Real-time Analysis of G Protein-coupled Receptor Reconstitution in a Solubilized System
2001; Elsevier BV; Volume: 276; Issue: 25 Linguagem: Inglês
10.1074/jbc.m009679200
ISSN1083-351X
AutoresTeresa Bennett, T. Alexander Key, Vsevolod V. Gurevich, Richard R. Neubig, Eric R. Prossnitz, Larry A. Sklar,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoReceptor based signaling mechanisms are the primary source of cellular regulation. The superfamily of G protein-coupled receptors is the largest and most ubiquitous of the receptor mediated processes. We describe here the analysis in real-time of the assembly and disassembly of soluble G protein-coupled receptor-G protein complexes. A fluorometric method was utilized to determine the dissociation of a fluorescent ligand from the receptor solubilized in detergent. The ligand dissociation rate differs between a receptor coupled to a G protein and the receptor alone. By observing the sensitivity of the dissociation of a fluorescent ligand to the presence of guanine nucleotide, we have shown a time- and concentration-dependent reconstitution of theN-formyl peptide receptor with endogenous G proteins. Furthermore, after the clearing of endogenous G proteins, purified Gα subunits premixed with bovine brain Gβγ subunits were also able to reconstitute with the solubilized receptors. The solubilizedN-formyl peptide receptor and Gαi3 protein interacted with an affinity of ∼10−6m with other α subunits exhibiting lower affinities (Gαi3 > Gαi2 > Gαi1 ≫ Gαo). TheN-formyl peptide receptor-G protein interactions were inhibited by peptides corresponding to the Gαi C-terminal regions, by Gαi mAbs, and by a truncated form of arrestin-3. This system should prove useful for the analysis of the specificity of receptor-G protein interactions, as well as for the elucidation and characterization of receptor molecular assemblies and signal transduction complexes. Receptor based signaling mechanisms are the primary source of cellular regulation. The superfamily of G protein-coupled receptors is the largest and most ubiquitous of the receptor mediated processes. We describe here the analysis in real-time of the assembly and disassembly of soluble G protein-coupled receptor-G protein complexes. A fluorometric method was utilized to determine the dissociation of a fluorescent ligand from the receptor solubilized in detergent. The ligand dissociation rate differs between a receptor coupled to a G protein and the receptor alone. By observing the sensitivity of the dissociation of a fluorescent ligand to the presence of guanine nucleotide, we have shown a time- and concentration-dependent reconstitution of theN-formyl peptide receptor with endogenous G proteins. Furthermore, after the clearing of endogenous G proteins, purified Gα subunits premixed with bovine brain Gβγ subunits were also able to reconstitute with the solubilized receptors. The solubilizedN-formyl peptide receptor and Gαi3 protein interacted with an affinity of ∼10−6m with other α subunits exhibiting lower affinities (Gαi3 > Gαi2 > Gαi1 ≫ Gαo). TheN-formyl peptide receptor-G protein interactions were inhibited by peptides corresponding to the Gαi C-terminal regions, by Gαi mAbs, and by a truncated form of arrestin-3. This system should prove useful for the analysis of the specificity of receptor-G protein interactions, as well as for the elucidation and characterization of receptor molecular assemblies and signal transduction complexes. G protein-coupled receptor guanine nucleotide-binding regulatory proteins ligand receptor G protein N-formyl peptide receptor fluorescein 5-isothiocyanate guanosine 5′-3-O-(thio)triphosphate antibody 1,4-piperazinediethanesulfonic acid GTP-binding regulatory protein-coupled receptors (GPCR)1 represent the largest class of cell surface receptors. A broad variety of physiological processes depend on this family of seven transmembrane proteins, making them prime targets for drug discovery (1Gudermann T. Nurnberg B. Schultz G. J. Mol. Med. 1995; 73: 51-63Crossref PubMed Scopus (178) Google Scholar). Several complementary approaches are being taken in this therapeutic effort. One approach is to define novel therapeutic agents including both agonists and antagonists for specific receptors, as well as molecules that block interactions