Determinants of Gi1alpha and beta gamma Binding
MEASURING HIGH AFFINITY INTERACTIONS IN A LIPID ENVIRONMENT USING FLOW CYTOMETRY*

Noune A. SarvazyanDagger §, Ann E. RemmersDagger , and Richard R. NeubigDagger par

From the Departments of Dagger  Pharmacology and  Internal Medicine/Hypertension, University of Michigan, Ann Arbor, Michigan 48109-0632

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

G protein heterocomplex undergoes dissociation and association during its functional cycle. Quantitative measurements of alpha and beta gamma subunit binding have been difficult due to a very high affinity. We used fluorescence flow cytometry to quantitate binding of fluorescein-labeled Gi1alpha (F-alpha ) to picomolar concentrations of biotinylated Gbeta gamma . Association in Lubrol solution was rapid (kon = 0.7 × 106 M-1 s-1), and equilibrium binding revealed a Kd of 2.9 ± 0.8 nM. The binding showed a complex dependence on magnesium concentration, but activation of F-alpha with either GDP/aluminum fluoride or guanosine 5'-O-(3-thiotriphosphate) completely prevented formation of the heterocomplex (Kd > 100 nM). The binding was also influenced by the detergent or lipid environment. Unlabeled beta gamma reconstituted in biotinylated phospholipid vesicles (pure phosphatidylcholine or mixed brain lipids) bound F-alpha ~2-3-fold less tightly (Kd = 6-9 nM) than in Lubrol. In contrast, beta gamma in ionic detergents such as cholate and 3-[(cholamidopropyl)diethylammonio]-1-propanesulfonate exhibited substantially lower affinities for F-alpha . Dissociation of F-alpha from beta gamma reconstituted in lipid vesicles was observed upon addition of aluminum fluoride or excess unlabeled alpha  subunit, indicating that myristoylated alpha  subunit has only a weak interaction with lipids without the beta gamma subunit. The kinetics of aluminum fluoride-stimulated dissociation were slower than those of the alpha  subunit conformational change detected by intrinsic fluorescence. These results quantitatively demonstrate G protein subunit dissociation upon activation and provide a simple but powerful new approach for studying high affinity protein/protein interactions in solution or in a lipid environment.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Receptor-mediated activation of heterotrimeric GTP-binding proteins is a common mechanism for transducing biological signals. Cell-surface receptors regulate G proteins by inducing a conformational change that catalyzes release of GDP from the alpha  subunit, allowing GTP to bind and activate the G protein. It is generally accepted that G protein activation in vitro involves alpha  subunit dissociation from beta gamma subunits and that dissociated alpha  and beta gamma subunits interact with effector proteins to modulate cellular responses (1-5). G protein deactivation is mediated by an intrinsic alpha  subunit GTPase activity that can be enhanced by GTPase-accelerating proteins (6, 7), followed by alpha  and beta gamma subunit reassociation. While G protein subunit association and dissociation have been extensively studied in detergent solutions, it has been difficult to obtain precise affinities for the subunits. The subunit interactions in a lipid environment are even less well understood.

Several areas of uncertainty remain in the mechanism of G protein subunit interactions with themselves and with membranes. The role of lipids and G protein subunit modifications in alpha /beta gamma interactions has not been defined quantitatively. The sequence of events in subunit activation and dissociation has recently been called into question (8). Also, it has been suggested that unphysiologically high magnesium concentrations are required for Gs subunit dissociation (9). Thus, we wished to test in a quantitative manner the binding of the alpha  and beta gamma subunits in both detergent solution and in a lipid environment to study factors regulating their affinity. One of the major limitations to the study of very high affinity protein/protein interactions is that the concentrations of proteins needed to evaluate affinities are often greater than the Kd for the binding interaction. This leads to artifacts and inaccurate Kd determinations. Sklar and co-workers (10) have used flow cytometry to study high affinity binding of fluorescent ligands to the surface of cells. More recently, Nolan et al. (11) used bead-based systems to study protein/DNA interactions in vitro. In this report, we describe the use of biotinylated Gbeta gamma (b-beta gamma )1 bound to avidin-coated polystyrene beads and fluorescein-labeled Gi1alpha (F-alpha ) to measure equilibrium constants and rates for alpha  and beta gamma binding in the picomolar range by flow cytometry. In addition, biotinylated lipids permit measurements of the binding of unlabeled beta gamma with F-alpha in a lipid environment. Mechanisms of alpha /beta gamma interactions and factors that modify those interactions are described. This simple and quantitative approach to the study of protein/protein interactions provides new information about G protein function and has great potential for the study of the assembly of membrane protein complexes in general.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Fluorescein 5-isothiocyanate was obtained from Molecular Probes, Inc. (Eugene, OR). Avidin-coated 6-µm polystyrene particles (catalog No. SVP-60-5) were purchased from Spherotech, Inc. (Liberville, IL). Maleimidobenzoyl-N-hydroxysulfosuccinimide ester and N-(6-((biotinoyl)amino)-hexanoyl)dipalmitoyl-L-alpha -phosphatidylethanolamine triethylammonium salt (EZ-LinkTMBiotin-LC-DPPE) were obtained from Pierce. Phosphatidylcholine (catalog No. A-30) was obtained from Doorsan Serdary Research Laboratories (Englewood Cliffs, NJ). Biotinamidocaproate N-hydroxysuccimide ester, omega -aminobutyl-agarose, and bovine brain membrane extract (catalog No. B-3635) were purchased from Sigma. The expression vectors pQE6 containing rat cDNA sequence for Gi1alpha and pBB131 containing yeast N-myristoyltransferase (12) were generously provided by Drs. Maurine Linder and Jeffrey Gordon (Washington University, St. Louis, MO), respectively.

