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Published as doi: 10.1096/fj.07-100388.
 (The FASEB Journal. 2008;22:1924-1932.)
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Antibodies that identify only the active conformation of Gi family G protein {alpha} subunits

J. Robert Lane*, David Henderson*, Ben Powney{dagger}, Alan Wise{dagger}, Stephen Rees{dagger}, Dion Daniels{dagger}, Chris Plumpton{ddagger}, Ian Kinghorn{ddagger} and Graeme Milligan*,1

* Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK;

{dagger} Screening and Compound Profiling, GlaxoSmithKline Research and Development, New Frontiers Science Park, Harlow, Essex, UK; and

{ddagger} Biological Reagents and Assay Development, GlaxoSmithKline Research and Development, Medicines Research Centre, Stevenage, Herts, UK

1Correspondence: Davidson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK. E-mail: g.milligan{at}bio.gla.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Production of antisera able to recognize individual heterotrimeric G protein {alpha} subunits resulted in rapid expansion of information on their distribution and function. However, no antibodies that specifically recognize the active state have been available. Four-way primary screening of 763 hybridomas generated from mice immunized with guanosine 5'-O-(3-thio)triphosphate-loaded G{alpha}i1 and isolated using an automated robotic colony picker identified three antibodies that interacted with the constitutively active, Q204L, mutant but neither the constitutively inactive, G203A, mutant nor wild-type G{alpha}i1. This profile extended to other closely related Gi family G proteins but not to the less closely related G{alpha}s and G{alpha}q/G{alpha}11 families. Each antibody was, however, also able to identify wild-type, GDP-bound Gi family G proteins in the presence of fluoroaluminate, which mimics the presence of the terminal phosphate of GTP and hence generates an active/transition state conformation. Stimulation of cells coexpressing a wild-type G{alpha}i subunit and the dopamine D2 receptor with the agonist ligand nor-apomorphine also allowed these conformationally selective antibodies to bind the G protein. Such reagents allow the specific identification of activated G proteins in a native environment and may allow the development of label-free screening assays for G protein-coupled receptor-mediated activation of Gi family G proteins.—Lane, J. R., Henderson, D., Powney, B., Wise, A., Rees, S., Daniels, D., Plumpton, C., Kinghorn, I., Milligan, G. Antibodies that identify only the active conformation of Gi family G protein {alpha} subunits.


Key Words: G protein-coupled receptor • ligand screening


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
G PROTEIN-COUPLED RECEPTORS (GPCRS) are the most tractable class of protein targets for the design and development of small molecule therapeutic medicines, and it has been estimated that some 30–50% of clinically available drugs target the function of GPCR family members (1 , 2) . Although certain signals initiated by GPCRs appear not to require a contribution of one or more members of the family of heterotrimeric guanine nucleotide-binding proteins (G proteins) (3) , GPCRs remain largely defined by their capacity to activate G proteins (4) . This activation process involves the release of GDP from a G protein {alpha} subunit and its replacement by GTP. Subsequently, either conformational rearrangements or physical separation of the GTP-bound {alpha} subunit from the β/{gamma} subunit complex (5 , 6) allows both of these elements to modulate the activity of either downstream effector enzymes that synthesize secondary messengers or various ion channels. The subsequent hydrolysis of the terminal phosphate of bound GTP by the GTPase activity that is intrinsic to the G protein {alpha} subunit acts to terminate these processes and functions as a kinetic restraint on signal generation (4) . Because all G proteins undergo this cycle of guanine nucleotide exchange and GTP hydrolysis, biochemical approaches have been used in a vast number of studies to measure one or more steps in the cycle as a marker of G protein activation (4) . However, both in efforts to identify activated G proteins in a cellular setting and in the context of ligand screening in the pharmaceutical and biotechnology industries, such assays present major challenges. First, although the greatest amount of information can be gathered from measures of ligand regulation of high-affinity GTPase activity, because they require the use of radiolabeled or fluorescent forms of GTP and are not easily adapted to a homogenous format (7 , 8) , such enzyme assays have not found favor outside academic laboratories. Second, although assays based on the binding of the poorly hydrolyzed analog of GTP, guanosine 5'-O-(3-thio)triphosphate ([35S]GTP{gamma}S), have been used widely (9 10 11) , the desire to move away from the use of radioactivity and to develop "label-free" assays is strong (12) . Fluorescence resonance energy transfer (FRET) sensors of G protein activation have also been developed recently. These have provided a great deal of detailed information on the kinetics of G protein activation and on conformational movements between the subunits of heterotrimeric G proteins (5 , 6) . However, there are concerns as to how the introduction of such FRET sensors might alter the pharmacology of GPCR-mediated G protein activation (5 , 6) because it is becoming clear that the use of promiscuous and/or chimeric G proteins, which are currently widely used in ligand screening campaigns to channel receptor responses to a single, defined end point may modify, or even mask, the pharmacology of certain ligands (13) . With these issues in mind we initiated a program to develop antibodies capable of identifying only the activated states of native G protein {alpha} subunits and concentrated on the Gi family of G proteins.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Myc-dopamine D2 receptor-enhanced yellow fluorescent protein (eYFP) fusion protein
Primers encoding the Myc epitope sequence were used to generate N-terminally tagged Myc-dopamine D2 receptor and remove the stop codon: sense, 5'-ACA GAC CCA AGC TTA TGG AAC AAA AAC TTA TTT CTG AAG AAG ATC TGG ATC CAC TGA ATC TGT CCT GG-3'; antisense, 5' CGG GGT ACC GCA GTG GAG GAT CTT CAG GAA 3'. [Underlined bases indicate introduced restriction sites (sense, HindIII; antisense, KpnI), and bases in bold indicate an introduced N-terminal Myc tag.] The polymerase chain reaction (PCR) fragment was digested using HindIII and KpnI. eYFP cDNA was obtained from Clontech Laboratories (Palo Alto, CA, USA). Primers were used to amplify eYFP cDNA with the desired 5' and 3' restriction sites: sense, 5' CGG GGT ACC ATG GTG AGC AAG GGC GAG GAG 3'; antisense, 5' TTT TCC TTT TGC GGC CGC TTA CTC GAT GTT GTG GCG GAT 3'. [Underlined bases indicate introduced restriction sites (sense, KpnI; antisense, NotI).] The PCR fragment was digested using KpnI and NotI. pcDNA3 was digested using the restriction endonucleases HindIII and NotI, and the Myc-dopamine D2 receptor and eYFP PCR fragments were ligated into the sites.

