Chapter 11. Subsecond analyses of G-protein coupled-receptor ternary complex dynamics by rapid mix flow cytometry.
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.
Buranda
Tione
T
Department of Pathology and Cancer Center, University of New Mexico Health Science Center, Albuquerque, New Mexico, USA.
Wu
Yang
Y
Sklar
Larry A
LA
D005434:Flow Cytometry / Q000379:methods*; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics; D020298:U937 Cells
D043562:Receptors, G-Protein-Coupled; D020962:Heterotrimeric GTP-Binding Proteins