (NOC), 1 µM both. Bmax (pmol/mg) of expressed receptors were 31 for OP1R-Galpha o[Q205L], and 8.1 for OP4R-Galpha o[C350I], when individually expressed, and 22 and 3.2, respectively, when co-expressed. Data are from one experiment, which was replicated two additional times by measuring GTPgamma S binding at 3 µM GDP. C, control HEK-293 cells (left) and a stable HEK-293 clone (right) expressing a fusion protein between delta -opioid receptor and the XTP-shifted alpha o mutant (OP1R-Galpha oX) were transfected with increasing (1.25-40 µg/flask) concentrations of cDNA encoding OP4R-Galpha o[C350I]. cDNA was mixed with non-coding plasmid to maintain the total amount constant. Cells were exposed to pertussis toxin before membrane preparation. Agonist stimulation of [35S]GTPgamma S binding (DDL or Noc, both 1 µM) was determined in the presence of 3 µM GDP using 2 µg of membrane proteins. The levels of the resident OP1R-Galpha oX and the transfected OP4R-Galpha o were measured by radioligand binding assays ("Experimental Procedures"). The expression of OP1R-Galpha oX (47.3 ± 6.2 pmol/mg) did not change significantly with OP4R-Galpha o[C350I] cDNA transfection, but the latter produced less expression in the permanent clone than in control cells (note the difference in axis scale between the graphs). The binding is plotted as a function of OP4R binding activity. Data are means of quadruplicate determinations of a single experiment in which control and clone cells were transfected and assayed simultaneously. The experiment was repeated two additional times using different protocols. In one, both fusion proteins were transiently transfected in COS-7 cells, whereas in the other increasing amounts of OP1R-Galpha oX cDNA were transiently transfected in a HEK293 clone permanently expressing OP4R-Galpha o[C350I]. Similar results were obtained in all cases.

Next, we investigated cross-interactions between a toxin-resistant version of Galpha o-fused nociceptin receptor (OP4R-Galpha o[C350I]) and a series of constructs consisting of OP1R linked to GTPase-defective mutants of alpha o. In some experiments OP4R-alpha o was co-expressed with OP1R-fused to the constitutively active Galpha o[Q205L] mutant (25). In pertussis toxin-treated membranes expressing only OP1R-Galpha o[Q205L] there was no DADLE-mediated enhancement of GTPgamma S (Fig. 8B), presumably because the mutation abolishes any change of GDP affinity in response to receptor activation. However, in membranes cotransfected with the two fusion proteins, both DADLE and nociceptin enhanced GTPgamma S binding, indicating that receptors from both fusion proteins can interact with the OP4R-tethered Galpha . There was no nonspecific stimulation of OP4 receptors by DADLE, as verified using cells expressing only OP4R-Galpha o[C350I] (Fig. 8B). Identical results (data not shown) were observed when the nociceptin receptor-Go construct was cotransfected with OP1R tethered to Galpha o[D273N], a mutant with reduced guanine nucleotide affinity (26). In still other experiments, the cross-interaction was monitored using OP1R joined to Galpha oX (the xanthine nucleotide-shifted mutant of alpha o resulting from the association the two mutations above) (27). When increasing concentrations of cDNA coding for OP4R-Galpha o[C350I] were transfected in a HEK-293 clone permanently expressing OP1R-Galpha oX, both DADLE and nociceptin-mediated enhancements of nucleotide exchange in toxin-treated membranes were increased in parallel (Fig. 8C), demonstrating that the alpha o-subunit of the co-expressed fusion protein can be activated by both tethered receptors.

A strategy that required no functional modifications of either the receptor or the alpha -subunit of the fusion construct was also used. Differentially amino-terminal-tagged fusion proteins consisting of FLAG-beta 2AR-Gs and dopamine D1R-Gs (carrying either a HA or a poly(His)6 sequence) were co-expressed. The activation of Gs (studied as agonist-enhanced ADP-ribosylation catalyzed by cholera toxin) was individually estimated for each fusion protein band by gel electrophoresis after solubilization and a selective pull-down procedure with affinity gels. To suppress cross-precipitation possibly due to receptor oligomerization or other protein-protein interactions, membranes were solubilized under strong denaturating conditions (0.5% SDS, 10 mM DTT at 95 °C) prior to affinity precipitation. As shown in Fig. 9A for co-expressed FLAG-beta 2AR-Gs and HA-D1R-Gs, the enhancement of ADP-ribose incorporation into the beta 2AR-Gs band immunoprecipitated by M1 antibody was stimulated by both isoproterenol and SKF-82958. Likewise, both agonists were capable of enhancing the ADP-ribosylation of D1R-Gs, which was selectively recovered by the anti-HA agarose. Similar results were obtained using membranes co-expressing FLAG-beta 2AR-Gs with (His6)-D1R-Gs, which was affinity-precipitated by nickel-agarose (Fig. 9A). Western blots of the M1-immunoprecipitate with anti-HA antibody or of the HA-immunoprecipitate with M1 antibody did not show any detectable cross-contamination (not shown). Although quantification of the labeled bands suggests some degree of preference (Fig. 9B), the data clearly indicate that each receptor can interact and activate both the homologous and the heterologous tethered Gs.