G protein {alpha} subunit constructs
Human wild-type G{alpha}i1, G{alpha}i1 Q204L, wild-type G{alpha}i2, wild-type G{alpha}o1, G{alpha}i2 Q205L, and G{alpha}o1 Q204L cDNA in the plasmid pcDNA3.1 were obtained from UMR cDNA Resource Center (University of Missouri–Rolla, Rolla, MO, USA). To change G203A and give a constitutively inactive human G{alpha}i1 mutant, cDNA site-directed mutagenesis was performed using the following primers and the wild-type human G{alpha}i1 in pcDNA3.1 as a template: sense, 5' CAT TTT AAA ATG TTT GAT GTG GGA GCT CAG AGA TCT CGG 3'; antisense, 5' CCG AGA TCT CTG AGC TCC CAC ATC AAA CAT TTT AAA ATG. (Bases in bold indicate changed bases.)

Expression of G{alpha}i family G proteins in Escherichia coli
cDNAs for all four rat G{alpha}i family members (G{alpha}i1, G{alpha}i2, G{alpha}i3, and G{alpha}o1) were subcloned into the prokaryotic expression vector pT7.7 and then transformed into E. coli strain BL21 DE3. Cultures of transformed bacteria were induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and incubated at 30°C for 4 h. Cells were then harvested by centrifugation for 30 min at 3220 g, supernatant was discarded, and pellets were frozen at –80°C. Cell pellets were thawed and resuspended in 20 ml of STE buffer (10 mM Tris-HCl, 150 mM NaCl, and 1 mM EDTA, pH 8.0). Cells were lysed with two passes through a French press set at 950 psi. Cell lysates were cleared by centrifugation at 10,000 g for 15 min, and the supernatant was removed and kept on ice. The protein concentration was determined using a bicinchoninic acid assay, and lysates were stored in 1-ml aliquots at –80°C.

Immunization—repetitive immunization at multiple sites (RIMMS)
A myristoylated preparation of recombinant rat G{alpha}i1 subunit (25 µg) was purchased from Calbiochem (Merck KGaA, Darmstadt, Germany). This was incubated with 100 µM GTP{gamma}S in a buffer containing 100 mM NaCl, 20 mM HEPES, 3 mM MgCl2, 1 mM EDTA, and 200 nM GDP (pH 8.0) for 30 min at room temperature. RIMMS was performed using a procedure simplified from that of Bynum et al. (14) . Over a period of 11 days, injections of 10 µg of protein (recombinant G{alpha}i1+10 µM GTP{gamma}S) emulsified in complete Freund’s adjuvant and RIBI adjuvants (Sigma-Aldrich, St. Louis, MO, USA) were given at six subcutaneous sites proximal to draining lymph nodes in two anesthetized female SJL mice (Harlan, Bicester, UK). After 6 and 11 days, mice were immunized with 5 µg of protein in RIBI adjuvant at four sites. Three days after the final boosts, a cell suspension harvested from popliteal, superficial inguinal, axillary, and brachial lymph nodes was prepared.