Purification of r-myr-alpha i1 and Brain beta gamma Subunits-- The pQE6 Gi1alpha and pBB131 N-myristoyltransferase vectors were cotransformed into the JM109 or BL21(DE3) strain of Escherichia coli. The resulting recombinant myristoylated G protein alpha i1 subunit (r-myr-alpha i1) was expressed and purified according to Mumby and Linder (13) with minor modifications. The specific activity was 15 pmol/µg based on [35S]GTPgamma S binding (14). Bovine brain Gbeta gamma subunits were isolated from purified Go/Gi as described by Katada et al. (15).

Labeling of alpha  and beta gamma Subunits-- Purified r-myr-alpha i1 was exchanged into DTT-free buffer containing 20 mM HEPES, pH 8.5, 1 mM EDTA, 100 mM NaCl, 0.5% cholate, 10 µM GDP, and AMF (20 µM AlCl3, 10 mM MgCl2, and 10 mM NaF) through consecutive Centricon-30 concentration and dilution. In a 5-ml reaction mixture, 50 nmol of r-myr-alpha i1 was incubated with a 10-fold molar excess of fluorescein 5-isothiocyanate for 2 h at room temperature in the dark with rocking. The reaction was terminated by addition of 0.5 ml of 1 M glycine, pH 8.5, and the majority of free dye was removed by dialysis using a Slide-A-Lyzer-10 (Pierce) overnight in 20 mM HEPES, pH 8.0, 1 mM EDTA, and 3 mM DTT in the dark at 4 °C. Active fluorescein-labeled r-myr-alpha i1 (F-alpha ) was affinity-isolated on a beta gamma -agarose column (16). This yielded ~64% of the originally loaded F-alpha with a specific activity of 11 pmol/µg of [35S]GTPgamma S binding and 0.9 mol of dye/mol of protein incorporated.

The procedure for the biotinylation of beta gamma by the amino group-specific derivative biotinamidocaproate N-hydroxysuccimide ester is a modification of published protocols (17, 18). Purified bovine brain heterotrimer (Go/Gi, 1 mg/ml) was incubated for 1 h at room temperature in HEDNML (50 mM HEPES, pH 8.0, 1 mM EDTA, 1 mM DTT, 100 mM NaCl, 1.2 mM MgSO4, and 0.1% Lubrol) with a 80:1 molar ratio of biotinamidocaproate N-hydroxysuccimide ester to protein. The reaction was quenched by addition of a 10-fold molar excess of glycine, pH 8.0, and the reaction mixture was applied to a Sephadex G-50-80 column. The b-beta gamma subunits were isolated from the heterotrimer by incubation with AMF for 20 min at 32 °C, followed by hydrophobic chromatography on a phenyl Poros PH/M column (PerSeptive Systems). b-beta gamma was further purified from the small fraction of heavily biotinylated alpha  subunit on a Mono Q HR5/5 column (Amersham Pharmacia Biotech). The resulting b-beta gamma subunit was 99% pure as evident from Coomassie Blue staining and Western blot analysis with peroxidase-labeled avidin.

Reconstitution of beta gamma into Biotinylated Lipid Vesicles-- Biotinylated phospholipid vesicles containing beta gamma subunits were prepared by a gel filtration procedure as described previously (19). EZ-LinkTMBiotin-LC-DPPE (biotin-labeled lipid), featuring a 2.7-nm spacer arm to avoid steric hindrance, was mixed with phospholipids in a 1:100 molar ratio to permit attachment of the vesicles to the avidin-coated beads. Phosphatidylcholine or bovine brain membrane extract (0.8 mg) was mixed with 8 µg of biotin-labeled lipid in chloroform/methanol (2:1), dried under N2, and resuspended in 0.2 ml of a 1.2% solution of sodium cholate. Mixtures were vortexed for 5 min at room temperature and sonicated for 30 min at 4 °C to clarity. Vesicles were prepared by gel filtration through a Sephadex G-25-50 column equilibrated with 20 mM HEPES, pH 8.0, 1 mM EDTA, 1 mM DTT buffer of the lipid/cholate solution with or without 80 µg of purified bovine brain beta gamma subunits. Fractions containing beta gamma were pooled, snap-frozen in aliquots in liquid nitrogen, and stored at -80 °C. The amount of vesicle-bound beta gamma accessible on the beads was estimated by immunostaining with beta -specific polyclonal antibody (MS/1, amino-terminal beta -specific rabbit antisera, NEN Life Science Products), followed by staining with a secondary fluorescein 5-isothiocyanate-conjugated antibody and detection by flow cytometry.