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Fig. 9.   Cross-activation of alpha -subunits from receptor tethered to distinct fusion proteins. A, ligand-induced enhancement of cholera toxin ADP-ribosylation by agonists of two different GPCR-Gs fusion proteins co-expressed in the same cells. Membranes were prepared from COS-7 cells transfected with pairs of cDNA (1:1 mix) encoding Gs-fused receptors tagged with different epitopes: FLAG-beta 2AR-Gs and HA-D1R-Gs (left), or FLAG-beta 2AR-Gs and His-D1R-Gs (right). Membranes were [32P]ADP-ribosylated by cholera toxin A-protomer in the absence (Bas) and presence of the indicated agonists, Iso, 1 µM; Skf, 100 nM; and equal aliquots (100 µg) were affinity-precipitated under denaturating conditions with the appropriate agarose-linked probe (anti-FLAG mouse monoclonal M1, anti-HA rat monoclonal, and nickel-NTA) to selectively recover each labeled fusion protein (see "Experimental Procedures"). B, radioactivity of the bands of immunoprecipitated fusion proteins (Instantimager) is averaged after normalization (ratios of incorporated cpm in the presence versus absence of agonist). Shown are means (± range) of four experiments for M1 precipitates and two experiments for nickel.

Effect of Polyanionic Inhibitors of Receptor-G Protein Coupling-- Molecules carrying rigidly spaced sulfate groups, such as heparin, suramin, or dextran sulfate, are potent inhibitors of receptor-G protein interaction (38-40). The mechanism has not been elucidated yet, but it is conceivably the result of electrostatic interactions between the charged polymer and polar residues (41) contributing to the binding of GPCR to alpha -subunit. We reasoned that if the fusion process converts the reaction between receptor and alpha -subunit from intermolecular to intramolecular, steric hindrance should reduce the efficacy of a very high molecular weight inhibitor. Thus, the potency of 500 kDa dextran sulfate to block G protein activation may significantly differ when receptor and Galpha interact as separated proteins or as partners of a fused construct.

In the case of Gs, the effect of dextran sulfate was examined using two different systems: in one, beta 2AR-Gs was confronted with co-expressed beta 2AR and OP1R-Gs, (Fig. 10A); in the other, D1R-Gs was compared with cotransfected D1R and beta 2AR-Gs (Fig. 10B). In the case of Go, membranes expressing either OP1R-Galpha o or a mixture of OP1R and V2TM1-Galpha o were examined (Fig. 10C). In all the experiments dextran sulfate inhibited agonist-induced nucleotide exchange at fused and non-fused interactions with virtually identical IC50 (Fig. 10, A-C). At the range of concentrations used the inhibitor did not affect the binding of GTPgamma S to recombinant Galpha o or to a Gi/o mixture purified from bovine brain (Fig. 10D), nor did it change GDP affinity (Fig. 10E), indicating that the block of agonist-enhanced nucleotide binding observed in transfected membranes does indeed reflect disruption of receptor-Galpha interactions. These data indicate that the surface of interaction between a GPCR and an alpha -subunit is equally accessible to bulky inhibitors either when they react as part of the same protein or as distinct molecular entities.