between receptors and their G protein transduction partners. In addition, the interactions between ligands and receptors are beginning to be thoroughly mapped through studies incorporating receptor mutagenesis as well as analysis of the binding and activity of ligand analogs (2Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1242) Google Scholar). Comparable efforts are being made to describe the molecular mechanisms that influence the specificity of coupling interactions between GPCR and their cognate heterotrimeric G proteins. Several experimental systems for reconstitution provide alternatives for studying receptors and G proteins, such as adding G protein to stripped membranes, pairing receptors and G proteins in phospholipid vesicles (3Cupo J.F. Allen R.A. Jesaitis A.J. Bokoch G.M. Biochim. Biophys. Acta. 1989; 982: 31-40Crossref PubMed Scopus (15) Google Scholar), and examining solubilized receptor-G protein interactions using gradient centrifugation (4Bommakanti R.K. Dratz E.A. Siemsen D.W. Jesaitis A.J. Biochim. Biophys. Acta. 1994; 1209: 69-76Crossref PubMed Scopus (8) Google Scholar). The kinetics of the interactions between ligand (L), receptor (R), and G protein (G) are described by the ternary complex model (5De L.A. Stadel J.M. Lefkowitz R.J. J. Biol. Chem. 1980; 255: 7108-7117Abstract Full Text PDF PubMed Google Scholar). The formation of LRG is required for the activation of the G protein and typically involves the formation of a high affinity receptor, release of GDP, and activation of the G protein through GTP binding (1Gudermann T. Nurnberg B. Schultz G. J. Mol. Med. 1995; 73: 51-63Crossref PubMed Scopus (178) Google Scholar). Extended ternary complex models have been developed to describe the transition of receptors from inactive to activated states (6Gether U. Kobilka B.K. J. Biol. Chem. 1998; 273: 17979-17982Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar). The assembly kinetics of these systems are not completely understood as the available methods do not provide the time resolution required to evaluate all of the steps in the activation process. Moreover, analysis of receptor-G protein interactions in cells is complicated by the difficulty in elucidating the G protein numbers and concentrations within the microdomains of the receptors and the transient association between receptors and G proteins. To further the understanding of GPCR-mediated processes, we have utilized the N-formyl peptide receptor (FPR), which couples to a pertussis toxin-sensitive G protein and is expressed predominantly on leukocytes (7Prossnitz E.R. Ye R.D. Pharmacol. Ther. 1997; 74: 73-102Crossref PubMed Scopus (228) Google Scholar). This receptor recognizes the bacterially generatedN-formyl peptides that act as potent chemoattractants for human phagocytes. The FPR is one of the better characterized receptors in the chemoattractant/chemokine subclass of GPCR (8Ye R.D. Boulay F. Adv. Pharmacol. 1997; 39: 221-289Crossref PubMed Scopus (105) Google Scholar). It modulates several cell functions including chemotaxis, superoxide formation, and degranulation, as well as influencing nuclear regulation via activation of MAPK cascades (7Prossnitz E.R. Ye R.D. Pharmacol. Ther. 1997; 74: 73-102Crossref PubMed Scopus (228) Google Scholar). It has been assumed that the FPR binds preferentially to a Gαi2 protein as this Giisoform is highly expressed in neutrophils, while Gαi3 is expressed at low levels and there is no expression of Gαi1 (9Gierschik P. Sidiropoulos D. Jakobs K.H. J. Biol. Chem. 1989; 264: 21470-21473Abstract Full Text PDF PubMed Google Scholar, 10Murphy P.M. Eide B. Goldsmith P. Brann M. Gierschik P. Spiegel A. Malech H.L. FEBS Lett. 1987; 221: 81-86Crossref PubMed Scopus (58) Google Scholar). A recent report using chimeric proteins containing the FPR fused to Gαi1, Gαi2, or Gαi3, expressed in Sf9 cells, suggested that the FPR couples to each of the Gi isoforms with similar efficiency (11Wenzel-Seifert K. Arthur J.M. Liu H.Y. Seifert R. J. Biol. Chem. 1999; 274: 33259-33266Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). However, several aspects of this interaction still remain unresolved. We have previously described a number of real-time assays of ligand-receptor interactions using flow cytometry and fluorescence that have been primarily directed toward viable cells or detergent-permeabilized cells (12Fay S.P. Posner R.G. Swann W.N. Sklar L.A. Biochemistry. 