Cell fusion
Lymph node cells were centrifuged with myeloma cells derived from the P3 x 63/Ag8.653 cell line(15) and fused using polyethylene glycol 1500 (Roche Applied Science, Indianapolis, IN, USA). After the fusion procedure the cell pellet was resuspended in JRH610 medium containing 10% hybridoma cloning factor (Sigma-Aldrich), 10% fetal calf serum (Hyclone Laboratories, Logan, UT, USA), penicillin/streptomycin, 1x hypoxanthine, aminopterin, and thymidine (HAT) (Invitrogen, Carlsbad, CA, USA), and 1.35% methylcellulose (Sigma-Aldrich). The fusion suspension was distributed between five Nunc OmniTrays and four Genetix PetriWell plates for selection and cloning in a single step. After 10–11 days of culture, colonies were imaged and picked using an automated robotic colony picker (ClonePix FL; Genetix, New Milton, Hampshire, UK). Individual monoclonal hybridoma colonies were deposited into Greiner 96-well plates containing 200 µl/well of the above medium without methylcellulose and 1x hypoxanthine and thymidine (HT) in place of HAT.

Cell culture and transfection for the generation of material for the fluorometric microvolume assay technology (FMAT) screening assay
HEK293T cells were grown to 80–90% confluence in 225 cm2 tissue culture flasks in Dulbecco’s modified Eagle’s medium + 10% dialyzed fetal calf serum (25 ml of tissue culture media/flask). Transfections were performed using an in-house transfection reagent, Gemini (GSC103 DOPE; GlaxoSmithKline, Stevenage, UK). Transfected cells were left at 37°C in a humidified atmosphere of air/CO2 (19:1) for 48 h before cell harvest. Cells were then washed once with 50 ml of PBS/flask and were harvested in 10 ml of PBS dissociation buffer/flask. Cells were pelleted by centrifugation at 450 g. Supernatant was discarded, and the resultant cell pellet was resuspended in 10% dimethyl sulfoxide/FBS and stored at –80°C in 1-ml aliquots until required.

Hybridoma screening
Spent medium from the hybridomas was assayed for appropriate activity after 3–4 days growth using FMAT (ABI 8200 cellular detection system; Applied Biosystems, Foster City, CA, USA). Each hybridoma well was tested in 4 wells of a 384-well FMAT assay plate (Applied Biosystems). Hybridoma supernatants (5 µl) were mixed with either a control HEK293T cell transfection or HEK293T cells transfected with wild-type G{alpha}i1 + 100 µM GTP{gamma}S, G{alpha}i1 Q204L, or wild-type G{alpha}i1 + 100 µM guanosine 5'-O-(2-thio)diphosphate (GDPβS) (5000 cells/well) suspended in PBS (Sigma-Aldrich) containing 1% BSA (Sigma-Aldrich), 0.1% sodium azide, and 0.2 µg/ml FMAT Blue (Applied Biosystems) -labeled donkey anti-mouse F(Ab')2 (Jackson ImmunoResearch Laboratories, Stratech, Newmarket, UK). All manipulations were performed using an automated liquid handling robot based on a Beckman BiomekFX (Beckman Coulter, Fullerton, CA, USA). Plates were read after at least 2 h of incubation at room temperature.

Hybridoma expansion and immunoglobulin purification
Hybridomas from wells of interest were expanded into 24-well plates (Greiner, Stonehouse, Gloustershire, UK), cryopreserved, and subcloned in semisolid medium to ensure stability. To generate purified monoclonal antibodies (mAbs), hybridomas were expanded further into 2 x T225 cm2 flasks in the above medium without hybridoma cloning factor and HT and cells were pelleted, resuspended in medium without serum, and cultured until cell viability dropped to ~10%. Immunoglobulins were purified from the conditioned medium using immobilized protein A (ProSep-A) and the Perbio buffer system (Perbio Science UK Ltd., Cramlington, Northumberland, UK) according to the manufacturer’s instructions. Purified mAbs were dialyzed against and stored in PBS (Sigma-Aldrich). Isotypes were determined using a typing stick kit from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). All mAbs were IgG1{kappa}.