Flow Cytometric Analysis-- Flow cytometric analyses of G protein subunit interactions were performed on a Becton Dickinson FACScan equipped with an air-cooled 15-watt argon ion laser. b-beta gamma was prebound to the avidin-coated beads within 3 h of each experiment. Beads (2 × 106/ml) were added to 2 nM b-beta gamma , incubated for 30 min at room temperature, washed to remove unbound b-beta gamma , and diluted in HEDNML (with 0.2 mM free Mg2+) to make the final beta gamma concentration 0.02-0.5 nM. For studies with lipids, vesicles were allowed to bind to avidin-coated beads in HEDNM (HEDNML without Lubrol) with 0.1% BSA and no detergent for 30 min at room temperature. In equilibrium binding experiments, 0.1-40 nM F-alpha was incubated in a 0.2-ml reaction volume with 105 beads/ml for 30 min at room temperature (21-24 °C) in the dark. Samples were run through the flow cytometer, and data were collected on the forward scatter (FSC), side scatter (SSC), and fluorescein (FL-1) channels. Data were gated on the singlet population of beads, which represents ~80% of all scattering events, and histograms of FL-1 fluorescence were obtained (see Fig. 1). Mean channel numbers for FL-1 were calculated from 3000 to 5000 events.

Association between 1 nM F-alpha and 0.1 nM b-beta gamma on the beads was monitored as the time-dependent increase in mean fluorescence channel number. Dissociation of subunits was initiated by addition of a 50-fold excess of unlabeled r-myr-alpha i1 over F-alpha . Both association and dissociation kinetics were approximated with a single exponential function, and the rate constants kon(app) and koff were determined, respectively. The Kd was calculated from the kinetic parameters as Kd = koff/kon, where kon was obtained from a slope and koff from the y intercept in a plot of kon(app) versus concentration of F-alpha (see Fig. 2, inset). Equilibrium binding parameters were determined from equilibrium data fit to a one-site binding hyperbola: [LR] = Bmax·[L]/(Kd + [L]).

G Protein Activation Kinetics-- The intrinsic fluorescence of alpha i(GDP)·beta gamma was measured using a stopped-flow spectrofluorometer (SX-17MV, Applied Photophysics Ltd., Leatherhead, United Kingdom). Purified r-myr-alpha i1 and bovine brain beta gamma subunits were mixed in equimolar concentrations in HEDNML and incubated for 20 min at 21 °C. Equal volumes of heterotrimer (200 nM) and a 2× stock of AMF (40 µM AlCl3, 20 mM MgCl2, and 20 mM NaF) in the same buffer were mixed, and the continuous time course of the fluorescence increase was measured with a photomultiplier tube behind a WG 320 filter (exciting at 290 nm with 9-nm slits).

Calculation of Free Magnesium-- All binding experiments were done with 0.2 mM free Mg2+ unless otherwise indicated. The final buffer composition was 50 mM HEPES, pH 8.0, 1 mM EDTA, 1 mM DTT, and 1.2 mM MgSO4. The concentration of free magnesium was calculated from total added MgCl2 using WinMAXC software (Stanford University, Pacific Grove, CA) using the BERS parameter set. The concentration of chelators, ionic strength, pH, and temperature of the buffer are taken into account by the analysis.

Data Analysis and Statistics-- Prism (GraphPad Software, San Diego, CA) was used for unweighted nonlinear least-squares fitting of all of the data.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

The use of flow cytometry for measuring high affinity F-alpha binding to b-beta gamma on avidin-coated beads is illustrated in Fig. 1A. Binding of 40 nM fluorescein-labeled Gi1alpha to 0.5 nM b-beta gamma (medium solid line) can be readily distinguished from nonspecific binding (thick solid line) in the histograms shown in Fig. 1B. Because the measured result is the mean of ~3000-5000 events, the mean channel numbers are very accurate, and replicates generally agreed within 2%. Nonspecific binding was only 15-20% of total binding even at 40 nM F-alpha . Avidin-coated beads without beta gamma gave essentially the same binding as beta gamma -bound beads in the presence of excess alpha subunit (data not shown). Accurate measurements of binding were routinely made with beta gamma concentrations as low as 100 pM (see Figs. 2-5), and even 20 pM beta gamma gave clear binding signals. The binding could be substantially decreased by preincubation of F-alpha with either GTPgamma S or AMF (Fig. 1B).


