Ligand-receptor-G-protein molecular assemblies on beads for mechanistic studies and screening by flow cytometry.
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-Gialpha2 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-Gialpha2 fusion protein for Gbetagamma, 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 Galphabetagamma disassembly.
Department of Pathology and Cancer Center, University of New Mexico HSC, Albuquerque, NM 87131, USA.
Peter C
PC
Simons
Mei
M
Shi
Terry
T
Foutz
Daniel F
DF
Cimino
Jeremy
J
Lewis
Tione
T
Buranda
William K
WK
Lim
Richard R
RR
Neubig
William E
WE
McIntire
James
J
Garrison
Eric
E
Prossnitz
Larry A
LA
Sklar
D005434:Flow Cytometry / Q000379:methods*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008863:Microspheres; D012995:Solubility; D014407:Tumor Cells, Cultured
D008024:Ligands; D019204:GTP-Binding Proteins