Immunocytochemistry
Cells were cultured and transfected as described above. Cells were washed one time with room temperature PBS and then incubated with Hoescht stain (1:1000 dilution in PBS) for 15 min at 37°C in the incubator. Fixation of cells was performed by incubation for 10 min with 10% formaldehyde in PBS solution at room temperature for exactly 10 min. Cells were then washed 3x with ice-cold PBS. Blocking and cell permeabilization was achieved in one step with a 10-min incubation in PBS + 3% nonfat milk + 0.15% Triton X-100. Primary antibodies were diluted in PBS-3% nonfat milk-0.15% Triton X-100, and coverslips incubated for 1 h at room temperature. Coverslips were then washed 3x with ice-cold PBS as before. Cells were incubated in secondary antibody solution (PBS+3% nonfat milk+0.15% Triton X-100) as follows: {alpha}-mouse Alexa 594, 1:400; {alpha}-rabbit Alexa 594, 1:600. Coverslips were mounted onto slides using Immuno-Mount solution and stored at 4°C until use.

Modified immunocytochemical method: fluoroaluminate (AlF4–) -stimulated whole cell assay
Cells grown on coverslips were incubated at room temperature for 1 h with AlF4– solution [GTP{gamma}S buffer+10 mM NaF, 0.03 mM AlCl3, 3% nonfat milk (w/v), and 0.2% saponin (v/v)] plus primary antibody. Coverslips were subsequently washed with PBS and fixed using 4% formaldehyde in PBS. Cells were incubated with the relevant secondary antiserum for 1 h at room temperature [PBS, 3% nonfat milk (w/v), and 0.2% saponin (v/v)]. Finally, cells were washed with ice-cold PBS and mounted using Immuno-Mount for microscopy.

Modified immunocytochemical method: nor-apomorphine (NPA) -stimulated GTP{gamma}S whole cell assay
All agonist-stimulated assays were performed in six-well plates. Cells were washed using GTP{gamma}S buffer. Cells were then incubated in GTP{gamma}S buffer + 0.2% (v/v) saponin/3% (v/v) nonfat milk/with 10 µM GTP{gamma}S and with or without 1 µM NPA, with an appropriate concentration of primary antibody, for 1 h at room temperature. Coverslips were then washed with PBS and fixed using 4% formaldehyde in PBS. The experiment was completed as above.

Epifluorescence microscopy
Formaldehyde-fixed cells, expressing the D2 receptor-eYFP fusion protein and appropriate G protein {alpha} subunit were imaged in two dimensions using an inverted Nikon TE2000-E microscope (Nikon Instruments, Melville, NY, USA) equipped with an x60 (NA=1.4) oil immersion Plan Fluor Apochromat lens, a z-axis linear encoder, and a cooled digital Cool Snap-HQ charge-coupled device camera (RoperScientific/Photometrics, Tucson, AZ, USA) as described previously (16) . For each experiment exposure and intensity were set using the positive control condition, and these settings were then used for all other conditions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Recombinant G{alpha}i1 was loaded with the GTP analog GTP{gamma}S and used to immunize mice. Antibody-producing plasma cells from these animals were used to generate hybridomas. Individual hybridomas were selected, cloned, and tested for production of antibodies with appropriate characteristics. Initial screens were performed with an ABI 8200 cellular detection system that uses FMAT. This is a fluorescence macroconfocal binding event analyzer that enables homogeneous assays with live cells. HEK293 cells transfected to transiently express various forms of G{alpha}i1 were mixed with hybridoma supernatant and fluorescent labeled secondary antibody in 384-well plates and allowed to settle. The helium/neon laser of the system automatically focuses and scans the cells on the inner bottom surface of the wells. Only particle-associated fluorescence is measured and reported, whereas unbound antibody remains in solution and is not detected. Supernatants from 763 hybridomas were screened in a quadrant pattern against permeabilized HEK293T cells expressing 1) wild-type G{alpha}i1 to which 100 µM GTP{gamma}S was added, 2) wild-type G{alpha}i1 to which 100 µM of the GDP analog GDPβS was added, 3) the constitutively active, Q204L G{alpha}i1 mutant that inhibits intrinsic GTPase activity and hence mimics the active state, or 4) mock-transfected HEK293 cells. Three hybridomas, designated 8A5, 8D11, and 6F12, displayed marked selectivity for samples 1 and 3 (Fig. 1 A). In a second set of preliminary experiments, each of these three antibodies failed to interact significantly with the constitutively inactive G203A G{alpha}i1 mutant that prevents the protein from being activated by GTP and hence is unable to adopt the active state. A number of hybridoma clones (29 in total), including 6B4 (Fig. 1B ) and 8F5 (not shown), displayed good G protein interaction but did not discriminate between active and inactive conformations of G{alpha}i1 (Fig. 1B ), whereas a series of other hybridomas, for example, 2B1, displayed little ability to detect G{alpha}i1 (Fig. 1B ).