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Fig. 1.   Measuring protein/protein interactions by flow cytometry. A, shown is a schematic representation of F-alpha binding to b-beta gamma coupled to 6-µm Spherotech avidin-coated polystyrene beads. Upon binding to beta gamma , dilute F-alpha is locally concentrated on the beads, and fluorescence is detected above the low solution fluorescence in the flow cytometer. B, b-beta gamma was prebound to avidin-coated beads as described under "Experimental Procedures." F-alpha (40 nM) subjected to the indicated treatments was added to the b-beta gamma -bound beads in HEDNML (105 beads/ml, 100 pM b-beta gamma final concentration) and incubated for 30 min at room temperature, and then samples were analyzed on the flow cytometer. Data are gated on forward and side scattering to detect only the singlet population of beads, which represents ~80% of all scattering events. Data are shown as frequency histograms, with the number of beads on the y axis and fluorescence in channel numbers on the x axis. Medium and thick solid lines show total (no unlabeled alpha i1 added) and nonspecific (0.5 µM unlabeled r-myr-alpha i1) fluorescence for GDP-bound F-alpha , respectively. Thin and dashed lines show the signal from F-alpha preincubated with 10 µM GTPgamma S (3 h at 30 °C) or AMF (30 min at room temperature), respectively.

The time course of association and dissociation of 1 nM F-alpha with 100 pM b-beta gamma is illustrated in Fig. 2. The data were generally fit well by a single exponential association and dissociation, but in some experiments, two kinetic phases of dissociation were evident. The koff was 0.047 ± 0.005 min-1 (0.00078 s-1) with a half-time of 14.8 min. The dissociation upon addition of AMF was much faster, being nearly complete by the first time point at 1 min. The apparent on-rate (kon(app)) was measured at several concentrations of F-alpha , and individual rate constants were determined. A secondary plot of kon(app) versus [F-alpha ] (Fig. 2A, inset) gave a kon of 0.7 × 106 M-1 s-1 and a koff of 0.0013 s-1. This yields a kinetic Kd of 1.9 nM.


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Fig. 2.   Association and dissociation kinetics of F-alpha binding to b-beta gamma . A, association kinetics. F-alpha (1 nM) was added to beta gamma -bound beads (100 pM beta gamma ) in HEDNML at 22 °C alone (black-square) or in the presence of AMF (black-triangle) or to blank beads with no beta gamma (square ). Binding was determined by analyzing 3000-5000 beads at the indicated times. Data are means ± S.E. of three experiments performed in triplicate. Inset, the apparent rate constant of binding (kapp) of F-alpha to 100 pM beta gamma was measured over a range of F-alpha concentrations. The solid line is a linear regression kapp versus concentration of F-alpha . Data are from a single experiment repeated once with similar results. B, dissociation kinetics. F-alpha (1 nM) was preincubated with 100 pM beta gamma on the beads for 30 min, and dissociation at 22 °C was initiated by dilution in an equal volume of unlabeled alpha  subunit (50 nM final concentration; black-square) or 2× AMF (black-triangle). Data are means ± S.D. of two experiments performed in triplicate. Solid lines are fits of the data to single exponential association or dissociation functions.

Since magnesium profoundly affects the interaction of alpha  and beta gamma subunits, we examined the effect of free magnesium on alpha /beta gamma binding in Lubrol. As expected, there was greater binding at lower magnesium concentrations (Fig. 3A). There appeared to be a biphasic curve with a plateau at the normal physiological magnesium concentration of 0.1-1 mM. Saturation curves with 0.2 or 9.8 mM free magnesium showed equivalent maximum binding capacities with an ~2-fold difference in Kd, 2.9 ± 0.8 (n = 7) and 4.4 ± 1.1 (n = 5), respectively (Fig. 3B). Other experiments with 20 ppm Lubrol or the monodisperse form C12E10 showed no significant difference in affinity from 0.1% Lubrol (data not shown).


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Fig. 3.   Effect of Mg2+ on the affinity between G protein subunits. A, binding of 500 pM F-alpha and 100 pM b-beta gamma was measured in the presence of the indicated concentrations of free Mg2+ (see "Experimental Procedures"). Nonspecific binding was subtracted, and data were normalized to the maximum value for each experiment (at no Mg2+ added). Nonspecific binding was essentially independent of Mg2+ and represented ~15% of maximum binding. Data are means ± S.E. of three independent experiments. The solid line is a nonlinear least-squares fit of the data to a two-site competition function with EC50 values of 2.5 µM and 8 mM. B, Scatchard plot of the binding of F-alpha (0.1-20 nM) in 0.2 (black-square) or 9.8 (black-triangle) mM free Mg2+ to 100 pM b-beta gamma . Lines are the result of linear regressions of the data. The data are from a single experiment repeated four times.