1991; 30: 5066-5075Crossref PubMed Scopus (87) Google Scholar, 13Nolan J.P. Chambers J.D. Sklar L.A. Babcock G. Robinson P. Cytometric Approaches to Cellular Analysis. Wiley-Liss, New York1998: 19-46Google Scholar, 14Sklar L.A. Finney D.A. Oades Z.G. Jesaitis A.J. Painter R.G. Cochrane C.G. J. Biol. Chem. 1984; 259: 5661-5669Abstract Full Text PDF PubMed Google Scholar). These studies characterized wild type and mutant receptors leading to a model of receptor-G protein coupling for the FPR (13Nolan J.P. Chambers J.D. Sklar L.A. Babcock G. Robinson P. Cytometric Approaches to Cellular Analysis. Wiley-Liss, New York1998: 19-46Google Scholar). More recently, we were able to assess the efficiency of solubilization of the FPR using fluorescence methods, and have shown that these receptors were able to reconstitute with G proteins in a non-cellular format (15Sklar L.A. Vilven J. Lynam E. Neldon D. Bennett T.A. Prossnitz E. BioTechniques. 2000; 28: 976-985Crossref PubMed Scopus (31) Google Scholar). In this report we have expanded our investigation of the detergent-solubilized FPR with emphasis on the receptor-G protein interaction. Using reconstitution assays, we demonstrate the ability of the receptor to couple with endogenous G proteins, as well as with exogenous G proteins containing specific Gα subunits. We were able to measure the affinities of the complexes and found that the FPR binds to a G protein heterotrimer containing the Gαi3 subunit with somewhat higher affinity than to heterotrimers containing Gαi2 or Gαi1 proteins. The individual α subunits and the βγ complex alone were unable to induce a change in the dissociation of ligand from the receptor indicating a necessity for the G protein heterotrimer. We were able to inhibit the G protein-receptor interaction with peptides derived from the Gαi subunits as well as with anti-Gαi antibodies. Finally, we have expanded the system to include analysis of GPCR-arrestin interactions. The methods described here provide an approach to study the mechanism of GPCR interactions with G proteins, arrestins, and other potential targets and at the same time provide a platform for identifying and characterizing novel therapeutic agents. The generation of U937 cells transfected with the FPR was previously described (16Prossnitz E.R. Quehenberger O. Cochrane C.G. Ye R.D. Biochem. J. 1993; 294: 581-587Crossref PubMed Scopus (42) Google Scholar). Plasticware was obtained from VWR Scientific Co. Chemicals and reagents were obtained from Sigma except where otherwise noted. Cells were grown in tissue culture-treated flasks (Corning) in RPMI 1640 (Hyclone) containing 10% fetal bovine serum (Hyclone), 2 mml-glutamine, 10 mm HEPES, with 10 units/ml penicillin and 10 μg/ml streptomycin. Cultures were grown in standard tissue culture incubators at 37 °C with 5% CO2, and passaged from subconfluent cultures every 3–4 days by reseeding at 2 × 105 cells/ml. Purified G protein α subunits Gαi1, Gαi2, Gαi3, and Gαo (functional, myristoylated, rat recombinant) were purchased from Calbiochem. The bovine brain βγ complex was isolated and purified as previously reported (17Neubig R.R. Connolly M.P. Remmers A.E. FEBS Lett. 1994; 355: 251-253Crossref PubMed Scopus (21) Google Scholar). Arrestins were expressed inEscherichia coli (strain BL21) and purified by sequential heparin-Sepharose and Q-Sepharose chromatography essentially as described (18Gurevich V.V. Benovic J.L. Methods Enzymol. 2000; 315: 422-437Crossref PubMed Google Scholar). Reagents for inhibition of reconstitution include three G protein α subunit blocking peptides composed of the last 10 amino acids of Gαi1,2, Gαi3, and Gαs, and anti-Gα antibodies recognizing the C terminus of Gαi1,2, Gαi3, Gαo, and an internal Gαi3 antibody (Calbiochem). U937 FPR cells were harvested, centrifuged at 200 ×g for 5 min, and resuspended in cavitation buffer (10 nm PIPES, 100 mm KCl, 3 mm NaCl, 3.5 mmMgCl2, 600 μg/ml ATP) at a density of 107cells/ml at 4 °C. The cell suspension was placed in a nitrogen bomb and pressurized to 450 psi using N2 gas for 20 min at 4 °C. Nuclei and cytoplasmic material were separated by centrifugation at 1000 × g for 5 min at 4 °C. The supernatant, containing the membranes, was washed 2 times by centrifugation at 135,000 × g for 30 min at 4 °C, then resuspended in HEPES sucrose buffer (200 mmsucrose, 25 mm HEPES, pH 7), aliquoted, and stored until use at −80 °C. Membranes were thawed and diluted to 1–2 × 108 membrane cell equivalents/ml (CEQ/ml) in binding buffer (30 mm HEPES, 100 mm KCl, 20 mm NaCl, 1 mm EGTA, 0.1% (w/v) bovine serum albumin, 0.5 mm MgCl2). Preparations were maintained at 4 °C throughout the extraction process. Membranes were centrifuged at 135,000 × g for 30 min and resuspended in 150 μl of binding buffer containing protease inhibitor mixture I and 1% n-dodecyl β-d-maltoside (Calbiochem). Preparations were incubated 60 min at 4 °C with agitation. The insoluble fraction was separated by centrifugation at 70,000 ×g for 5 min in a Beckman Airfuge. The supernatant, containing the solubilized fraction, was removed and this extract was used for experimentation within 6 h. Membranes (1 × 108cell equivalents) were solubilized and applied to 1 ml of a 5–20% linear sucrose gradient prepared in binding buffer plus 1%n-dodecyl β-d-maltoside. Gradients were centrifuged at 40,000 rpm in a TLS-55 rotor (Beckman) for 14 h and fractionated into 20 × 50-μl fractions. To establish the distribution of receptor, 25 μl of each fraction was incubated with 10 nm formyl-Met-Leu-Phe-Lys-fluorescein 5-isothiocyanate (fMLFK-FITC, Peninsula Laboratories) for 2 h on ice. Fractions were subjected to spectrofluorometric analysis, as outlined below. Gradients containing 5 μg of bovine serum albumin (4.4 S) and rabbit immunoglobulin (7.7 S) were centrifuged in parallel. These fractions were analyzed by SDS-polyacrylamide gel electrophoresis, followed by Coomassie staining. Based on the sedimentation of these standard proteins, the migration of 4 S and 7 S proteins was calculated to peak at fractions 9 and 15, respectively. Purified individual α subunits were mixed in an equimolar ratio with the βγ complex and incubated on ice for 15 min to form the heterotrimeric complex (17Neubig R.R. Connolly M.P. Remmers A.E. FEBS Lett. 1994; 355: 251-253Crossref PubMed Scopus (21) Google Scholar). Solubilized FPR (5–10 μl of ∼10 nm receptor in 1% n-dodecyl β-d-maltoside prepared as above), was then incubated with the specific G-protein heterotrimers or with a mixture of bovine brain Gi/Go heterotrimer (Calbiochem) at a concentration of up to 3 μm for up to 2 h on ice in the presence of 10 nm fMLFK-FITC. Blocked samples were incubated with 1 μm formyl-Met-Leu-Phe-Phe (fMLFF, CBI) for 15 min prior to the addition of fMLFK-FITC. Samples were prepared in small volumes (typically 15 μl) to maximize receptor concentration. Control samples were prepared in the presence of appropriate buffer(s). The Gα subunit buffer contained 100 mm NaCl, 20 mm HEPES, 3 mmMgCl2, 1 mm EDTA, pH 8.0. The Gβγ buffer contained 50 mm HEPES, 1 mm EDTA, 1 mm dithiothreitol, 0.5% sodium cholate, pH 8.0, The arrestin buffer contained 10 mm Tris, 100 mmNaCl, 2 mm EDTA, 2 mm EGTA, 2 mmphenylmethylsulfonyl fluoride, 10 μm leupeptin, 0.7 μg/ml pepstatin A, 10 μm chymostatin, pH 7.5. Reconstitution incubations were carried out in the presence of 0.8 to 0.85% detergent (final concentration). The activity and concentration of the Gαo subunit was verified using a BODIPY FL GTPγS binding assay titrating the subunit (1 nm to 300 nm) against 50 nmnucleotide and recording the resulting changes in fluorescence on the spectrofluorometer (19McEwen, D. P., Gee, K. R., Kang, H. C., and Neubig, R. R. (2001) Methods Enzymol., in press.Google Scholar, 20Draganescu A. Hodawadekar S.C. Gee K.R. Brenner C. J. Biol. Chem. 2000; 275: 4555-4560Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The derived value for the Gαo subunit concentration was consistent with the value reported by the supplier (data not shown). Fluorescence associated with fMLFK-FITC was measured by a SLM 8000 spectrofluorometer (SLM Instruments, Inc.) using the photon counting mode in acquisition. The sample holder was fitted with a cylindrical cuvette adapter to permit measurements in stirred volumes of 200 μl using small cylindrical cuvettes (Sienco) and 2 × 5-mm stir bars (Bel-Art). Excitation was fixed at 490 nm, and stray light was reduced with a 490 