Figure 1
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  		Figure 1. FMAT screen of 763 hybridoma clones. Hybridoma supernatants were screened in an FMAT assay using the following four conditions: 1) HEK293T cells transfected with wild-type (WT) G{alpha}i1 cDNA, permeabilized in PBS + 1% BSA + 0.1% NaN3 (FMAT buffer), and incubated with 100 µM GTP{gamma}S. 2) HEK293T cells transfected with wild-type G{alpha}i1 cDNA, permeabilized in FMAT buffer, and incubated with 100 µM GDPβS. 3) HEK293T cells transfected with constitutively active G{alpha}i1 Q204L cDNA, permeabilized in FMAT buffer. 4) HEK293T cells transfected with empty pcDNA3, permeabilized in FMAT buffer. A) This screen of 763 hybridoma clones revealed 3 showing selectivity toward conditions in which G{alpha}i1 was expressed and in the active (GTP-bound) conformation. These were 8A5, 8D11, and 6F12. B) A number of hybridoma clones, for example. 6B4, displayed good G protein interaction but did not discriminate between active and inactive conformations of G{alpha}i1, whereas a series of other hybridomas, for example, 2B1, displayed little ability to detect G{alpha}i1 selectively.

In Western blots on membranes of E. coli transformed to express the {alpha} subunits of each of the pertussis toxin-sensitive Gi family G proteins G{alpha}i1, G{alpha}i2, G{alpha}i3, and G{alpha}o1, a series of the nonconformationally selective antibodies, including both 8F5 and 6B4, identified only G{alpha}i1, whereas a rabbit polyclonal antipeptide antiserum (1319) raised against the C-terminal decapeptide of transducin {alpha} identified each of these G protein subunits to varying degrees (Fig. 2 ). In contrast and as anticipated for potentially conformationally selective antibodies 6F12, 8A5, and 8D11 (Fig. 2) were unable to identify the denatured G protein {alpha} subunits.


Figure 2
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  		Figure 2. Conformationally selective antibodies 8A5, 6F12, and 8D11 do not recognize members of the G{alpha}i/o G protein family denatured by SDS-PAGE. Nonconformationally selective antibodies 6B4 and 8F5 selectively recognize G{alpha}i1 over other members of the G{alpha}i/o family. Chemically competent BL21 E. coli cells were transformed with the vector pT7.7 containing the cDNA of the relevant G{alpha} subunit. Expression of G{alpha} subunit was induced with the addition of 1 µg/ml IPTG. Cell lysate proteins (0.5 µg/well) were separated by SDS-PAGE and Western blotting was performed using the putative conformationally selective antibodies 6F12, 8AD, and 8D11 or the nonconformationally selective antibodies, 6B4 and 8F5. An in-house antiserum generated against the C-terminal decapeptide of transducin {alpha} (1319), which identifies G{alpha}i1, G{alpha}i2, and G{alpha}i3, was used as a control.

To confirm the conformational selectivity of antibodies 8A5, 8D11, and 6F12 for activated G{alpha}i1, we performed a series of immunocytochemistry-based studies. HEK293 cells were transfected to express a form of the dopamine D2 receptor C-terminally tagged with eYFP (D2 receptor-eYFP), to allow visualization of positively transfected cells, in parallel with either wild-type G{alpha}i1, constitutively active Q204L G{alpha}i1, or constitutively inactive G203A G{alpha}i1. In concert with Hoechst staining to identify cell nuclei, each of the antibodies—8A5, 8D11, and 6F12—identified only cells expressing Q204L G{alpha}i1 (Fig. 3 A and data not shown). In contrast, in equivalent experiments, the nonconformationally selective antibodies 8F5 and 6B4 identified each of the following: wild-type G{alpha}i1, Q204L G{alpha}i1, and G203A G{alpha}i1 (Fig. 3B and data not shown). This was also true for the C-terminal antipeptide antiserum 1319 (see later).