There have been questions raised recently about whether GTP analogs can fully dissociate subunits in low magnesium solutions (9). In our system, the binding of F-alpha was completely eliminated after full occupancy with GTPgamma S at 0.2 mM free Mg2+ (Fig. 4). Initial experiments showed evidence of some residual affinity, but this was due to incomplete occupancy of the alpha  subunit with nucleotide.2 Similarly, aluminum fluoride in the presence of 10 mM magnesium resulted in a Kd for alpha /beta gamma association of >100 nM.


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Fig. 4.   Saturation binding with GDP-, GTPgamma S-, and AMF-liganded F-alpha . Fluorescein-labeled Gi1alpha (80 nM) was preincubated for 3 h at 30 °C in HEDNML with 10 µM GDP (black-square) or GTPgamma S (bullet ) or for 30 min at room temperature with AMF (black-triangle). Equilibrium binding to 100 pM b-beta gamma was then determined as described under "Experimental Procedures." Data are means ± S.E. of three (GDP) or mean ± range of two (GTPgamma S and AMF) experiments. The solid line is a nonlinear least-squares fit to a hyperbolic binding function with a Kd of 2.9 nM, and the dashed lines are linear least-squares fits.

Interestingly, the affinity of subunits changed significantly with different detergents. Preliminary saturation experiments showed Kd values for F-alpha of 3, 30, and 50 nM for Lubrol, CHAPS, and cholate, respectively. Due to the lower affinity and higher nonspecific binding, the saturation data with cholate and CHAPS were less reliable. Thus, IC50 values for r-myr-alpha i1 were measured in competition with F-alpha (Fig. 5 and Table I). As in the direct binding studies, r-myr-alpha i1 competed ~10 and 30 times less well in CHAPS and cholate, respectively. Although the ratio of affinities in the three detergents were conserved, IC50 values for competition by r-myr-alpha i1 were consistently lower than Kd values determined in saturation experiments for F-alpha binding to beta gamma . This may reflect slightly weaker beta gamma binding by the labeled alpha  subunit or a heterogeneity of the beta gamma pool purified from bovine brain tissue. In the latter case, there could be a higher affinity in competition experiments due to the selective occupancy of high affinity beta gamma sites by the low labeled ligand concentrations.


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Fig. 5.   Competition binding of F-alpha to beta gamma in different detergents. b-beta gamma was bound to avidin-coated beads and exchanged by centrifugation in HEDNM containing three different detergents: 0.1% Lubrol (black-square), 0.4% cholate (bullet ), or 0.7% CHAPS (black-triangle). Unlabeled r-myr-alpha i1 (0.2-1000 nM) was premixed with either 1 nM F-alpha (Lubrol) or 2 nM F-alpha (cholate or CHAPS). The mixture of alpha  subunits was combined with the indicated beads and incubated for 1 h at room temperature. Binding of F-alpha was determined as described under "Experimental Procedures." Nonspecific binding expressed as mean channel numbers (3.0 in Lubrol, 7.1 in CHAPS, and 4.0 in cholate) was subtracted from the total fluorescence in the absence of competitors (23 in Lubrol, 14 in CHAPS, and 7.5 in cholate), and then data were normalized to maximum specific fluorescence. Data are means of three independent experiments, and solid lines are nonlinear least-square fits to a one-site competition function. Average IC50 values derived from the fits are summarized in Table I.

                              
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Table I
IC50 values for r-myr-alpha i1 binding to beta gamma in different environments
IC50 values were determined from one-site fits of competition binding curves (Fig. 5). Data represent means ± S.E. of at least three experiments in each group.

To examine G protein subunit binding affinity in a lipid environment, we prepared bovine brain lipid vesicles containing 1 mol % biotin-labeled lipid with and without unmodified beta gamma subunit. Incorporation of beta gamma into the lipid vesicles and vesicle binding to avidin beads were monitored by immunostaining with a beta -specific polyclonal antibody. There was very little competable binding of F-alpha to the beads with biotin-labeled lipids alone, but lipids containing beta gamma showed substantial specific binding (Fig. 6A). Similarly, when the biotin-labeled lipids were left out of the reconstitution mixture, there was no specific binding of F-alpha to the beads (provided BSA was included in the incubation and wash buffers). This demonstrates the requirement for biotin-labeled lipids and rules out nonspecific absorption of lipid vesicles to the avidin-coated beads. The affinity for specific F-alpha binding to beta gamma in brain lipid vesicles (Kd = 8.6 ± 1.8 nM; Fig. 6B) was slightly lower than the affinity in Lubrol (Kd = 2.9 ± 0.8 nM). The affinity of F-alpha for beta gamma in vesicles prepared from pure dimyristoylphosphatidylcholine was similar to that in bovine brain lipids (Kd = 6.8 ± 1.4 nM; data not shown). In competition studies, the IC50 of r-myr-alpha i1 for beta gamma in brain lipids was also a factor of 2 greater than that in Lubrol (Table I).


