Chapter 11. Subsecond analyses of G-protein coupled-receptor ternary complex dynamics by rapid mix flow cytometry

Abstract

The binding of full and partial agonist ligands (L) to G-protein-coupled receptors (GPCRs) initiates the formation of ternary complexes with G-proteins (LRG complexes). We describe the assembly of detergent-solubilized LRG complexes on beads. Rapid mix flow cytometry is used to analyze the subsecond dynamics of guanine nucleotide-mediated ternary complex disassembly. Ternary complexes were assembled with three formyl peptide receptor constructs (wild type, FPR-Galpha(i2) fusion, and FPR-GFP fusion) and two isotypes of the alpha subunit (alpha(i2) and alpha(i3)) and betagamma dimer (beta(i)(1)gamma(2) and beta(4)gamma(2)). Experimental evidence suggests that thermodynamic stability of ternary complexes depends on subunit isotype. Comparison of assemblies derived from the three constructs of FPR and G-protein heterotrimers composed of the available subunit isotypes demonstrate that the fast step is 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

Schematic of small volume rapid mix device. Mixing and delivery of fluid samples to the flow cytometer are initiated with the three syringes labeled, “Buffer” and “Double Barrel” (Syringes1 and 2 define cell/bead and ligand/reagent loading respectively). The volumes shown refer to the maximum capacity of the syringes. Samples are loaded into Syringes 1 and 2 through the sample-loading paths of the loading “Y” connectors. The Buffer Syringe3 is used to push samples from the delay line into the flow cytometer. A computer program is used to control the sequence in which syringe pumps and valves are activated. Syringe4 is used to clean the delay line without affecting the loaded samples in the double barrel syringes after each run. Two three-way Teflon solenoid, mixing valves (1 and 2) are used to isolate the delay line from the rest of the sample lines. Mixing Valve 1 is used to switch flow between the Double Barrel syringes and the Buffer syringe. A 13.2 cm (60μL) delay line is used to carry the mixed samples. Mixing Valve 2 is positioned at the end of the delay line and is used to direct sample flow either into the flow cytometer or to waste.

Figure 2

Schematics of modular molecular assemblies of ternary complexes on beads. Numbered arrows refer to junction points that are likely to break in the when complexes are activated by guanine nucleotides. A socket and plug connecter is utilized to depict the very high affinity interaction of the epitope tag and βγ-subunits of the G protein (circles labeled with β and γ) that are fused with a FLAG epitope tag, which recognizes the biotinylated M2 antiFLAG antibodies on streptavidin-coated beads. The modular setup of G protein heterotrimers allows for α_i-subunits for capturing receptors (R). Fluorescent components such as GFP or ligand are indicated in green. See text for details.