Figure 3
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  		Figure 3. Immunocytochemistry using the antibody 6F12 demonstrates its selectivity toward the constitutively active variant Q204L G{alpha}i1. HEK293T cells were grown on poly-D-lysine-coated coverslips. These cells were cotransfected with cDNA of a D2 receptor-eYFP fusion protein and either wild-type G{alpha}i1 (G{alpha}i1W-T), Q204L G{alpha}i1 active variant (G{alpha}i1 Q204L), or the inactive variant G203A G{alpha}i1 (G{alpha}i1 G203A). Immunocytochemistry was performed using the conformationally selective antibody 6F12 (A) or the nonconformationally selective antibody 6B4 (B) at a final dilution of 1:200. The fluorescently labeled secondary antibody anti-mouse Alexa fluor 594 was used at a final dilution of 1:400. Fluorescence detected from i) D2 receptor-eYFP, ii) Alexa Fluor 594, and iii) Hoechst stain is shown.

G{alpha}i1 is highly related in sequence and therefore presumably in structure to G{alpha}i2, G{alpha}i3, and G{alpha}o1 but substantially less so to the other major G protein subfamilies, G{alpha}s and G{alpha}q/G{alpha}11. We next assessed the capacity of the conformationally selective antibodies to identify wild-type and constitutively active, Q to L, mutants of G{alpha}i2 and G{alpha}o1. These antibodies did not recognize wild-type G{alpha}i2 or G{alpha}o1 but did identify both Q205L G{alpha}i2 and Q204L G{alpha}o1 (Fig. 4 A, B). As controls we confirmed expression of both wild-type G{alpha}i2 and G{alpha}o1 in parallel immunocytochemistry experiments using rabbit polyclonal antipeptide antisera against the corresponding C-terminal decapeptide sequences (Fig. 4A, B ). Unlike the situation with Q205L G{alpha}i2 and Q204L G{alpha}o1, none of the conformationally selective anti-G{alpha}i1 antibodies were able to identify either equivalent constitutively active (Q227L G{alpha}s or Q209L G{alpha}q) or constitutively inactive (G226A G{alpha}s or G208A G{alpha}q) forms of G{alpha}s or G{alpha}q (Fig. 5 ), although in all cases staining with rabbit polyclonal antipeptide antisera against the corresponding C-terminal decapeptide sequences confirmed expression of the relevant G protein polypeptide (Fig. 5) .


Figure 4
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  		Figure 4. The conformationally selective antibody 6F12 shows selectivity for the constitutively active mutants Q205L G{alpha}i2 and Q204L G{alpha}o1. HEK293T cells were grown on poly-D-lysine-coated coverslips. The ability of 6F12 to recognize constitutively active variants of G{alpha}i2 (A) and G{alpha}o1 (B) was assessed using the following conditions. For G{alpha}i2, cells were cotransfected with cDNA of the D2 receptor-eYFP fusion protein and either wild-type G{alpha}i2 (G{alpha}i2 W-T), Q205L G{alpha}i2 (G{alpha}i2 Q205L), or Q204L G{alpha}i1 (G{alpha}i1 Q204L). For G{alpha}o1, cells were cotransfected with cDNA of the D2 receptor-eYFP fusion protein and either wild-type G{alpha}o1 (G{alpha}o1 W-T), Q204L G{alpha}o1 (G{alpha}o1 Q204L), or Q204L G{alpha}i1 (G{alpha}i1 Q204L). As a control HEK293T cells were cotransfected with cDNA of the D2 receptor-eYFP fusion protein and pcDNA3. The antibody 6F12 was used at a final dilution of 1:200. Anti-mouse Alexa Fluor 594 was used at a final concentration of 1:400. Fluorescence detected from i) D2 receptor-eYFP, ii) Alexa Fluor 594 bound to 6F12, and iii) Hoechst stain is shown. To demonstrate expression of transfected G{alpha} protein a parallel set of immunocytochemistry experiments was performed using rabbit polyclonal antipeptide antisera raised against the corresponding C-terminal decapeptide sequences of transducin {alpha} (1319), which identifies G{alpha}i2 strongly (see Fig. 2 ) or G{alpha}o1 (OC2; ref. 26 ).