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Fig. 6.   Binding of F-alpha to beta gamma in lipid vesicles. Biotin-labeled lipid-containing vesicles with and without non-biotin-labeled beta gamma (bg) were prepared by gel filtration as described under "Experimental Procedures." Vesicles were allowed to bind to avidin-coated beads in HEDNM (with 0.2 mM free Mg2+) with 0.1% BSA and no detergent for 30 min at room temperature. A, total and nonspecific F-alpha binding to the beta gamma -containing lipid vesicles versus lipid vesicles alone. F-alpha (20 nM) was incubated with beads coupled to beta gamma -containing vesicles, or the corresponding amount of lipid vesicles without beta gamma was added (solid bars). Nonspecific binding was defined by addition of 1 µM unlabeled r-myr-alpha i1 to the vesicles before F-alpha was added (hatched bars). Data are from a single experiment repeated two times with similar results. B, saturation binding of F-alpha to beta gamma in brain lipid vesicles. Equilibrium binding of F-alpha in HEDNM (with 0.1% BSA) to 0.1 nM beta gamma in lipid vesicles was measured as described under "Experimental Procedures." Data are means ± S.E. of three independent experiments. Nonspecific binding in the presence of a 50-fold excess of unlabeled r-myr-alpha i1 was subtracted and fit to a hyperbolic binding function (dashed line).

With the ability to measure G protein subunit interactions in lipid membranes, we wanted to determine whether G protein subunit dissociation would lead to rapid Galpha dissociation from the membrane. Heterotrimer was assembled by preincubation of F-alpha with the beta gamma -containing biotinylated bovine brain lipid vesicles coupled to the beads. Dissociation of F-alpha was initiated by adding a 50-fold excess of unlabeled r-myr-alpha i1 or by activation with aluminum fluoride. Both treatments resulted in F-alpha dissociation from the vesicles (Fig. 7). In agreement with the dissociation rates for the heterotrimer in Lubrol solution, AMF-induced release of F-alpha was rapid (koff = 0.047 min-1). As expected from the lower affinity between subunits in the lipid environment than in Lubrol, the dissociation rate from the surface of the vesicles was ~2-5-fold faster than that in Lubrol (koff = 0.28 min-1). These data show that r-myr-alpha i1 can rapidly dissociate from a lipid vesicle surface following G protein activation. The incomplete dissociation of F-alpha upon AMF activation may be due to some small residual affinity of AMF-bound alpha  subunits for beta gamma or lipids (Figs. 4 and 6A).


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Fig. 7.   Dissociation kinetics of F-alpha from beta gamma in brain lipid vesicles. Unlabeled beta gamma subunits were reconstituted into the bovine brain lipid vesicles with 1% biotinylated lipid as described under "Experimental Procedures." Vesicles were preincubated with avidin-coated beads for 30 min at room temperature in HEDNM with 0.1% BSA. Particles were then centrifuged for 2 min in an Eppendorf microcentrifuge, and the supernatant was exchanged two times to remove any unbound material. Fluorescein-labeled Gi1alpha (2 nM) was added to the beta gamma -containing vesicles coupled to the beads and incubated for 30 min at room temperature. Dissociation of F-alpha from beta gamma was initiated at 22 °C either by competing with a 50-fold excess of unlabeled r-myr-alpha i1 (black-triangle) or by sequential addition of AMF (black-square). Data are means ± S.D. of duplicate determinations from a single experiment representative of two independent experiments.

By combining two real-time measurements, namely spectroscopic analysis of intrinsic tryptophan fluorescence of the heterotrimer and flow cytometric detection of physical separation of the subunits, the sequence of the events involved in G protein activation could be resolved. The increase in Galpha tryptophan fluorescence has been used as a measure of conformational changes accompanying G protein activation (20). Rapid kinetics of the alpha i1 intrinsic fluorescence increase were recorded using a stopped-flow spectrofluorometer. Addition of AMF resulted in a 10-15% increase in fluorescence of the alpha i1·beta gamma heterotrimer. Controls using HEDNML instead of AMF or mixing of boiled r-myr-alpha i1·beta gamma with AMF showed no change in the intensity of the fluorescence. The AMF activation-induced dissociation of F-alpha from the F-alpha ·b-beta gamma heterotrimer assembled on the avidin-coated beads was analyzed by continuous acquisition mode on the flow cytometer. The rate of Galpha activation monitored by intrinsic fluorescence was ~3-fold faster than the rate of subunit dissociation by flow cytometry (Fig. 8). This indicates that an activating conformational change in the alpha  subunit precedes subunit dissociation (see "Discussion"). This was not a difference between labeled and unlabeled protein as similar rates of AMF-induced fluorescence change were seen with F-alpha ·beta gamma . If there was any difference, F-alpha gave slightly faster kinetics (data not shown).


