Ligand-Receptor-G-Protein Molecular Assemblies on Beads for Mechanistic Studies and Screening by Flow Cytometry

Abstract

G protein-coupled receptors form a ternary complex of ligand, receptor, and G protein heterotrimer (LRG) during signal transduction from the outside to the inside of a cell. Our goal was to develop a homogeneous, small-volume, bead-based approach compatible with high-throughput flow cytometry that would allow evaluation of G protein coupled receptor molecular assemblies. Dextran beads were derivatized to carry chelated nickel to bind hexahistidine-tagged green fluorescent protein (GFP) and hexahistidine-tagged G proteins. Ternary complexes were assembled on these beads using fluorescent ligand with wild-type receptor or a receptor-Gi␣2 fusion protein, and with a nonfluorescent ligand and receptor-GFP fusion protein. Streptavidin-coated polystyrene beads used biotinylated anti-FLAG antibodies to bind FLAG-tagged G proteins for ternary complex assembly. Validation was achieved by showing time and concentration dependence of ternary complex formation. Affinity measurements of ligand for receptor on particles, of the ligand-receptor complex for G protein on the particles, and receptor-Gi␣2 fusion protein for G␤␥, were consistent with comparable assemblies in detergent suspension. Performance was assessed in applications representing the potential of these assemblies for ternary complex mechanisms. We showed the relationship for a family of ligands between LR and LRG affinity and characterized the affinity of both the wild-type and GFP fusion receptors with G protein. We also showed the potential of kinetic measurements to allow observation of individual steps of GTP-induced ternary complex disassembly and discriminated a fast step caused by RG disassembly compared with the slower step of G␣␤␥ disassembly.GPCRs interact with extracellular stimuli, such as photons, hormones, neurotransmitters, and odorants (Gilman, 1995). These stimuli cause conformational changes in the receptor, leading to binding of intracellular G protein heterotrimers, each with one copy of a guanyl nucleotide binding ␣ subunit and a ␤␥ dimer (Neer, 1995). After stimulation, the ␣ subunit binds GTP, which promotes dissociation of the ␣ subunit from the ␤␥ dimer, exposing new surfaces to cytoplasmic effectors, such as adenylyl cyclase and phospholipase C. The human genome contains ϳ600 GPCR genes, 27 ␣, 5 ␤, and 13 ␥ (Venter et al., 2001), with smaller numbers of these G proteins (17, 5, and 12, respectively) found to date. With such large numbers, determining how productively any given GPCR couples to a particular ␣␤␥ heterotrimer is daunting (1020 ␣␤␥ combinations alone). The assembly of a high agonist-affinity complex is a good criterion of productive partners (Gilman, 1987).The formyl peptide receptor (FPR) responds to the presence of N-formyl methionine-containing peptides resulting from bacterial and mitochondrial protein synthesis, as well as other hydrophobic peptides (Gao et al., 1994). This receptor has served as a model for signal transduction in phagocytic cells and for inflammatory and autoimmune diseases (Prossnitz and Ye, 1...

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

“…Bead-based studies have compared ternary complexes assembled with chimeric FPR fusion proteins [13]. The results, interpreted according to a ternary complex model [15], were consistent with the idea that the dissociation of the α and βγ subunits in the presence of GTPγS was slow compared to the typical kinetics of intracellular signals such as intracellular Ca 2+ elevation and therefore could not likely account, kinetically, for cell activation.…”

“…α i2 and α i3 G-protein subunits were purchased from Calbiochem (La Jolla, CA) and were used without further purification. FLAG-his tagged βγ subunits (β 1 γ 2 or β 4 γ 2 ) were prepared and purified as described previously [13,20]. The subunits were stored at −80°C in 2-5µL volume aliquots depending on stock concentration (3-7µM).…”

“…The prototypical ligand, N-formyl-Met-Leu-Phe is capable of initiating leukocyte inflammatory responses such as adhesion, chemotaxis, superoxide generation and degranulation [11]. The receptor, cloned and overexpressed in tissue culture cells, has been solubilized and assembled with a formyl peptide ligand and G proteins to form a high agonist-affinity ternary complex, both in solution [12], or on beads, where it has been investigated by flow cytometry [13]. The detergent solubilized FPRs preserve native ligand binding affinity, and exhibit evidence of appropriate G protein assembly that discriminates full and partial agonists [14].…”

“…In earlier studies, FPR ternary complexes reconstituted in detergent were analyzed in terms of the sensitivity of ligand dissociation kinetics to the presence of guanine nucleotides [12,13]. For clarity we will summarize the results of the two principal studies here.…”

“…The results of that study showed that the dissociation kinetics of a fluorescent ligand from a G protein coupled FPR (τ 1/2 ≤ 100sec) were much slower than the uncoupled FPR (τ 1/2 ≈ 20sec). A follow up study involved the assembly of ternary complexes of detergent solubilized GPCRs on beads designed to examine individual steps associated with GTP-mediated disassembly of ternary complexes [13]. These studies did not have adequate time resolution to define precisely the disassembly kinetics.…”