nm, 10-nm band pass filter (Corion). FITC fluorescence emission was monitored using a 520 nm, 10-nm band pass interference filter (Corion) and a 3–70 orange glass, 500-nm long pass filter (Kopp). Additions to samples during kinetic measurements were performed using 10-μl glass syringes (Hamilton) adding reagents through an injection port on the SLM 8000 above the sample cuvette. Following preparation at 4 °C, samples were brought up to a volume of 200 μl with binding buffer plus 0.1% n-dodecyl β-d-maltoside, equilibrated to 22 °C, and placed into the spectrofluorometer with constant stirring. Data were acquired for 180–210 s in 1-s intervals. Typically, total fluorescence was obtained for the first 20 s, then, an anti-fluorescein antibody was added to the sample. The antibody binds free fMLFK-FITC with high affinity and results in essentially complete quenching of the fluorescence associated with free ligand (14Sklar L.A. Finney D.A. Oades Z.G. Jesaitis A.J. Painter R.G. Cochrane C.G. J. Biol. Chem. 1984; 259: 5661-5669Abstract Full Text PDF PubMed Google Scholar). Thus, the remaining fluorescence represents the receptor-bound. In G protein experiments, GTPγS (100 μm) sensitivity was used to assess the coupling between receptors and G proteins based on characteristic ligand dissociation rates. Experiments were performed using a detergent concentration slightly above the critical micelle concentration. Data were analyzed and graphed using Prism software (Graph Pad Software Inc.). To determine the dissociation characteristics of the receptor preparations, the fluorescence over time in blocked control samples was subtracted point by point from the fluorescence over time of ligand binding samples as described previously (14Sklar L.A. Finney D.A. Oades Z.G. Jesaitis A.J. Painter R.G. Cochrane C.G. J. Biol. Chem. 1984; 259: 5661-5669Abstract Full Text PDF PubMed Google Scholar). The data were then normalized and the relative fluorescence was fit by nonlinear regression using single (Equation 1) or double (Equation 2) exponential decay equations. I=A*e(−kt)+plateauEquation 1 I=A1*e(−k1t)+A2*e(−k2t)+plateauEquation 2 Where I = fluorescence intensity in arbitrary units, kx is the off rate of the receptor state in s−1, t is time in seconds, plateau is the fluorescence when all peptide has dissociated and Axis the fraction of receptor in the state with the rate ofkx. Rates are given as mean ± S.E. Membrane preparations were solubilized in detergent as outlined above. The sample was then incubated with 20 μl of anti-Gαi1,2,3antibody (Calbiochem) for 45 min on ice. To remove antibody-substrate conjugates, 100 μg of Protein A-agarose was added to the sample. After a 30-min incubation, the sample was centrifuged at 14,000 ×g for 30 s and the supernatant was removed. The process was then repeated. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Gelman) with a semi-dry transfer apparatus (Owl Scientific). Membranes were blotted with antibody (rabbit) against either Gαi1,2,3 or βγ (Calbiochem) followed by an horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Sigma). The blots were developed using ECL Plus (Amersham Pharmacia Biotech) and imaged using a PhosphorImager (Molecular Dynamics). Receptor-G protein interactions have been characterized in intact and permeabilized cells, membranes, phospholipid vesicles, and in detergent using gradients (3Cupo J.F. Allen R.A. Jesaitis A.J. Bokoch G.M. Biochim. Biophys. Acta. 1989; 982: 31-40Crossref PubMed Scopus (15) Google Scholar,21Klotz K.N. Krotec K.L. Gripentrog J. Jesaitis A.J. J. Immunol. 1994; 152: 801-810PubMed Google Scholar, 22Kurose H. Regan J.W. Caron M.G. Lefkowitz R.J. Biochemistry. 1991; 30: 3335-3341Crossref PubMed Scopus (88) Google Scholar, 23Prossnitz E.R. Schreiber R.E. Bokoch G.M. Ye R.D. J. Biol. Chem. 1995; 270: 10686-10694Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). We have extended the use of detergent-solubilized FPR and G proteins, adding fluorescence detection, to study receptor-G protein complexes in real-time. We have taken advantage of the fact that the ligand dissociation rate of the LR complex of the FPR (in the absence of G protein or in the presence of GTPγS) is many times faster than ligand dissociation from LRG (12Fay S.P. Posner R.G. Swann W.N. Sklar L.A. Biochemistry. 