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Fig. 8.   Kinetics of activation-induced conformational change and dissociation of the heterotrimer. The time courses of the AMF-induced increase in intrinsic tryptophan fluorescence and of the release of F-alpha from b-beta gamma attached to beads are compared on the same time scale. Purified r-myr-alpha i1 and bovine brain beta gamma were mixed in an equimolar ratio in HEDNML and incubated for 20 min at room temperature (22 °C). Equal volumes of 400 nM heterotrimer and 2× AMF were mixed in the stopped-flow spectrofluorometer, and tryptophan fluorescence of alpha i1(GDP)·beta gamma was measured by exciting at 290 nm and monitoring emission at wavelengths above 320 nm (see "Experimental Procedures"). Data were collected every 2 ms, and are averages of eight traces from a single experiment representative of four independent experiments. The fluorescence increase is ~15% of the starting fluorescence. The AMF-induced dissociation of F-alpha from the F-alpha ·b-beta gamma heterotrimer assembled on the beads was monitored by flow cytometry. Fluorescein-labeled Gi1alpha (2 nM) was incubated for 20 min at room temperature in HEDNML with 100 pM b-beta gamma precoupled to avidin-coated beads as described under "Experimental Procedures." Fluorescence from the beads was measured to provide the value of maximum binding, and then the samples were diluted with equal volumes of 2× AMF in the same buffer. The data were collected in a continuous "list mode" data acquisition option of the flow cytometer. With the flow cytometric data, sample addition and mixing prevented acquisition of data earlier than 10 s. More than 10,000 data points were accumulated during the 200-s time course, so the data were smoothed by averaging every 10 time points (black-square). Data shown are from a single measurement repeated at least two times in each of three independent experiments. The kinetics for both G protein activation and subunit dissociation were best fit by a two-exponent function. Both phases were faster for the intrinsic fluorescence change. The weighted mean t1/2 of Galpha activation was 15 s, and that for subunit dissociation was 48 s.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The results described here extend our knowledge of G protein subunit interactions and how they relate to the lipid environment and G protein activation mechanisms. This novel flow cytometric approach permitted us to address quantitatively the role of detergent, lipid environment, and regulatory factors affecting G protein alpha  and beta gamma subunit binding. Also, we show that Galpha conformational changes precede subunit dissociation and that upon G protein activation in lipid vesicles, a myristoylated alpha  subunit rapidly dissociates from the membrane.

Previous attempts to determine affinities between G protein subunits using different experimental approaches have resulted in a range of the dissociation constants. Kohnken and Hildebrandt (21) used Western blots to detect binding of the alpha  subunit to biotinyl-beta gamma immobilized on streptavidin-agarose. The apparent Kd values in 0.05% Lubrol-PX were ~20 nM for alpha 41 and ~350 nM for alpha 39. The last value was at least three times larger that the EC50 measured indirectly from the beta gamma -supported ADP-ribosylation of alpha 39 by pertussis toxin (22, 23). Helmreich and co-workers (24) used steady-state fluorescence energy transfer to examine the interaction between fluorescein-labeled Goalpha and rhodamine-labeled brain beta gamma subunits. In 0.25% Lubrol, the apparent Kd for specific GTPgamma S-sensitive binding was 10 nM. The signal-to-noise ratio in these studies was limited as only 20% quenching of fluorescence was seen. In comprehensive studies by Ueda et al. (25), the inhibitory effect of six different beta gamma forms on the steady-state GTPase activity of alpha  subunits was determined in 0.001-0.05% C12E10. Both Gsalpha and Goalpha showed EC50 values of 2 nM for beta 1gamma 1 and 0.2-0.5 nM for all other beta gamma complexes. Gi2alpha GTPase was inhibited with an EC50 of ~0.4 nM by all beta gamma subunits tested. In our analysis of resonance energy transfer between fluorescein-labeled Goalpha and eosin-labeled beta gamma by stopped-flow kinetics, we observed only 5% quenching of F-alpha fluorescence and could only say that the equilibrium Kd was <60 nM (26). Thus, the present data showing a dissociation constant of 1-3 nM in 0.002-0.1% Lubrol are generally consistent with previous literature reports. The advantages of this approach are as follows: 1) direct measurement of the binding interaction rather than an indirect functional measure; 2) the simplicity of the method and the reproducibility of results; and 3) its easy application to a wide variety of experimental conditions, which has allowed us to test the effects of many factors that regulate subunit interaction.

Flow cytometry is equally suitable for continuous analysis of real-time binding or dissociation kinetics (10, 11, 27). We defined the on- and off-rate constants for alpha -GDP and beta gamma , and the binding is quite fast; kon is 0.7 × 106 M-1 s-1. The dissociation of the alpha i·beta gamma complex upon AMF activation has been shown previously by its ability to reverse the aggregation of alpha i in the presence of beta gamma (28). Cerione and co-workers (8) proposed that the dissociation of transducin subunits by AMF precedes the tryptophan fluorescence change usually attributed to activation. A potential problem with that study is the indirect measurements of dissociation by subunit exchange and fluorescence energy transfer. Our direct determination of the dissociation rate of the alpha i·beta gamma heterotrimer found it to be 3-fold slower than the rate of the G protein conformational change accompanying activation. This suggests a simpler model in which the conformational change occurs first, followed by dissociation.