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Fig. 10.   Inhibitory effect of dextran sulfate at receptor and Galpha reacting as fused partners or as separated proteins. Concentration-response curves for the inhibition by 500-kDa dextran sulfate (DS) of agonist-mediated enhancement of GTPgamma S binding in the presence of GDP (3 µM). Each panel (A-C) shows a comparison between fused and non-fused interactions, using membranes prepared from cells, either individually transfected with a GPCR-Galpha fusion proteins or cotransfected with the same proteins in non-fused condition. Data were fitted (lines) to a 4-parameter logistic function (30) to compute the IC50 (± S.E.). A, HEK-293 cells expressing beta 2AR-Gs (IC50, 233 ± 35 pM) or beta 2AR together with OP1R-Gs (IC50, 328 ± 48 pM). B, COS-7 cells expressing D1R-Gs (IC50, 214 ± 13 pM) or a combination of D1R and beta 2AR-Gs (IC50, 113 ± 19 pM). C, HEK-293 cells (treated with pertussis toxin before membrane preparation) expressing OP1R-Go[C350I] (IC50, 204 ± 5 pM) or the combination of OP1R and V2TM-Go[C350I] (IC50, 305 ± 18 pM). Data points (means of triplicate determinations) are net agonist-stimulated binding (after subtraction of the binding measured in the absence of ligand at each dextran sulfate concentration) and are representative of experiments that were replicated at least twice with identical results. Panels D and E, lack of effect of DS on the nucleotide-binding site of purified G proteins. D, binding of [35S]GTPgamma S (1 nM) to myristoylated alpha o (recombinant, 1 nM) or Gi/o heterotrimers (from bovine brain, 100 ng/tube) at the indicated concentrations of DS. E, binding of [35S]GTPgamma S to Gi/o at the indicated concentrations of GDP in the absence or presence of 100 nM DS.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We show in this study that the fusion between GPCR and alpha -subunit, although ensuring their expression as tethered units in the plasma membrane, does not force their interaction to the 1:1 stoichiometry that molecular proximity would imply. This conclusion is based on a variety of diverse experimental evidences. (a) We found that agonists acting through a fused GPCR can activate with identical EC50 both fused and endogenous alpha -subunits, which suggests that the receptor of the fusion protein can bind to either alpha -subunit with equal efficiency. (b) The characteristic enhancement of GTPgamma S stimulation observed in fusion proteins could be mimicked when a non-fused GPCR and a alpha -subunit tethered to a single transmembrane polypeptide are co-expressed in the membrane, which indicates that this enhanced stimulation does not require molecular proximity but simply that the alpha -subunit be inserted in the membrane via a transmembrane handle. (c) Similarly, the GPCR-tethered alpha -subunit of a fusion protein can be activated by a second non-tethered GPCR receptor cotransfected in the same membrane, and the effects are not additive when both agonists at saturating concentrations are present. This means that tethered and not GPCR can compete for the same receptor-linked alpha -subunit. (d) Moreover, we found extensive cross-reactions between the GPCR of one fusion protein and the alpha -subunit of another, when cotransfected in the same membrane. This cross-reaction was observed regardless of whether in one of the cotransfected pair of fusion proteins the alpha -subunit was made inactive or constitutively active. It was also verified by individually measuring Galpha activation in two Gs-attached co-expressed receptors. (e) Finally, the fact that a bulky polyanionic inhibitor blocks with identical potency receptor and G protein reacting as fused or as separate partners is additional evidence that the interacting surfaces are equally accessible in both cases.

Taken collectively, these data provide compelling evidence that when GPCR and the alpha -subunit are linked into one protein, interactions can occur with equal chance within the same or between separate macromolecules. Therefore, the improved "coupling efficiency" generally observed for fusion proteins (1-4) does not result from the tethered status of the reactants. These results are surprising and suggest two equally possible but radically different interpretations.

One interpretation comes from the same intuitive argument that leads us to assume 1:1 stoichiometry of interaction for the fused construct: the reaction between receptor and Galpha linked by a covalent bond should be thermodynamically favored over that between unattached molecules. Since it is not so, it means that when GPCR and the alpha -subunit are part of the same polypeptide backbone their binding surfaces cannot easily make contact. Hence, binding to partners belonging to adjacent molecules is just as possible or even predominant, and the interactions between fused and non-fused species appear equally probable simply because they are intermolecular in both cases. While this a rational explanation, the reason why the fused status would exert hindrance to intramolecular but not to intermolecular contacts is, however, not obvious.

A trivial consideration is that the peptide bond between the alpha -subunit and the receptor carboxyl terminus may significantly reduce the degree of freedom of Galpha , compared with its native lipid-tethered status. This may impose enough rotational constraints to make the proper alignment of the electronegative binding surfaces of Galpha with the footprint of its tethered GPCR difficult, without however limiting its orientation with respect to a neighboring GPCR molecule. As long as orientation is a crucial factor for receptor-Galpha interaction, this is a sound explanation. Alternatively, it may be the binding to beta gamma that makes the interactions within the fused construct unfavorable. We have verified, in agreement with others (1, 5, 7, 12), that GPCR-tethered alpha -subunits can interact with beta gamma , and do so just as effectively as purified alpha -subunits, when judged by ADP-ribosylation. There is ample evidence that the binding surface of the G protein for the receptor comprises residues from both alpha - and beta gamma -subunits (42-46). It is possible that the formation of the heterotrimer with membrane-attached beta gamma attracts the tethered alpha -subunit away from its intramolecular GPCR and facilitates the interaction with that of a nearby molecules, thus largely offsetting the entropic advantage of their tied condition. Unfortunately, we have not been able to design an experiment capable of distinguishing between these two possibilities, although a fusion protein that includes a GPCR and both beta - and alpha -subunits, if still signaling-competent, might help.