1991; 30: 5066-5075Crossref PubMed Scopus (87) Google Scholar, 24Posner R.G. Fay S.P. Domalewski M.D. Sklar L.A. Mol. Pharmacol. 1994; 45: 65-73PubMed Google Scholar, 25Sklar L.A. Mueller H. Omann G. Oades Z. J. Biol. Chem. 1989; 264: 8483-8486Abstract Full Text PDF PubMed Google Scholar). Thus, the presence of the G protein imparts a higher affinity for ligand to the receptor. When GTP is not available, the G protein remains bound to the FPR. The addition of GTP, or its non-hydrolyzable analog GTPγS, putatively induces the dissociation of G protein from the receptor and results in a decrease in the affinity of the ligand, increasing its dissociation rate. The change in the ligand dissociation rate is likely to represent a switch from the slowly dissociating LRG to the rapidly dissociating LR as the activated G protein uncouples from the FPR. The assay is based upon a fluorescein-conjugated ligand used along with an anti-fluorescein antibody that rapidly quenches the fluorescein of the free ligand upon binding. The antibody is able to interact only with the fluorescein on the free ligand, as FPR-ligand complexes sterically inhibit the antibody from binding the fluorescein (14Sklar L.A. Finney D.A. Oades Z.G. Jesaitis A.J. Painter R.G. Cochrane C.G. J. Biol. Chem. 1984; 259: 5661-5669Abstract Full Text PDF PubMed Google Scholar). In this way we were able to determine the quantity of FPR-bound ligand (Fig. 1) immediately following the addition of the anti-fluorescein antibody. We were also able to follow the ligand dissociation kinetics as the excess antibody further quenches ligand released from the receptor. These methods were based on assays that have been fully characterized using permeablized whole cells (12Fay S.P. Posner R.G. Swann W.N. Sklar L.A. Biochemistry. 1991; 30: 5066-5075Crossref PubMed Scopus (87) Google Scholar, 13Nolan J.P. Chambers J.D. Sklar L.A. Babcock G. Robinson P. Cytometric Approaches to Cellular Analysis. Wiley-Liss, New York1998: 19-46Google Scholar, 14Sklar L.A. Finney D.A. Oades Z.G. Jesaitis A.J. Painter R.G. Cochrane C.G. J. Biol. Chem. 1984; 259: 5661-5669Abstract Full Text PDF PubMed Google Scholar). The dissociation data was analyzed as described under “Experimental Procedures.” The data were fit to one or two rates, the slow rate apparently representing L dissociation from RG and the fast rate apparently representing L dissociation from R. The human FPR was previously stably transfected into the undifferentiated human myeloid cell-line, U937 (16Prossnitz E.R. Quehenberger O. Cochrane C.G. Ye R.D. Biochem. J. 1993; 294: 581-587Crossref PubMed Scopus (42) Google Scholar). The U937 cells expressing the wild type FPR react specifically to formylated peptides with calcium and chemotactic responses (26Kew R.R. Peng T. DiMartino S.J. Madhavan D. Weinman S.J. Cheng D. Prossnitz E.R. J. Leukoc. Biol. 1997; 61: 329-337Crossref PubMed Scopus (61) Google Scholar). Membrane fractions were prepared using N2 cavitation and the work presented is data from both intact membranes or membranes that have been solubilized with 1.0% detergent solution. The extent of ligand binding to the FPR is similar for membranes and the solubilized membrane extract (15Sklar L.A. Vilven J. Lynam E. Neldon D. Bennett T.A. Prossnitz E. BioTechniques. 2000; 28: 976-985Crossref PubMed Scopus (31) Google Scholar). However, in the membrane samples it appeared that G proteins were either pre-coupled to, or interacted almost immediately with, the receptor after the addition of ligand, but once the membranes were solubilized, no G protein interaction was observed over the same time frame (Fig. 1 A). In earlier studies we demonstrated the ability of the solubilized receptor to interact with an exogenous G protein, if the receptor was incubated in the presence of ligand for 2 h (15Sklar L.A. Vilven J. Lynam E. Neldon D. Bennett T.A. Prossnitz E. BioTechniques. 