Figure 5
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  		Figure 5. The conformationally selective antibody 6F12 is unable to identify either active or inactive mutants of Gs or Gq. HEK293T cells were grown on poly-D-lysine coated coverslips. The inability of 6F12 to recognize active (Q209LG{alpha}q, Q227LG{alpha}s) or inactive (G208A G{alpha}q, G226A G{alpha}s) variants of G{alpha}q or G{alpha}s was assessed as in the legend to Fig. 4 . A positive control for antibody 6F12 was expression of Q204L G{alpha}i1. Antibody 6F12 was used at a final dilution of 1:200. Anti-mouse Alexa Fluor 594 was used at a final concentration of 1:400. Fluorescence detected from i) D2 receptor-eYFP, ii) Alexa fluor 594 bound to 6F12, and iii) Hoechst stain is shown. iv) To demonstrate expression of transfected variants of G{alpha}s and G{alpha}q parallel sets of immunocytochemistry experiments were performed using rabbit polyclonal antipeptide antisera raised against the corresponding C-terminal decapeptide sequences of G{alpha}s (CS; ref. 27 ) or G{alpha}q (CQ; ref. 28 ).

AlF4– is a substitute for and can mimic the {gamma} phosphate of GTP. As such, addition of AlF4– to permeabilized cells expressing a wild-type, GDP-bound G protein should convert the G protein to a mimic of the active state. After expression of wild-type G{alpha}i2 in HEK293 cells, along with the D2 receptor-eYFP construct, addition of AlF4– (10 µM AlCl3+10 mM NaF) resulted in recognition of G{alpha}i2 by each of the three conformationally selective antibodies, including 6F12 (Fig. 6 ).


Figure 6
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  		Figure 6. The conformationally selective antibody 6F12 recognizes the complex G{alpha}i2-GDP-AlF4–. HEK293T cells cotransfected with cDNA of the D2 receptor-eYFP fusion protein and wild-type G{alpha}i2 were incubated with (G{alpha}i2 W-T+AlF4–) or without (G{alpha}i2 W-T–AlF4–) the addition of AlF4– (10 µM AlCl3+10 mM NaF). As positive and negative controls HEK293T cells were cotransfected with cDNA of the D2 receptor-eYFP fusion protein and the active variant G{alpha}i2 (G{alpha}i2 Q205L) or pcDNA3. Fluorescence detected from i) D2 receptor-eYFP, ii) Alexa Fluor 594 bound to 6F12, and iii) Hoechst stain is shown.

Finally, we wished to assess whether the conformationally selective antibodies would be able to trap and identify agonist-activated G protein {alpha} subunits. HEK293 cells transiently transfected to coexpress D2 receptor-eYFP and wild-type G{alpha}i2 were exposed to the dopaminergic agonist NPA (1 µM) in the presence of 10 µM GTP{gamma}S, saponin (0.1% v/v), and an assay buffer (50 mM Tris, 5 mM MgCl2, 100 mM NaCl, and 0.2 mM EGTA) previously used by others to develop an in-cell [35S]GTP{gamma}S binding assay (17) . In these conditions the conformationally selective antibodies identified G{alpha}i2 only in cells stimulated by agonist (Fig. 7 ). These data indicate that the conformationally selective antibodies described herein may be used to identify activated Gi family G proteins in a cellular setting.