G protein subunit interactions are strongly influenced by magnesium (21, 24, 29-31). We observed a 2-fold reduction in alpha /beta gamma binding affinity by increasing the free MgCl2 concentration from 0.2 to 10 mM, and binding was reduced even further in 40 mM MgCl2. This nucleotide-independent dissociation at very high magnesium is important to keep in mind in evaluating G protein subunit interactions. Because of these observations, all other studies in this paper used a physiologically relevant free Mg2+ concentration of 200 µM. The effect of GTPgamma S to completely prevent formation of the F-alpha ·b-beta gamma heterocomplex even in the presence of physiological concentrations (0.2 mM free MgCl2) is at odds with a recent report for alpha s that indicated that very high magnesium concentrations were required for subunit dissociation (9). It will be interesting to examine alpha s/beta gamma interactions with the flow cytometric method to further evaluate this possible discrepancy.

One aim of this study was to quantitate interactions between G protein subunits in detergent micelles and in lipid vesicles under similar conditions. Several frequently used detergents had significant effects on the affinity of heterocomplex formation. Sodium cholate (0.4%) and CHAPS (0.7%) reduced the affinity of alpha  and beta gamma by >10-30-fold. Reconstitution of beta gamma into biotinylated phospholipid vesicles appeared to preserve the high affinity interaction with fluorescently labeled r-myr-alpha i1 with an excellent signal-to-noise ratio. The affinity for alpha /beta gamma binding on the surface of lipid vesicles was 2-3-fold lower than that observed in 0.1% Lubrol, which is consistent with the difference previously reported for alpha o using resonance energy transfer (Kd = 10 nM in 0.2% Lubrol versus 30 nM in lipid vesicles) (24).

Dissociation of myristoylated F-alpha from the membrane surface following G protein activation by AMF is consistent with the earlier observations where activation of the heterotrimer led to a slow, partial release of alpha o and alpha i from the cell membranes (32, 33) or phospholipid vesicles (28). The incomplete dissociation of F-alpha shown in Fig. 7 could indicate that a fraction of the F-alpha pool is not activated by AMF, or it could reflect low affinity binding of F-alpha to lipid-incorporated beta gamma in the presence of AMF. The failure of GTPgamma S treatment to induce alpha  release in other systems (34, 35) suggested that alpha  subunits are tightly attached to cell membranes through a mechanism that may be independent of interaction with the beta gamma complex. Recent data indicate that dual fatty acid modification of alpha  contributes to a membrane association of the subunit, with palmitoylation providing stronger association with membrane lipids (36-38) and myristoylation enhancing the affinity of alpha  for beta gamma subunits (12, 39). The discrepancy in the binding of alpha  to native membranes versus lipid vesicles could be explained by differences in lipid modification of alpha  subunits or by binding to some other proteins, distinct from the beta gamma subunit, which could anchor alpha  more tightly to the membrane.

The simplicity and sensitivity of the flow cytometric approach for measuring G protein subunit interactions in both detergent and lipid environment permit testing of several important questions. The ability to analyze the dynamics of heterotrimer complex formation and disassembly upon activation with guanine nucleotides and receptors in reconstituted systems is an exciting future direction.

    ACKNOWLEDGEMENTS

We gratefully acknowledge Drs. Larry A. Sklar (University of New Mexico) and John P. Nolan (Los Alamos National Laboratory) for the suggestion of flow cytometry and consultation support from the National Flow Cytometry Resource supported by National Institutes of Health Grant RR01315. We are grateful to Drs. Maurine E. Linder and Jeffrey I. Gordon for generously providing plasmids for the Gi1alpha protein and N-myristoyltransferase. We thank Dr. Latham J. Claflin (Department of Microbiology, University of Michigan) for help in flow cytometry.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant GM 39561 (to R. R. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by Research Fellowship Award 25F967 from the American Heart Association, Michigan Affiliate.

par To whom correspondence should be addressed: Dept. of Pharmacology, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0632. Tel.: 734-763-3650; Fax: 734-763-4450; E-mail: RNeubig{at}umich.edu.

1 The abbreviations used are: b-beta gamma , biotinylated Gbeta gamma ; F-alpha , fluorescein-labeled Gi1alpha ; r-myr-alpha i1, recombinant myristoylated G protein alpha i1 subunit; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); DTT, dithiothreitol; BSA, bovine serum albumin; CHAPS, 3-[(cholamidopropyl)diethylammonio]-1propanesulfonate.

2 Even with 90% saturation of the alpha  subunit with GTPgamma S, there was sufficient unoccupied alpha  subunit to give an apparent Kd of 20 nM (versus 2 nM for GDP-bound alpha  subunit).

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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