2000; 28: 976-985Crossref PubMed Scopus (31) Google Scholar). Thus it was possible that the solubilized receptor would reconstitute with endogenous G proteins over time if ligand was present. As shown in Fig. 1 B, there was indeed a time dependence to the interaction. Additionally, when assays were prepared in larger volumes, that diluted the concentration of both receptor and G protein, reconstitution was not observed (data not shown). This dependence on receptor and G protein concentration in solution suggests that while within the membranes, the receptor-G protein interactions take place rapidly in two dimensions, the solubilized proteins exist in three dimensions requiring extra time for the proteins to associate. To confirm that our receptor preparations were solubilized in the presence of 1% detergent (n-dodecyl β-d-maltoside), a sucrose velocity sedimentation assay was performed. This assay determines the extent of solubilization and complex formation of proteins. Briefly, the solubilized receptor fraction is layered onto a 5–20% sucrose gradient. Following centrifugation, the gradients are fractionated, and the position of the FPR within the gradient was determined by spectrofluorometric analysis. The FPR was found in fractions 8–10, sedimenting as a 4 S species, as expected for a monodisperse receptor (Fig.2). Also, this further confirmed that the solubilized FPR was not initially coupled to a G protein. In order to generate a system in which the affinities of specific exogenous G protein subunits could be determined for the FPR, we first needed to remove the endogenous G protein from our solubilized receptor solution. This was accomplished by incubating the sample with an anti-Gαi1,2,3 antibody then adding protein A-agarose to clear the antibody-substrate conjugates. Our solubilized receptor sample, when treated in this manner, lost all sensitivity to GTPγS and the non-linear regression fit of the data indicated only a single exponential decay corresponding to LR. This suggested that the endogenous G proteins had indeed been removed from the sample (Fig. 3 A). Western blot analysis of the sample prior to, and after antibody treatment, confirmed that this procedure effectively cleared the endogenous Gi proteins from the system. It appeared that the Gi proteins were present in the heterotrimeric form as the anti-Gαi antibody pulled down both the α subunits and the βγ complex (Fig. 3 B). A receptor preparation treated in this way would be appropriate for the analysis of specific receptor-G protein interactions. It has been shown that the FPR interacts with pertussis toxin-sensitive Gi proteins in neutrophils and U937 cells (9Gierschik P. Sidiropoulos D. Jakobs K.H. J. Biol. Chem. 1989; 264: 21470-21473Abstract Full Text PDF PubMed Google Scholar, 10Murphy P.M. Eide B. Goldsmith P. Brann M. Gierschik P. Spiegel A. Malech H.L. FEBS Lett. 1987; 221: 81-86Crossref PubMed Scopus (58) Google Scholar). The predominantly expressed Gi protein in neutrophils has a Gαi2 subunit. Thus it has been assumed that the FPR preferentially binds to the Gαi2 proteins. Until now it has been difficult to directly assess the affinity between G proteins and receptors. We have previously demonstrated the ability of solubilized receptors to reconstitute with a bovine brain G protein mixture with an ED50 of ∼10−6m under conditions where the ligand and receptor concentrations are ∼10 nm(15Sklar L.A. Vilven J. Lynam E. Neldon D. Bennett T.A. Prossnitz E. BioTechniques. 2000; 28: 976-985Crossref PubMed Scopus (31) Google Scholar). Expanding this study, we examined this interaction using specific, purified G protein α subunits (Gαi1, Gαi2, Gαi3, and Gαo). The individual α subunits were first mixed in an equal molar ratio with bovine brain βγ to form a G protein heterotrimeric complex (17Neubig R.R. Connolly M.P. Remmers A.E. FEBS Lett. 1994; 355: 251-253Crossref PubMed Scopus (21) Google Scholar). Solubilized receptor, that had been depleted of endogenous G protein, was incubated with the individually prepared G protein heterotrimers in the presence of fluorescein-conjugated ligand for 2 h. The resulting plots, as seen in Fig. 4,A-D, demonstrate the preference of the FPR for the Gαi subunits as the Gαo protein does not appear to induce the high ligand affinity state of the receptor. Non-linear regression of the first phase (t = 20–50 s) of the Gαi curves (*) fit to a double exponential (after subtraction of the free component), indicating that two different rates were present. The slower rate was 0.0065 ± 0.0020 s−1, representing ligand dissociation from RG (high affinity state), and t
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