Figure 7
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  		Figure 7. The conformationally selective antibody 6F12 recognizes the agonist-induced complex G{alpha}i2.GTP{gamma}S. HEK293T cells were cotransfected with cDNA of the D2 receptor-eYFP fusion protein and either cDNA of wild-type G{alpha}i2 were incubated in the presence of 10 µM GTP{gamma}S with (G{alpha}i2 W-T+NPA) or without (G{alpha}i2 W-T–NPA) the addition of the dopamine D2 receptor agonist NPA (1 µM). As positive and negative controls HEK293T cells were cotransfected with cDNA of the D2 receptor-eYFP fusion protein and the active variant G{alpha}i2 (G{alpha}i2 Q205L) or pcDNA3. Fluorescence detected from i) D2 receptor-eYFP, ii) Alexa Fluor 594 bound to 6F12, and iii) Hoescht stain is shown. iv) To demonstrate expression of transfected G{alpha} protein, a parallel immunocytochemistry experiment was performed using rabbit polyclonal antipeptide antiserum against the C-terminal decapeptide sequence of transducin {alpha}.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Receptor-activated exchange of GTP for GDP on the {alpha} subunit is the key step in the activation of heterotrimeric G proteins and the initiation of downstream signal transduction pathways (4) . This is reversed by the GTPase activity that is intrinsic to the G protein {alpha} subunit and restores GDP to the nucleotide binding pocket (4) . Assays based either on the binding of poorly hydrolyzed analogs of GTP or on measures of the rate of GTP hydrolysis are widely used to monitor this cycle. FRET-based reporters that monitor the dissociation or reorientation of the {alpha} subunit and β/{gamma} complex have also been used to study the kinetics of G protein activation (5 , 6) . The availability of atomic level structures of both isolated {alpha} subunits and heterotrimeric G protein complexes (18 19 20 21) has also provided detailed insights into conformations associated with each of the inactive, active, and transition states, particularly for the retinal G protein transducin and the related, inhibitory G protein Gi1. Immunological reagents directed against specific G protein subunits have been invaluable in understanding the tissue distribution of different members of the G protein family (22 23 24 25 26 27 28) , and certain antipeptide antisera have also been of great use in delineating sites of contact between G protein subunits and interacting proteins such as GPCRs (29 30 31 32) . However, antibodies able to specifically identify G protein {alpha} subunits in the active state have not been available. In an attempt to develop such reagents we immunized mice with GTP{gamma}S-loaded recombinant G{alpha}i1 and, after hybridoma generation, screened for the production of antibodies with the desired characteristics. The key to success was the development of stringent screening and selection criteria. To move forward to detailed analysis, each hybridoma supernatant was required to bind in cells expressing wild-type G{alpha}i1 supplemented with the poorly hydrolyzed GTP analog GTP{gamma}S and cells expressing the Q204L active state mutant of G{alpha}i1 but not those expressing wild-type G{alpha}i1 that were supplemented with the GDP analog GDPβS. Furthermore, a combination of RIMMS, which leads to generation of affinity-matured monoclonal antibodies in a reduced time frame (33) , and the use of semisolid media (34) that facilitated isolation of large numbers of monoclonal colonies in a single step with decreased manipulations was supplemented by using a robotic imaging and picking platform.

Q204L G{alpha}i1 was selected for the first-pass screen because, in the GTPase hydrolytic mechanism, Q204 in G{alpha}i1 functions to stabilize the phosphate of the leaving group in the transition state of the reaction complex and orientates the attacking water molecule to allow GTP hydrolysis. Mutation of this residue thus traps the active-state, GTP-bound form. It is also known that AlF4– is able to substitute for the terminal phosphate of GTP and hence interacts with inactive, GDP-bound G protein {alpha} subunits to generate an active, transition state that is not a substrate for hydrolytic attack. In the second-phase screens we thus added AlF4– to permeabilized HEK293 cells transfected to express wild-type G{alpha}i1, because we wished to be able to identify the switch from inactive, GDP-bound subunits to active, GTP-mimetic bound forms. The final requirement for useful antibodies was that they would identify wild-type G proteins that became activated in response to agonist occupation of a cell surface GPCR. Coexpression of a dopamine D2 receptor tagged with eYFP had the dual benefit of easy identification of positively transfected cells and the fact that this receptor is known to activate all G{alpha}i isoforms (16) . In these assays, we added GTP{gamma}S to trap G protein activated by the receptor in an active conformation and used buffer conditions derived from the development of in-cell [35S]GTP{gamma}S binding studies (17) .

Although G{alpha}i1 was use as the immunogen for these studies, detailed analysis demonstrated each of the conformationally selective antibodies to also selectively identify mutationally active conformations of G{alpha}i2 and G{alpha}o1 but not of G{alpha}s or G{alpha}q. Each of the pertussis toxin-sensitive G{alpha}i subunits is highly similar in sequence and presumably, therefore, in structure. Although the epitopes identified by these antibodies are unclear, comparisons of the available atomic level structures of activated and ground state forms may provide difference maps that will prove insightful because the relevant epitopes are certainly three-dimensional.

In recent times, antibodies able to selectivity identify the active state of the serotonin 5-HT2c receptor have been produced by a strategy similar to that used herein (35) , whereas antibodies raised against linear peptide sequences from the N-terminal domain of a number of GPCRs have been indicated to selectively identify the agonist-bound state (36) . Although enormously useful reagents, each GPCR to be studied requires the generation of an individual antibody. The conformationally selective antibodies generated in these studies should usefully report activation of Gi{alpha} family G proteins produced in a receptor- or, indeed, nonreceptor-mediated fashion and are likely to have particular utility in the development of label-free assays of G protein activation.


   ACKNOWLEDGMENTS
 
J.R.L. thanks the Biotechnology and Biosciences Research Council for a Co-operative Award in Science and Engineering studentship, and D.H. thanks the Medical Research Council for a Master of Research award.

Received for publication October 15, 2007. Accepted for publication December 6, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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