Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides

Abstract

We have used rapid-mix flow cytometry to analyze the early subsecond dynamics of the disassembly of ternary complexes of G protein-coupled receptors (GPCRs) immobilized on beads to examine individual steps associated with guanine nucleotide activation. Our earlier studies suggested that the slow dissociation of Galpha and Gbetagamma subunits was unlikely to be an essential component of cell activation. However, these studies did not have adequate time resolution to define precisely the disassembly kinetics. Ternary complexes were assembled using three formyl peptide receptor constructs (wild type, formyl peptide receptor-Galpha(i2) fusion, and formyl peptide receptor-green fluorescent protein fusion) and two isotypes of the alpha subunit (alpha(i2) and alpha(i3)) and betagamma dimer (beta(1)gamma(2) and beta(4)gamma(2)). At saturating nucleotide levels, the disassembly of a significant fraction of ternary complexes occurred on a subsecond time frame for alpha(i2) complexes and tau(1/2)< or =4s for alpha(i3) complexes, time scales that are compatible with cell activation. beta(1)gamma(2) isotype complexes were generally more stable than beta(4)gamma(2)-associated complexes. The comparison of the three constructs, however, proved that the fast step was associated with the separation of receptor and G protein and that the dissociation of the ligand or of the alpha and betagamma subunits was slower. These results are compatible with a cell activation model involving G protein conformational changes rather than disassembly of Galphabetagamma heterotrimer.

Figure 1. Assembly of G protein and ternary complex

A. Binding curve of fluorescein labeled Gα_i3^fl subunits to βγ subunits on beads. Total binding (filled circles), specific binding (open squares), and non-specific binding (filled squares) are shown in curves a, b, and c, respectively. The FLAG epitope-tagged βγ subunits are attached to the beads via M2 antibodies. Beads displaying M2 without βγ subunits were used as controls for nonspecific binding. The K_d was 32 nM (insert). B. Comparison of ternary complex formation with Gα_i3 and Gα_i2 subunits. Titration of ligand (fMLFF) activated FPR-GFP on G beads of the type β₁γ₂•α_i2 or β₁γ₂•α_i3.

Figure 2. A. Stability of bead-based assemblies

Plot of mean channel fluorescence (MCF) of G-beads versus time. The arrow indicates the addition of 0.1mM GTPγS to Gα_i3^fl bearing G beads. Data show that GTPγS does not cause subunit dissociation, when the nucleotide-binding pocket is blocked by high affinity binding GDP. B. (a) Dissociation of Gα_i3^fl•GDP•AlF₄⁻ from the βγ subunit on G beads in the presence of AlF₄⁻. The G beads appear to bear two distinct populations of G proteins corresponding to the biphasic dissociation of Gα_i3^fl•GDP•AlF₄⁻. These characteristics have been previously described separately in the literature. (b) Self-exchange of Gα_i3^fl with unlabeled Gα_i3 subunits. C. Dissassembly of L•FPR^GFP-Gαβγ after manual addition of GTPγS to G beads on a flow cytometer. Data show (a) the fluorescence time course of the complex after dilution with buffer, (b) dissociation from beads of LFPR^GFP after rapid mixing with 0.2mM GTPγS, and (c) background fluorescence of L•FPR^GFP-Gαβγ prepared in the presence of GTPγS. The data corresponding to the rapid kinetics of disassembly are lost during the 10 second deadtime.

Figure 3. Rapid mix disassembly of FPR-Gα_i2 ternary complex

Red arrows in the scheme show junction points of disassembly, with the larger arrow indicating the weaker link that actually is shown to break in the experiment. A. Data show (a) filled squares: the fluorescence time course of the complex after dilution with buffer, (b) open squares: dissociation from beads of fMLFK-FITC (L^F) after rapid mixing with 0.2 mM GTPγS, and (c) filled circles: background fluorescence of L^FR-α_i2β₁γ₂ prepared in the presence of GTPγS. B. Dilution and background corrected data showing measurements of L^F dissociation kinetics from Gα_i2-FPR complexed to β₁γ₂ subunits on beads after the addition of 0.1 mM GTPγS. The data were fit to a single exponential model; the half time τ_1/2 of GDP is ≈ 34 sec (data not shown), compared to that of GTPγS (≈ 18 sec). C. Manual mix flow cytometry measurements of the putative dissociation of L^F from Gα_i2-FPR on G beads on beads after the addition of GTPγS. Data sets show (a) open squares: effect of dilution on L^FR-α_i2β₁γ₂ in DHPSM buffer, (b) dotted line: represents the apparent inhibition of L^F dissociation by 20 nM L^F from the ternary complex following addition of 0.1 mM GTPγS which uncouples the receptor ligand high affinity interaction from the G protein. (c) crosses: 0.1 mM GTPγS added to ternary complex. (d) filled circles: background fluorescence of L^FR-α_i2β₁γ₂ prepared in the presence of GTPγS.

Figure 4. Rapid mix disassembly of FPR-GFP (R^F) ternary complex

Red arrows in the scheme show junction points of disassembly, with the larger arrow indicating the weaker link that actually is shown to break in the experiment. A. Representative data show (a) the fluorescence time course of the complex after dilution with buffer, (b) dissociation from beads of R^F after rapid mixing with 0.2 mM GTPγS, and (c) background fluorescence of LR^Fα_i2β₁γ₂ prepared in the presence of GTPγS. B. Dilution and background corrected data showing measurements of R^F dissociation kinetics from G beads following the addition of 0.1 mM GDP (a) or 0.1 mM GTPγS (b). The data were fit to a two-phase exponential model. The effect of the two nucleotides is distinguished by the half time τ_1/2 of the fast components: for GDP the half time is ≈ 1.2 sec, compared to ≈ 0.7 sec for GTPγS. The halftime values for the subsecond decay are estimates, constrained by the time resolution of the data.

Figure 5. Representative disassembly of wildtype FPR ternary complex

Red arrows in the scheme show junction points of disassembly, with the large arrow indicating the weaker link that actually is shown to break in the experiment. A. Data show (a) the fluorescence time course of the complex after dilution with buffer, (b) dissociation from beads of L^F after rapid mixing with 0.2 mM GTPγS, and (c) background fluorescence of L^FRα_i2β₁γ₂ prepared in the presence of GTPγS. B. Dilution and background corrected data showing measurements of L^FR dissociation kinetics from Gα_i2β₁γ₂ beads after the addition of 0.1 mM GTPγS. C. Dilution and background corrected data showing measurements of L^FR dissociation kinetics from Gα_i3β₁γ₂ beads after the addition of 0.1 mM GTPγS.