To question the occurrence of intramolecular coupling in fusion proteins, Small et al. (12) reported that the signaling properties of beta 2AR-Gs expressed at physiological levels in cells were not consistent with a significant shift of the intrinsic equilibrium of the receptor toward the active form. Yet, if the covalently linked alpha -subunit could interact with the tethered GPCR, that propensity should induce a high degree of constitutive activation (12).

A second possible interpretation of the results is that the supramolecular architecture that organizes the interaction between receptors and G proteins in the membrane (47) plays such a dominant role in driving their reaction, that the effect of a covalent linkage between interacting partners is irrelevant. A variety of diverse experimental evidences suggests that the interaction between receptor and G proteins occurs between preassembled units rather than between random encounters floating long distances within the plane of the lipid bilayer (47, 48). It is not known yet what mechanism ensures efficient "precoupling" of receptor and G proteins in the membrane. However, data suggesting the formation of stable oligomeric forms for either Galpha subunits (48) and GPCR (49, 50), including opioid (51) and beta 2-adrenergic (52) receptors, have been reported. This process may represent a primary mechanism of concentrating interactions for such signaling macromolecules. If receptors and alpha -subunits interact normally as dimeric or higher order oligomeric forms, and if the assembly of oligomers is not disturbed by the covalent linkage between GPCR and alpha -subunit, the spatial proximity that such an arrangement can generate may render the effect of fusion virtually unnoticeable.

Apart from what inhibits exclusive interactions between the tethered members of RG fusion proteins, an additional question concerns the enhancement of signaling efficiency generally observed with such constructs. If there is no entropic gain in the interaction of covalently linked receptor and alpha -subunit why do they appear to react more efficaciously than the cotransfected native molecules?

We found that the cotransfection of non-fused receptor with alpha o tethered to a transmembrane polypeptide (but not with native alpha o) can reproduce the enhancement of agonist-induced nucleotide exchange observed when the same GPCR and alpha -subunit are expressed as a fusion protein. Similar observations were made following the cotransfection of beta 2AR or D1R with a single transmembrane domain-tethered form of Gs (53). Therefore, it is the alpha -helical anchored status of the alpha -subunit, not fusion to the receptor, which causes the increase in receptor coupling. Yet, the reason for this enhancement remains unclear.

Restricted membrane mobility, enhanced hydrophobic interactions with receptor transmembrane domains, improved cytoskeletal interactions (53), or prevention of Galpha release from the membrane (2, 7, 53) are all possible mechanisms that have been proposed to explain the enhanced coupling efficiency of transmembrane-tethered alpha -subunits. Here we offer an additional suggestion. Unlike native G proteins, either V2TM-alpha o (22) and the OP1R and beta 2AR-tethered alpha -subunits used in this study2 were found excluded from low density Triton-resistant vesicles, representing cholesterol-rich microdomains of the plasma membrane (54). Moreover, it was shown that a docked GPCR provides the sole means of membrane insertion for the otherwise cytosolic acylation-defective mutants of alpha -subunits (55, 17). Thus, an interesting possibility is that the transmembrane anchor of the alpha -subunits, by targeting such molecules to the plasma membrane outside of the caveolin-rich rafts where they are naturally located, can shield them from a constitutive inhibitory influence and favor receptor interactions.

    ACKNOWLEDGEMENTS

We thank Dr. Marco Parenti (University of Milan, Bicocca) for kindly supplying the transmembrane tethered Go and to Dr. Patrick J. Casey (Duke University) for the generous gift of purified beta gamma -subunits.

    FOOTNOTES

* This work was supported in part by Telethon Grant D129 and the EU BIOMED 2 Programme.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.

Dagger To whom correspondence should be addressed. Tel.: 39-06-49902386; Fax: 39-06-49387104; E-mail: tomcosta@iss.it.

Published, JBC Papers in Press, February 21, 2003, DOI 10.1074/jbc.M300731200

2 M. C. Gro and T. Costa, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; AR, adrenergic receptor; OP1R and OP4R, opioid receptors type 1 and 4, also known as delta and nociceptin receptors, respectively; V2R, vasopressin 2 receptor; D1R, dopamine 1 receptor; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; NTA, nitrilotriacetic acid; HEK, human embryonic kidney cell; CHO, chinese hamster ovary; DTT, dithiothreitol; RIA, radioimmunoassay; WGA, wheat germ agglutinin; DDL, [D-Ala2,D-Leu5]enkephalin; HA, hemagglutinin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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